WO2007042776A1

WO2007042776A1 - Modulators of the purinergic signalling pathway for treating sodium homeostatsis, hypertension and aldosteronism

Info

Publication number: WO2007042776A1
Application number: PCT/GB2006/003738
Authority: WO
Inventors: Christopher F. Edwards; Yuri Korchev
Original assignee: Imperial College Innovations Limited
Priority date: 2005-10-07
Filing date: 2006-10-06
Publication date: 2007-04-19
Also published as: GB2446735A; GB0520405D0; JP2009511457A; GB0807031D0

Abstract

The invention relates to agents and methods for preventing and/or alleviating a condition in an individual associated with abnormal sodium homeostasis wherein the compound is capable of modulating the activity of a component of the purinergic signalling pathway. In particular, the invention relates to agents and methods for preventing and/or alleviating a condition in an individual selected from the group comprising: primary aldosteronism; secondary aldosteronism; heightened sensitivity to aldosterone and/or essential hypertension.

Description

MODULATORS OF THE PURINERGIC SIGNALLING PATHWAY FOR TREATING SODIUM HOMEOSTATSIS, HYPERTENSION AND ALDOSTERONISM

hi mammals, levels of sodium in the plasma are controlled by the kidneys. Typically, the normal plasma sodium concentration in man is 140 mM, meaning that approximately 25,000 mmol of sodium are filtered by the kidneys per day. To maintain overall sodium balance excretion must equal dietary salt intake, which is usually 100-200 mmol/day, and the kidney must therefore reabsorb almost all the filtered sodium. This is accomplished by an integrated system of ion channels, exchangers and transporters (Schrier, 2003): 60% of the filtered sodium is reabsorbed in the proximal tubule of the nephron, largely by Na⁺ZH⁺ exchange; 30% is reabsorbed in the thick ascending limb of Henle's loop by Na -K -2Cl^" co- transport; 7% is reclaimed by Na⁺-Cl^" co-transport in the distal convoluted tubule; and the last 2% is reabsorbed via the epithelial Na⁺ channel (ENaC) in the cortical collecting tubule (CCT), which also called the cortical collecting duct (CCD)

^■ (Garty & Palmer, 1997; Lifton et al, 2001). Whilst this last step accounts for only a small fraction of salt reabsorption, it is the principal site at which the net salt •balance is normally determined, as the activity of ENaG is highly regulated -by the renin-angiotensin-aldosterone system (RAAS) (Fraser et al, 1981; Gross, 1958; Lifton et al, 2001). The excess secretion of the final endocrine signal in RAAS, namely aldosterone, may result in human lrypertension (Brown et al, 1972; Connell et al, 2001; Fraser et al, 1981).

The sodium homeostasis regulatory hormone aldosterone was first identified in 1953 (Simpson et al, 1953) and is now known to promote the retention of sodium,

^■ the loss of magnesium and potassium, S3'mpathetic activation, paras3Ηipathetic inhibition, myocardial and vascular fibrosis, baroreceptor dysfunction, vascular damage and impairment of arterial compliance. Early studies on the biochemical mechanism of action were reviewed b}^ Edelman and Pimognari (1968). TMs work was greatly helped by the introduction by Ussing and Zerahn (1951) of the short-circuit current method for measuring sodium transport. Jean Crabbe first demonstrated the effect of aldosterone on sodium transport across toad bladder and skin and drew attention to the latent period of 60-90 minutes hi the action of aldosterone (Crabbe, 1961; 1963). He postulated the synthesis or activation of an intermediate by aldosterone. Work by Edehnan's group showed that inhibition of DNA-dependent KNA synthesis and of protein synthesis blocked the aldosterone effect (Edelman et ah, 1963). Edelman went on to demonstrate that aldosterone activation of sodium transport was critically dependent on ATP production (Edehnan et al, 1963). In substrate-depleted toad bladders aldosterone had little or no effect but the response was restored by adding glucose or pyruvate to the medium. Edehnan suggested that a possible explanation of this absolute dependence on substrate was that hi the energy-depleted system the rate of activation of sodium transport was hmited by the local concentration of ATP. However he felt that this was questionable given that substrate-depleted toad bladders responded to vasopressin and to amphotericin B with a normal increase in sodium transport. This work became known as the ATP generation hypothesis of aldosterone action with ATP inter alia increasing the amount of energy available for the sodium pump.

A number of conditions have been associated with abnormal levels of, or sensitivity to, aldosterone, in particular: primary aldosteronism; secondary aldosteronism; heightened sensitivity to aldosterone; and essential hypertension.

Primary aldosteronism was first described by Conn hi 1955 (Conn, 1955) and removal of a benign adrenal tumour resulted hi resolution of the hypertension and hypokalemia. Conn originally thought that 20% of patients with essential hypertension might have this condition (Conn et al., 1964) but this was recognised to be a gross over-estimate. More recent work using the measurement of the aldosterone-renin ratio has suggested that approximately 10% of patients with hypertension seen hi primary care might have this condition (Lim et ah, 1999). There are a number of distinct causes of primary aldosteronism (Edwards, 2001). Of these, the molecular mechanism has only been defined in glucocorticoid- suppressible hyperaldosteronism (Edwards, 2001). The other causes include primary aldosteronism due to a tumour of the zona glomerulosa, idiopathic zona glomerulosa hyperplasia (IHA)₅ primary adrenal hyperplasia and a familial condition described by Gordon and colleagues (Edwards, 2001). Padfield has suggested that IHA is part of a continuum with low renin essential hypertension (Padfield et ah, 1981). One potential explanation for IHA and LREH is that the zona glomerulosa is excessively sensitive to angiotensin II (Witzgall et άl., 1985).

Primary aldosteronism has been treated either surgically by the removal of an adrenal tumour or medically by drugs which either antagonise the action of aldosterone (e.g. spironolactone) or block renal tubular ionic transport (e.g. amiloride) (Edwards, 2001). Side-effects with spironolactone are common and include gynaecomastia and menstrual dysfunction. In appropriate dose in patients with adrenal tumour, spironolactone is effective in normalising the plasma potassium and lowering blood pressure. Inhibitors of renal tubular ionic transport are usually less effective in controlling blood pressure.

Patients with IHA should be treated individually and are usually given either spironolactone or amiloride in the first^' instance. The blood pressure response is often suboptimal and other drugs such as calcium channel blockers or ACE inhibitors are frequently added.

Secondary aldosteronism is a common condition found in patients with activation .. of the renin-angiotensin-aldosterone axis. Diseases associated with this include congestive cardiac failure, accelerated or malignant phase hypertension, cirrhosis of the liver and the nephrotic syndrome. Rare causes include a renin-secreting tumour. Current treatment involves either aldosterone antagonists or inhibitors of renal tubular ionic transport. A particular focus on the role of aldosterone emerged from the RALES study in which patients with severe congestive heart failure on optimal medical therapy were given spironolactone or placebo (Pitt et ah, 1999). The spironolactone group showed a 25% reduction in mortality. The causes of most conditions in which, there is apparent primary aldosteronism and of low renin essential hypertension remain unclear. Considerable evidence supports the idea that in essential lrypertension the blood pressure 'follows the kidney' (for example, transplantation of a lddney from a normotensive donor to a hypertensive one may result in normal blood pressure - Curtis et al._t 1983; Meneton et al, 2005). Indeed, many patients in whom there is no currently defined mechanism for their hypertension frequently have a family history of raised blood pressure. The treatment for essential hypertension is dependent on its severity but often includes diuretics, beta-blockers, ACE inhibitors and calcium channel blockers.

All this raises the possibility that there may be conditions in which the abnormality relates to an exaggerated sensitivity of the renal tubule to aldosterone (i.e. heightened sensitivity to aldosterone) resulting in excessive sodium retention with consequent inhibition of renin secretion. The end result would be a change in the aldosterone-renin ratio which could be diagnosed as primary aldosteronism.

On the treatment side current therapies for aldosterone excess are unsatisfactory either because of side effects or lack of efficacy.

The present inventors have now surprisingly discovered that aldosterone exerts its effect on sodium import by stimulating the release of ATP from the basolateral side of kidney epithelial cells which acts in a autocrine/paracrine system to activate a purinergic signalling pathway that stimulates sodium import. Since the purinergic pathway plays a key role in aldosterone action then inhibition of this pathway offers a novel way of treating conditions either with aldosterone excess or enhanced renal tubular sensitivity to aldosterone. Thus, the purinergic signalling pathway represents a therapeutic target for regulating conditions in which there is abnormal sodium homeostasis and also the biological effects of aldosterone.

Thus, in a first aspect the invention provides a use of a compound for preventing and/or alleviating a condition in an individual associated with abnormal sodium homeostasis wherein the compound is capable of modulating the activity of a component of the puriαergic signalling pathway.

By "abnormal sodium homeostasis" we mean those conditions in which there is either abnormal sodium retention (as in, for example, primary aldosteronism or other mineralocorticoid excess syndromes) or in which there is activation of the renin-angiotensin-aldosterone axis (as in, for example, diuretic therapy, congestive heart failure).

It will be understood that many conditions associated with abnormal sodium homeostasis could be- prevented and/or alleviated by modulating the activity of a component of the purinergic signalling pathway. For example, modulating the purinergic signalling pathway could be used to treat, among others, forms of aldosteronism, hypertension, cardiac-related disorders, congestive cardiac failure, accelerated or malignant phase hypertension, cirrhosis of the liver and the nephrotic syndrome, all of which are well known to those skilled in the relevant medical field.

Preferably, the invention provides a use wherein the condition in an individual is selected from the group comprising: primary aldosteronism; secondary aldosteronism; heightened sensitivity to aldosterone or other mineralocorticoids and/or essential hypertension, wherein the compound is capable of modulating the activity of a component of the purinergic signalling pathway.

By "primary aldosteronism" we include the condition caused by aldosterone production that is excessive to the needs of the individual's body and relatively autonomous of its normal chronic regulator, the renin-angiotensin system, which is suppressed. In early cases of primary aldosteronism, hypokalemia was thought to be present in the majority of patients, but in more recent studies the majority of patients are normokalemic. Very occasionally patients are normotensive and in some cases normal levels of plasma and urinary aldosterone have been found. Thus, "primary aldosteronism" may or may not include hypokalemia and/or patients that are normotensive. By "secondary aldosteronism" we include conditions resulting from activation of the renin-aήgiotensin-aldosterone axis. ^!

By "heightened sensitivity to aldosterone" we include conditions in which the renal tubule is abnormally or excessively responsive to the sodium-retaim^'ng action of aldosterone or other mineralocorticoids.

By "essential hypertension" we include the condition in those patients in whom there is no currently defined mechanism for their hypertension but who frequently have a family history of raised blood pressure.

The individual is preferably a human, but may be any mammal such as a domesticated mammal, preferably of agricultural significance including a horse,, pig, cow, sheep, dog and cat.

By "compound" we include any purified or isolated natural or chemically- synthesised molecule includrng a polynucleotide and/or a polypeptide and/or a small chemical molecule.

By "purinergic signalling pathway" we include the intracellular and extracellular machinery, components and events that are involved in the production of ATP or its metabolites and/or the generation, amplification;, transmission and subsequent inhibition of a cellular signal from the purinergic receptor agonist and purinergic receptors to the cellular and nuclear machinery responsible for bringing about a change in cell behaviour. The "purinergic signalling pathway" may or may not include those cellular steps involved in binding of aldosterone to the aldosterone receptor and its subsequent activation resulting in the release of ATP from the basolateral side of epithelial kidney cells. In particular, we include the purinergic signalling pathway in kidney cells or a cell in which there is a mineralocorticoid receptor.

Thus, by "component of the purinergic signalling pathway" we include a cellular signalling molecule involved in the generation, amplification, transmission and subsequent inhibition of a signal via the purinergic signalling pathway. It will be understood that such components may include one or more purinergic receptor agonist, purinergic receptor (for ^■ example, the Pl and/or P2 receptor), phosphatidylinositol-3-ldnase (also known as PD kinase or PDK)₅ calcium channel and/or transcription factor. The phrase "component of the purinergic signalling pathway" may or may not include one or more sodium channel such as the epithelial sodium channel, ENaC.

It will be understood that the activity of a component of the purinergic signalling pathway can be modulated by inducing and/or increasing or inhibiting and/or preventing the cellular function or the expression level of that component.

It will be understood by those skilled in the relevant medical field that abnormal sodium homeostasis can be determined by measuring plasma potassium levels, blood pressure and aldosterone-renin ratio, all of which are routine techniques to a person skilled in the relevant field of medicine or biochemistry. The level of sodium in the plasma is generally a poor indicator of sodium homeostasis because cellular mechanisms generally compensate for any imbalance in plasma sodium levels.

In a second aspect, the invention provides a use of a compound for inhibiting and/or reducing sodium import into one or more cell, wherein the compound is capable of modulating the activity of a component of the purinergic signalling ■pathway. The compound may or may not directly modulate the activity of a sodium channel, such as the epithelial sodium channel, ENaC. ^■ - - ^■

By "sodium import" we include the process of transport of sodium ions (for example, Na⁺) and/or sodium-containing compounds across a cell membrane into a cell. It will be understood that import is usually mediated by channels, such as ion channels, located in the cell membrane.

Down-regulation of Na^"1" absorption by extracellular ATP through P2 receptors on the apical membrane appears to be a general principle in renal epithelia (Kunzelmann et ah, 2001; McCoy et άl., 1999). However, there are also many reports that demonstrate an ATP-induced increase of Na⁺ transport across epithelia. Leipziger and colleagues (Leipziger et aϊ., 1997) have observed the stimulation OfNa⁺ transport in rat distal colonic mucosa, mediated by basolateral ATP. Brodin and Nielsen (Brodin & Nielsen, 2000) have reported basolateral ATP up-regulated Na⁺ absorption by P2Y and P2X purinergic receptors via an increase in cytosolic Ca2⁺-concentration in frog skin epithelium. Recently Fronius and co-workers (Fronius et ah, 2004) have shown that extracellular ATP also stimulates amiloride sensitive Na⁺ transport m ^' Xenopus lung epithelium.

It will be appreciated by a person skilled in the relevant art that this aspect of the invention includes a use of a compound for inhibiting and/or reducing sodium import into one or more cell in vivo (for example, in an individual) and/or ex vivo (for example, outside the body of an individual) and/or in vitro (for example, in a cell culture).

Preferably, the invention provides a use wherein the compound is capable of inhibiting and/or reducing the activity of a component of the purinergic signalling pathway.

Preferably, the invention provides a use wherein the compound is capable of activating and/or increasing the activity of a component of the purinergic signalling pathway.

It will be understood that activating and/or increasing the activity of a component of the purinergic signalling pathway could be used to modulate sodium import.

For example, activation of the P2 Y purinergic receptors on the apical surface of the renal epithelial cells is known to inhibit sodium transport. Thus, P2Y antagonists (such as suramin and/or pyridoxal phosphate-6-azo(benzene-2,4- disulfonic acid)) could be used to treat conditions in which there is sodium retention, for example caused by apical ATP excess.

Alternatively, increasing the activity of a component of the purinergic signalling pathway could be used to stimulate and/or increase sodium import, thereb}' providing an approach for the treatment of conditions in which there is reduced sodium import leading τo abnormal sodium homeostasis.

Preferably, the invention provides a use wherein the component of the purϊnergic signalling pathway is a purinergic receptor.

Purinergic receptors (also known as "purinoceptors") are cell surface receptors capable of binding to, and being activated by, extracellular adenosine triphosphate (ATP) and which are located on numerous cell types including epithelial cells, platelets, neutrophils, fibroblasts, smooth muscle cells and cells of the pancreas. Two receptor types have been described, termed Pl and P2, which differ in their specificity and affinity for ATP. P2 receptors are thought to have the higher affinity for ATP.

A number of classes of purinergic receptors have been identified and described. For example, P2 purinergic receptor classes include: P2Y, P2X, P2X4 and P2X6.

P2X receptors are controllers of ligand-gated cation channels (Jans et al., 2002). P2Y receptors are G-protein coupled receptors (GPCRs). Localisation studies performed by Wolff et al. (2005) have shown that P2Y(1), P2Y(11), P2Y(12), P2Y(14) reside in the basolateral membrane and P2Y(2), P2Y(4), P2Y(6) at the apical membrane. The localisation of P2Y(13) is not yet known (Wolff et ah, 2005).

Most studies of the P2 purinergic receptors of the distal tubule have been performed on cell lines, such as the A6 cell line which exhibits transport properties similar to the mammalian distal tubule (Turner et al., 2003).

Banderali et al. (1999) showed that apical application of nucleotides in A6 cells gave a transient increase in chloride secretion mediated via a P2Y2 like receptor. In rat, Turner et al. (2003) could only detect P2X4 and P2X6 receptors in the basolateral segment of the rat distal tubule and a low level of P2X5 in the cortical collecting duct. In a rat model of autosomal dominant polycystic kidney disease the cells lining the cysts show an excess of P2Y2 and P2Y6 receptors and P2X7 receptors. This suggests that locally-released ATP activates P2 receptors in cyst-lining cells causing cyst expansion from increased fluid secretion (Turner et ah, 2004).

Recent studies have demonstrated that P2 receptors derived from human and rat may differ in their sensitivity to antagonists. For example, P2X4 receptors are much less sensitive to antagonism by suramin and pyridoxal 5-phosphate-6-azo- 2\4'-disulfonic acid (PPADS) (North and Surprenant, 2000). In addition, it has been shown that the human P2X4 receptor displays a very similar agonist potency profile to that of rat P2X4 but has a notably higher sensitivity for the antagonists suramin and pyridoxal 5-phosphate-6-azo-2',4'-disulfonic acid. The regions responsible for this different sensitivity have been mapped (Garcia-Guzman et ah, 1997).

Purinergic receptors are known to exist as a monomer (i.e. consisting of a single purinergic receptor subunit, such as a single P2 receptor), or as a multimer {i.e. consisting of a number of purinergic receptor subunits, such as a dimer or trimer of Pl and/or P2 purinergic receptors).

Preferably, the invention provides a use wherein the purinergic receptor is a monomeric purinergic receptor; conveniently a monomeric purinergic receptor comprising or consisting of a Pl purinergic receptor or a P2 purinergic receptor. In a preferred embodiment, -the -monomeric purinergic receptor comprises or consists of the P2X4 purinergic receptor.

Alternatively, the purinergic receptor is a multimeric purinergic receptor (i.e. comprising multiple purinergic receptor subunits - that is, more than one purinergic receptor subunit), preferably a dimeric purinergic receptor (i.e. formed of two purinergic receptor subunits) or a trimeric purinergic receptor (i.e. formed of three purinergic receptor subunits). Preferably, the purinergic receptor comprises the Pl purinergic receptor and/or the P2 purinergic receptor and, more preferably, comprises the P2X4 purinergic receptor. In one embodiment, the multimeric ptirinergic receptor is selected from the group comprising or consisting of: i) a dimer comprising or consisting of the P2X4 purinergic receptor and the P2X5 purinergic receptor; ii) a trimer comprising or consisting of the P2X4 purinergic receptor and the P2X5 purinergic receptor and the P2X6 receptor; and ϋi) a multimer comprising or consisting of the P2X4 receptor and a Pl purinergic receptor.

Preferably, the invention provides a use wherein the compound capable of reducing and/or inhibiting the activity of the purinergic receptor is an antagonist of the purinergic receptor. Conveniently, the antagonist is an antagonist of the Pl purinergic receptor, such as 8-cyclopentyl-l,3-dipropyl-xanthine (DPCPX), which may be purchased from Sigma (Dorset, UK). Other antagonists will be known to a person skilled in the relevant art.

Alternatively, the antagonist is an antagonist of the P2 purinergic receptor, such as an antagonist selected from the group comprising or consisting of: pyridoxal phosphate-6-azo(benzene-2_:,4-disulfonic acid) and suramin. Other antagonists will be known to a person skilled in the relevant art.

Preferably, the antagonist is an antagonist of the P2X4 purinergic receptor; more preferably, an antagonist selected from the group comprising or consisting of: cibacron blue; bromophenol blue; and brilliant blue G, all of which can be purchased from Sigma (Dorset, UK). Cibacron blue is known to reduce and/or inhibit activity of the P2X4 receptor with an IC50 of 40-140μM, and bromophenol blue with an IC50 of 50μM. Brilliant blue G can be purchased from Sigma (Dorset, UK: catalogue number B5133).

Preferably, the invention provides a use wherein the component of the purinergic signalling pathway is phosphatidylinositol-3 -kinase (also known as phosphoinositide-3 kinase, PI3 kinase or PI3K). Phosphatidylinositol-3 -kinase (PDKs) are heterodimeric enzymes that are known to regulate many signal transduction pathways and .that comprise a p85 regulatory subunit and a pi 10 subunit capable of phosphorylating inositol lipids that modify the activity of downstream signalling machinery.

More preferably, the invention provides a use wherein the compound, capable of reducing and/or inhibiting the activity of PDK is an antagonist of PDK. Even more preferably, the compound is 2-(4-morpholmyl)-8-phenil-l(4H)-benzopyran- 4-one hydrochloride (LY-294002) which can be purchased from Sigma (Dorset, UK). Other antagonists will be known to a person skilled in the relevant art.

Preferably, the invention provides a use wherein the component of the purinergic signalling pathway is a calcium channel.

Ion channels are typically located in cellular membranes and facilitate the: transport of ions, such as sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺) and chloride (Cl^"), across lipid membranes. By "calcium channel" we include an ion channel capable of transporting calcium and/or calcium ions across a cellular membrane.

More preferably, the invention provides a use wherein the compound capable of reducing and/or inhibiting the activity of the calcium channel is a calcium channel blocker.

Calcium channel -blockers inhibit the slow inward current induced by the entry of extracellular calcium through the cell membrane, especially in cardiac and arteriolar smooth muscle. They act by lowering myocardial oxygen demand, reducing arterial pressure, and reducing contractility. Some agents induce a reflex tachycardia (for example, nifedipine, nicardipine, amlodipine) and are best administered in combination with a β-adrenoceptor antagonist. By contrast, diltiazem and verapamil are suitable for patients who cannot tolerate a β-blocker because they inhibit conduction through the atrioventricular (AV) node and tend to cause bradycardia. All calcium antagonists reduce myocardial contractility and may aggravate heart failure. Dmydropyridine calcium-entry blockers should be employed with β-blockers in acute coronary syndromes to avoid reflex tachycardia. In patients unable to tolerate β-blockers, a heart rate-slowing calcium antagonist may be appropriate. Short-acting dihydropyridines should not be used in isolation in acute coronary syndromes.

Preferably, the calcium channel blocker is selected from the group consisting of: nifedipine, nicardipine and amlodipine. Other antagonists will be known to a person skilled in the relevant art.

Preferably, the invention provides a wherein the component of the purinergic signalling pathway is a sodium channel selected from the group comprising: the epithelial sodium channel (ENaC).

Ion channels are typically located in cellular membranes and facilitate the transport of ions, such as sodium (Na⁺), potassium (K⁺), calcium (Ca²⁺) and chlorine (Cl^"), across lipid membranes. By "sodium channel" we include an ion channel capable of transporting sodium and/or sodium ions across a cellular membrane.

The single-channel unitary conductance for ENaC is 4-5 pS at room temperature when Na⁺ is the major conducting ion in the outer solution (Garry & Palmer, 1997; Palmer & Frindt, 1986b). Unlike the Na⁺ channels observed in excitable tissues, the current- voltage relationship of ENaCs does not show a strong voltage- dependent gating, which reveals a slight increase in open probability in response to the membrane hyperpolarization (Canessa et al, 1994b; Garty & Palmer, 1997). The ENaC channels are characterized by about 20 times greater selectivity for Na⁺ over K⁺ (Benos et al, 1980; Palmer, 1982; Taylor et al, 1999). Another distinguishing feature of this channel is the slow kinetics of gating, with long periods of opening and closing at room temperature (Palmer & Frindt, 1986a; Taylor et al, 1999).

More preferably, the invention provides a use wherein the compound capable of reducing and/or inhibiting the activity of the sodium channel is a sodium channel antagonist: even more preferably amiloride. It will be understood that increasing the activity of cellular components that negatively regulate the purinergic signalling pathway will reduce and/or prevent signalling and thereby decrease and/or prevent sodium import. For example, increasing the activity of cellular components responsible for internalising and/or dόwnregulating the activity of the Pl and/or P2 purinergic receptors, and/or increasing the activity of a component responsible for downregulating PI3K, such as PTEN, will effectively reduce and/or prevent signalling through the pathway and reduce and/or prevent sodium import.

For example, activation on of the P2Y purinergic receptors on the apical surface of the renal epithelial cells is known to inhibit sodium transport. Thus, P2Y antagonists could be used to treat conditions in which there is sodium retention, for example caused by apical ATP excess.

Preferably, the invention provides a use wherein the component of the purinergic signalling pathway is an apical P2Y receptor, more preferably an antagonist of the apical P2Y receptor. More preferably the antagonist is suramin and/or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS). Other antagonists will be known to a person skilled in the relevant art.

Preferably, the invention provides a use wherein the cell is a kidney cell or other cell containing mineralocorticoid receptors. Other suitable cells will be known to a person skilled in the relevant art.

By "kidney cell" we include any cell type derived from a kidney, including cells of the cortical and/or medullary collecting tubule, especially the principal cells. More preferably, the invention provides a use wherein the kidney cell is a basolateral epithelial cell.

In a third aspect, the invention provides a use of a compound capable of modulating the activity of a component of the purinergic signalling pathway in the manufacture of a medicament for preventing and/or alleviating a condition in an individual associated with abnormal sodium homeostasis.

In a fourth aspect, the invention provides a method of inhibiting and/or reducing sodium import into one or more cell in vitro, wherein the method comprises treating one or more cell with a compound capable of modulating the activity of a component of the purinergic signalling pathway.

In a fifth aspect, the invention provides a method of preventing and/or alleviating a condition in an individual associated with abnormal sodium homeostasis, wherein the method comprises administering an effective amount of a compound capable of modulating the activity of a component of the purinergic signalling pathway to an individual in need thereof.

Preferably, the invention provides a use and/or a method wherein the condition is selected from the group comprising: primary aldosteronism; secondary aldosteronism; heightened sensitivity to aldosterone and/or essential hypertension.

It will be understood to a person skilled in the relevant medical field that an effective amount of the compound of the invention may be administered by any appropriate delivery method or route, including by oral, nasal, parenteral or intravenous adnήnistration. Such a person will be aware of the appropriate formulation for the administration route. Preferably, the compound of the invention may be delivered orally, as is known with other drugs used in the treatment of aldosteronism, such as spironolactone.

By "effective amount" we include an amount that is sufficient to inhibit and/or reduce sodium import in one or more cell and thereby prevent and/or alleviate a condition selected from the group comprising: primary aldosteronism; secondary aldosteronism; heightened sensitivity to aldosterone and/or essential hypertension in an individual. It will be understood that an effective amount of the compound of the invention could be determined by the measurement of the effect of the compound on plasma potassium levels, blood pressure and aldosterone-renin ratio, all of which are routine techniques to a person skilled in the relevant field of medicine or biochemistry. Elevated aldosterone levels or activity result in an increase in blood pressure, an imbalance of potassium plasma levels and, depending on the cause of the aldosterone excess, a change in the aldosterone- renin ratio.

In a sixth aspect, the invention provides a method of identifying a compound capable of modulating the activity of a component of the purinergic signalling pathway comprising the steps of:

(i) providing a compound to be tested;

(ϋ) providing a component of the purinergic signalling pathway; (iii) treating the component of the purinergic signalling pathway with the compound and measuring and/or determining the activity of the component of the purinergic signalling pathway; (iv) identifying the compound as capable of modulating the activity of a component in the event that the activity of the component is altered.

Preferably, the invention provides a method wherein the compound is capable of inhibiting and/or reducing the activity of a component of the purinergic signalling pathway. More preferably, the component of the purinergic signalling pathway is a purinergic receptor as defined herein. Conveniently, the component comprises or consists of the P2X4 purinergic receptor.

Alternatively, the component of the purinergic signalling pathway is phosphatidylinositol-3 -kinase or a calcium channel or the epithelial sodium channel (ENaC) as defined herein.

Preferably, the invention provides a method wherein the compound is capable of activating and/or increasing the activity of a component of the purinergic signalling pathway. Conveniently, the component of the purinergic signalling pathway is the apical P2Y receptor. It will be understood that screening for compounds capable of modulating the activity of components in the purinergic signalling pathway can be performed using in vitro, in vivo or ex vivo biochemical and/or physiological assays.

For example, signalling through the purinergic signalling pathway may be monitored and/or measured using the models and experiments described in, but not limited to, the accompanying Examples. As an alternative to screening for activity of the purinergic signalling pathway in the A6 cell line, other primary or transformed cell lines may be used, such as cell lines derived from mammalian kidney cells.

For example, the model and experiments described in the Examples that have been used to test the effects of Suramin, PPADS and ATP may be used to screen for compounds capable of modulating the activity of the purinergic signalling pathway from the basolateral side of the cell monolayer. Such an approach may be used to screen for compounds capable of modulating the activity of, for example, P2X4 purinergic receptors and sodium transport, which may be assessed by measuring transepithelial electrical resistance (TEER).

Preferably, the invention provides a method further comprising the step: (v) making a compound identifiable by any of the methods of the invention. Preferably, the invention provides a method further comprising the step: (vi) formulating the compound with a pharmaceutically-acceptable carrier to form a pharmaceutical composition. A skilled person would be aware of the methods and approaches suitable for performing steps (v) and (vi) of the method of the invention.

In a seventh aspect, the invention provides a pharmaceutical composition comprising a compound capable of modulating the activity of a component of the purinergic signalling pathway and a pharmaceutically-acceptable carrier.

It will be understood that a pharmaceutical composition of the invention may additionally comprise an effective amount of other compounds capable of modulating the activity of the purinergic signalling pathway and/or the activity of aldosterone. For example, a pharmaceutical composition of the invention may farther comprise an effective amount of two or more compounds of the invention. Alternatively, or in addition, a pharmaceutical composition of the invention may further comprise an inhibitor of aldosterone and/or an antagonist of an aldosterone receptor. It will be understood that it may not be desirable to totally block aldosterone action as this could lead to hyperkalemia.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

Figure 1: (A) Schematic diagram of SICM Setup. The A6 monolayer grown on a membrane filter insert is mounted on a 3D Piezo Scanner controlled by a feedback & scan control system. The modulated glass nanopipette scans the apical side of the monolayer and obtains topographical images of the cell membrane. (B) SICM image of living A6 cells cultured on a membrane filter insert. (C) SEM image of a fixed A6 cell monolayer cultured on a membrane filter.

Figure 2: Effect of osmotic-stress and aldosterone on the transepithelial electrical conductance (G_t) and the topography of A6 cells monolayer. (A) Typical time-dependent effect of basolateral hypo-osmotic stress on the amiloride- sensitive G₁ ^51"1*¹⁰ of A6 cell monolayer. (B) SICM images of a selected area of A6 cells monolayer cultured on a membrane filter scanned before (Control) and 1- hour after basolateral hypotonic stress (+Hypo). (C) Typical time-dependent effect of aldosterone on the G_t of A6 cell monolayer. (D) SICM images of a selected area of A6 cell monolayer cultured on a membrane filter scanned before - (Control) and 2 hours after aldosterone stimulation (+Aldosterone).

Figure 3: Aldosterone induced cell contraction and sodium transport. (A) Topographical images of A6 cell monolayer, illustrating the cell contraction (observe cells 1, 2 and 3), obtained from two successive scans of continuous imaging separated by 15 min interval during aldosterone stimulation. (B) Effect of aldosterone (•) (n=8), aldosterone plus amiloride (Θ) (n=5)₃ aldosterone plus LY-294002 (H) (n=7) and hypo-osmotic stress (A) (n=7) or without any stimulation (O) (n=5) on the percentage of A6 cells contracted. All data were expressed as the mean values ± S. E. (C-D) Smart patch-clamp recordings of ENaC in the cell attach configuration from a contracted A6 cell (lower panel) and non-contracted A6 cell (upper panel), during aldosterone stimulation. The image at the left side of the recordings illustrates the morphology of the cell patched. (E) High-resolution SICM images showing the fine structure of the apical membrane of a non-contracted (upper) and contracted (lower) A6 cell during aldosterone stimulation. (F) Schematic model of aldosterone induced cell contraction and ENaC activation. The apical membrane of the contracted A6 cell (lower) lifts up, possibly activating the ENaCs. In the non-contracted cell (upper), the typical microvilli structure of the apical membrane is maintained and there is no ENaC activation.

Figure 4: Aldosterone induces ATP release and changes the morphology of A6 cells. (A) Effect of aldosterone (n=8) and basolateral hypotonic stress (n=7) on the amiloride-sensitive transepithelial electrical conductance (G^⁰) of A6 cell, monolayer. (B) Rate of ATP release (RA_TP) from A6 cell monolayers during aldosterone stimulation (n=7) and basolateral hypotonic stress (n=5). All data in (A) and (B) were expressed as the average of experiments (mean± S. E.). Asterisk denotes value statistically different from the control based on paired Student t test (* P < 0.05; ** P < 0.005). (C) Effect of basolateral ATPγS on the topography of A6 cells. Two SICM topographical images of a selected area of a living A6 cell monolayer before (Control) and after (+ATP) stimulation with ATPγS. (D) Effect of ATPγS on the transepithelial electrical conductance (G_t) of A6 cell monolayer. 50 μM ATPγS was added to basolateral side. (E) Effect of hexokinase on the aldosterone-induced G_t of A6 cell monolayer. Aldosterone, hexokinase and glucose were added together to the basolateral compartment at the time marked by the arrow, removing the hexokinase 90 rnin later. (F) Hexokinase and glucose were added to the basolateral compartment of an aldosterone-stimulated A6 cell monolayer at the time marked by the arrow (upper graph). 100 nM DPCPX₅ a Pl receptor antagonist, was added to the basolateral compartment of an aldosterone stimulated A6 cell monolayer at the time marked by the arrow (lower graph). The thick lines on the bottom of Figure (D). (E) and (F) indicate the application of amiloride to the apical side of the A6 cell monomer. Figure 5: A model of aldosterone stimulation of sodium transport and cell contraction in epithelial cells.

Figure 6: A6 cell morphology changes in response to basolaterάl 2MeSATP. (A) Six images chosen from a time-lapse series (18 minute time gap between scans) SICM scans of a selected 40 mm x 40 mm area of an A6 cell monolayer exposed to basolateral 50 mM 2MeSATP. The time marked on the frames represents hour: minutes.

(B) Typical morphological changes (frame 0:18 to 1:12) of a selected A6 cell marked by asterisk in (A) frame 0:00. The topographical profile drawing which represents the scan along the dash line across the tight-junction-joints "a" and "b" of the asterisk marked cell in (A) frame 0:18 (upper trace) and frame 1:12 (lower trace).

Figure 7: Effects of basolateral 2MeSATP on the transepithelial electrical conductance (Gt) of A6 cell monolayers. Typical time-dependent effect of the basolateral addition of 50 mM 2MeSATP on Gt increases of the A6 monolayer. The major part of increases in Gt was amiloride-sensitive. 2MeSATP was applied for the duration indicated by the solid line above the trace. The thick lines on the bottom of figure indicate the application of amiloride to the apical side of the A6 cell monolayer.

Figure 8: Graph of effects of various P 2X agonists (50 mM) on the amiloride- sensitive Gt changes of A6 monolayer. Compared with control the basolateral addition of selected P2X agonists, including 2MeSATP₅ ATPγS, βγmeATP, αβmeATP and CTP, all significantly increased amiloride sensitive GtENaC changes of cell monolayer. 2MeSATP was the most potent agonist among these chosen agonists. All P2X agonist induced changes in ΔGtENaC of A6 monolayers were normalized with the control experiments. The columns and the bars represent mean ± SEM. * P < 0.01 vs. Control, # P < 0.05 vs. 2MeSATP.

Figure 9: Zn^~+, suramin and PPADS induced potentiation on 2MeSATP -evoked Gf changes of A6 monolayer. Addition of 50 mM 2MeSATP to the basolateral compartment of A6 cell monolayer increased amiloride-sensitive Gt of cell monolayer. Adding 10 mM ZnCl₂ to basolateral side potentiated the 2MeSATP- induced Gt increase. 100 mM suramin and PPADS not only did not reduce but further increased Gt of A6 monolayer. 2MeSATP₅ Zn²⁺, suramin and PPADS applied for the duration indicated by the marked solid line above the trace. The thick lines on the bottom of figure indicate the application of amiloride to the apical side of the A6 cell monolayer.

Figure 10: Ivermectin potentiated and BBG reduced the 2MeSATP -induced changes in AGtENaC of A6 monolayer. (A) Compared with 50 mM 2MeSATP treatment only, pre-incubation of the basolateral side of A6 monolayers with 10 mM ivermectin had a significant potentiating action on 2MeSATP -induced changes in ΔGt ENaC of A6 monolayer. All changes in ΔGt ENaC of A6 monolayers were normalized with the control experiments. The columns and the bars represent mean ± SEM. * P < 0.05 vs. 2MeSATP Control. (B) Pre-incubation of the basolateral side A6 cells with 10 mM BBG did not totally inhibit the 2MeSATP-induced increase in ΔGt ENaC of A6 monolayer, but significantly reduced the effect of 2MeSATP on ΔGt ENaC. All changes in ΔGt ENaC of A6 monolayers were normalized with the control experiments. The columns and the bars represent mean ± SEM. * P < 0.05 vs. BBG Control, # P < 0.001 vs. 2MeSATP.

Figure 11: Increase in [Ca²⁺J₁ evoked by 2MeSATP and Suramin addition of individual fluo-4 loaded A6 cells. Representative fluorescence intensity graph showing normalized fluorescence intensity (F/F0) changes of individual A6 cells in response to 50 μM 2MeSATP without (A, n=8) and with 100 μM suramin (B, n=10). Each different colour trace is from a different cell.

EXAMPLE 1 - Experimental Data

1.1. Identification of the purinergic pathway

Materials and methods

Cell culture

The experiments used a single A6 cell line (kindly provided by Dr P. DeSmet - Karnolieke Universiteit, Belgium) carried out between 127-134 passages. Cells were cultured on membrane filters as described previously (Gorelik et al., 2004; Kemendy et al, 1992).

The A6 cell line is derived from Xenopus laevis kidney and was first established by Rafferty (Rafferty & Sherwin, 1969) and has been shown to be a useful model for studying the action of aldosterone in vitro (WatUngton et al., 1982). Our A6 cell line (kindly provided by Dr P. DeSmet, Katholieke Universiteit, Leuven, Belgium) was used in all the experiments, carrying out between 127-134 passages.

The Scanning Ion Conductance Microscope (SIClVf) set up

This method has been previously described (Korchev et al., 1997b; Hansma et al., 1989). Briefly, the sensitive SICM probe consists of a glass nanopipette filled with electrolyte (Fig. IA). An Ag/AgCl electrode plugged into it is connected to a current amplifier that measures the ion current passing through the pipette tip. The probe, mounted on a 3-axis piezo translation stage is modulated vertically 100 nm at 200 Hz when close to the sample surface. The modulated current is amplified and fed into a lock-in amplifier tuned to the modulation frequency. The output is connected to a DSP card to generate a feedback signal to maintain the probe-sample separation distance by moving the translation stage on which the sample is mounted in the vertical direction. The control/data acquisition electronics record both the lateral and vertical positions of the probe and generate the topographical image. Smart patch technique

The SICM set-up was also adapted for patch clamping (Gu et al._s 2002). After obtaining the topographic images the system positions the nanopipette (now used as a patch pipette) precisely over an area or point of interest for patch recording. The feedback control is switched off, the pipette is lowered and suction applied to form a GΩ-seal. Ion channel recording is then performed by conventional patch clamp method in the cell-attach configuration.

L- 15 medium was used as the bath and pipette backfill solution for the SICM and patch clamp recordings. The micropipette when backfilled with Ll 5 medium had an average tip resistance of 150 MΩ. For patch-clamp recording, currents were sampled at 10 kHz and filtered at 2 kHz (—3 dB₅ 4-pole Bessel) using an Axopatch 200B amplifier and pClamp 8.2 (Axon Iαstruments, Inc., Union City, CA, USA), which was also used to generate the pulse protocols and analysed the single^ channel activity. A total of 60 patches were performed, equally probing three different types of the cells- contracted (in a total of 15 patches) and non-contracted (in a total of 45 patches) after acute stimulation by aldosterone.

Aldosterone stimulation

A modified Kemendy protocol (Kemendy et ah, 1992) tested the effects of aldosterone. Briefly the mature A6 cell monolayer was maintained in the growth medium together with 10% fetal calf serum as above, but supplemented with 1.5μM aldosterone (Sigma, Dorset, UK). After 24 hours, the monolayer was incubated in a serum-free medium in the presence of aldosterone for another 24 hours. The cells were then grown in serum- and aldosterone-free culture medium and incubated in a humidified incubator with 1% COj in air for a further 48 hours.

During the experiments assessing aldosterone action, 1.5 μM aldosterone was re- added to both sides of the monolayer (Kemendy et al., 1992). To determine whether any observed effect of aldosterone involved a direct or indirect action via Na+ channels we used 10 μM amiloride (Sigma) on the apical side of the cell monolayers.

Measurement of Transepithelial Electrical Conductance (G_t)

Transepithelial electrical conductance (G_t) was measured with an EVOM ohmmeter (World Precision Instruments, Hertfordshire, UK) using the intact epithelium cultured on filters of 23.1 mm diameter and with 4.19 cm effective growth area of membrane. Changes in Na⁺ permeability were determined as changes in the G_t across filters mounted in an Endohm 24 chamber (World Precision Instruments) from successive measurements of the transepithelial electrical resistance (TEER)₅ as G_t=l/TEER. G_t of whole monolayer was divided by the effective growth area of membrane and expressed per cm of cells.

ATP release

Monolayers on the membrane filters were gently superfused with L- 15 medium and equilibrated for 60 minutes in an Endohm 24 chamber, maintaining the apical and basolateral solution volumes at 2 ml. We quantified ATP release using an ATP bioluminescent assay kit (Sigma) following a modified method previously reported (Jans et al, 2002). Briefly, a 500 μl sample was taken from the basolateral compartment and added in a mixture of 100 μl luciferin/luciferase assay reagent and 500 μl L-15 medium in a Petri dish. The dish was inserted into a light-tight compartment, and the ATP luminescence intensity was detected by an integrated photomultiplier-tube (814 Photomultiplier Detection System, Photon Technology International, inc., West Sussex. UK). In order to account for constituents in the sample medium, which affect the sensitivity of the ATP assay, calibration curves were constructed by adding incremental concentrations of ATP standard to the same sample for each measured point in each series of studies.

ATP release was quantified using a commercial ATP bioluminescent assay kit (FL-AA, Sigma, Dorset, UK) according to the manufacturer's instructions and following a modified method previously reported (Jans et al., 2002). ATPγS and Hexokinase

50 μM ATPγS (a non hydrolysable ATP analog) (Sigma, Dorset, UK) was added to the basolateral side of cell monolayer to assess the role of ATP on sodium reabsorption and cell morphology changes. In order to trap free ATP from the basolateral side, 6 Unit/ml Hexokinase (Sigma, Dorset, UK) and 5 mM Glucose (Sigma) was added to the basolateral compartment.

Results and discussion

We have used SICM to image living A6 cells growing on filter supports in physiological conditions (Gorelik et ah, 2004) to study the mechanism of aldosterone action. Figure IB presents a typical SICM topographical image of the . apical membrane of A6 cells in a monolayer cultured on a membrane filter insert. For comparison a scanning electron microscopy (SEM) image of the same, preparation reveals similar structures (Fig. 1C). Microvilli projections and cell borders are clearly visible and comparable on both images.

The effects of localised basolateral hypo-osmotic stress and aldosterone on sodium transport were studied. Figure 2A and C show that both stimuli stimulated an increase in amiloride-sensitive sodium transport with hypo-osmotic stress producing a more marked activation than aldosterone. We then studied the effects of these two stimuli on cell morphology. The SICM images demonstrated that hypo-osmotic stimulation resulted in prolonged cell morphology changes (Fig. 2B) that remained even after the regulatory volume decrease (RVD). These topographical changes with only a proportion of cells with apical membrane expansion are most likely the result of cell contraction. Previously, we have demonstrated that some A6 epithelial cells show slow lateral contraction under basal conditions (Gorelik et ah, 2004). Figure 2D shows topographical images of A6 cells before and after stimulation with aldosterone. Remarkably, aldosterone produced similar morphological changes as hypotonic stress; which occurred not in every cell, but rather in separate clusters of cells. We next examined the relationship between cell contraction and sodium transport

(Fig. 3). During aldosterone stimulation^' successive 15 minute scans of the same cells showed the contraction of individual cells (Fig. 3A₅ see cells marked 1,2,3).

The progressive recruitment of contracted cells in response to aldosterone or hypo-osmotic stress (Fig. 3B) had a similar time course to that of the increase in

^■ G_t (Fig. 2A₅ C). However in contrast to conductance the aldosterone-induced contraction was not blocked by amiloride but was by LY-294002 (PI-3-K inhibitor) (Fig. 3B).

To test whether cell contraction is directly associated with sodium transport we individually studied non-contracted (Fig. 3C₅ upper panel) and contracted (Fig. 3C₅ lower panel) cells during aldosterone stimulation. The SICM images of non- contracted and contracted cells along with cell-attach patch-clamp single-channel, recordings using our scanning patch-clamp technique (Gu et άl., 2002) are presented in Figure 3C and D. Channel activity was only found in contracted cells-; (Fig. 3D, lower panel). Further high resolution SICM scans showed that contraction alters the cell membrane/cytoskeleton link with the loss of surface projection of microvilli (Fig. 2E, lower panel). Given the mechano-sensitivity of ENaC (Awayda and Subramanyam, 1998) and ENaC activity F-actin cytoskeleton dependency (Cantiello et aϊ., 1991) these changes may result in the opening of the sodium channels (Fig. 3F).

However, it is unclear what causes cell contraction during hypo-osmotic or aldosterone stimulation. A known modulator of epithelial cell contraction is purinergic stimulation that acts via intracellular Ca²⁺ elevation (Nakano et al.i 1997). As already discussed others have demonstrated that hypotonic-induced sodium transport _^correlates with _. ATP release from epithelial cells into the basolateral compartment and this stimulates a rise in intracellular Ca²⁺ (Jans et ah, 2002). It is well known that ATP rapidly hydrolyses to adenosine via ADP and ANCP. This together with the wide distribution of renal epithelial purinergic receptors (Scwiebert and Kishore, 2001) suggests a possible common purinergic mechanism in ENaC activation by cell contraction. To determine whether aldosterone, similar to hypotonicity, releases ATP from the basolateral side of A6 cells we measured the change in transepithelial electrical conductance (G_t) (Fig. 4A) and the relationship of the rate of ATP release (RATP) (Fig. 4B) to amiloride-sensitive G^⁰. Aldosterone increased amiloride-sensitive Q_t ^ENaC _as expected and this was associated with a marked increase in ATP release.

To test whether ATP stimulation per se contracted epithelial cells we imaged the response of A6 cell monolayer to basolaterally delivered ATPγS. Figure 4C shows that ATP induced contraction in approximately 75% of the monolayer cells.

The basolateral addition of non-hydrolysable ATPγS produced an increase in amiloride-sensitive transepithelial conductance (Fig. 4D), which suggested that ATP release and G_t ^ENaC were likely to be linked. We then added the ATP consumption system, hexokinase/glucose, to the basolateral compartment. Aldosterone had no effect on conductance in the absence of available extracellular. ATP (Fig. 4E). When, however, hexokinase and glucose were removed by washing the basolateral compartment there was a rapid increase in G_t (Fig. 4E). In another complementary experiment, when hexokinase and glucose were added to an aldosterone pre-stimulated A6 monolayer, the amiloride-sensitive G_t was reduced gradually (Fig. 4F, upper graph).

Knowing that ATP rapidly hydrolyses to adenosine via ADP and AMP, we also checked the sodium transport effect of ADP and adenosine. ADP had no effect on conductance in contrast with the expected increase with adenosine (Jans et ah, 2002) (data not shown). The Pl receptor antagonist 8-cyclopentyl-l,3-diprop3d- xanthine (DPCPX) reduced aldosterone-stimulated sodium transport (Fig. 4F, bottom graph): suramin and/or Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic

< acid) (PPADS) (P2 receptor blockers) not only did not reduce conductance but suramin markedly increased it. Inhibition of PI-3-K by LY-294002 (2-(4- Morpholinyl)-8-phenil-l(4H)-benzopyran-4-one hydrochloride) abolished sodium conductance by ATP or adenosine (data not shown). Our experiments thus indicate that neither suramin, not PPADS inhibits aldosterone action. One possible explanation is that the aldosterone action is via P2X4 purinergic receptors which are insensitive to these two antagonists in the rat (North and Surprenant, 2000). Studies have shown that rat distal tubule (i.e. the site of action of aldosterone) contains P2X4 receptors. The human P2X4 receptor displays a very similar agonist potency profile to that of rat P2X4 but has a notably higher sensitivity for the antagonists suramin and pyridoxal 5-phosphate- 6-azo-2',4'-disulfonic acid. The regions responsible for this different sensitivity have been mapped (Gaxcia-Guzman et al., 1997). Thus, P2 receptors, particularly P2X4 receptors, are likely to be involved in the purinergic signalling pathway and of therapeutic importance in humans.

Using these results we propose a new model of aldosterone-induced epithelial sodium transport (Fig. 5). Aldosterone enters the cell and, by mechanisms yet to be determined, stimulates ATP release. ATP is then excreted from the cell and, as has been demonstrated in many cell types, signals via an autocrine/paracrine system via P2 purinergic receptors (Burnstock and Williams, 2000). Excreted ATP rapidly hydrolyses to ADP, AMP and adenosine. Adenosine being also a purinergic stimulator acts via its own Pl receptors. Our experiments demonstrate that both ATP and adenosine induced Na⁺ transport can be blocked by LY-294002 indicating the importance of PI-3-K pathway in ENaC activation. Many studies confirm that both ATP and adenosine increase intracellular calcium (Jans et al., 2002). Macala and Hayslett suggested that renal sodium transport might be controlled by locally produced adenosine acting as an autacoid, and hence induces a local physiological effect (Macala and Hayslett, 2000). We postulate that this produces the cell contraction that we have observed. A similar mechanism has been reported in myoepithelial cells (Nakano et al., 1997). The contraction profoundly alters the apical membrane topography with the loss of normal microvillar architecture. Given the mechano-sensitivity of ENaC (Awayda and Subramanyam, 1998) and ENaC activity F-actin cyto skeleton dependency (Cantiello et ah, 1991) these changes may also facilitate the opening of the sodium channels (Fig. 5). It is important to recognize the very different effects of ATP when released into the apical as compared to the basolateral compartment. Thus, in experiments on guinea pig distal colon ATP released into the luminal side by hypotonic stimulation exerted an inhibitory effect on sodium absorption thought to be via a P2Y₂ receptor (Yamamoto and Suzuki, 2002).

Traditionally the effect of aldosterone is divided into an early response (0.5-3 hours) with a 2- to 3 -fold increase in sodium reabsorption and a later phase (3-24 hours) where the increase is up to 20-fold (Alvarez et ah, 2000). Although the later stages are well studied the dominant early effect of modulating Epithelial Sodium Channel (ENaC) activity before any increase in ENaC subunit mRNA or protein is poorly understood (Chen et ah, 1999). There are two potential ways to increase sodium transport: augmenting the number of cell membrane channels, or. enhancing the open probability of the existing ENaCs. Some studies suggest that aldosterone does not regulate the sodium channel pool in the early stages. (Kleyman et ah, 1992). Others conclude that the effect is due exclusively to an increase in channel density (Hehnan et ah, 1998), and that aldosterone, vasopressin and insulin act on sodium transport by promoting the incorporation of channels in the apical membrane (Alvarez and Canessa, 2003). Conversely several groups have suggested that aldosterone increases the ENaC open probability (Kemendy et ah, 1992), possibly by post-translational modification (Kleyman et ah, 1992; Eaton et ah, 2001). It has been suggested that regulation of . trafficking and/or functioning of sodium channels is controlled by aldosterone- induced modulatory proteins and kinases (Snyder et ah, 2002). One regulatory cascade proposed acts via aldosterone-induced expression of small G-protein K- ■ Ras2A with subsequent activation of phosphatidylinositol 3-kinase (PI-3-K) which produces phosphoinositol-3,4₅5-trisphosphate (PP₃) and that this is a direct regulator of ENaC open probability (Eaton et ah, 2001). This regulation also involves serum- and glucocorticoid-kinase (SGKl) that prevents ENaC degradation and mitogen-activated protein kinase (MAPK) that in contrast stimulates channel degradation (Pearce et ah, 2000: Tong et ah, 2004). In A6 cells hypotonicity as well as aldosterone induces SGKl expression (Rozansky et ah, 2002). However studies in the SGKl knockout mouse demonstrated that renal salt handling is normal when the animals are on a standard salt intake but impaired with dietary salt restriction suggesting that SGKl participates in, but does not full account for, mineralocorticoid regulation of sodium reabsorption (Wulff et ah, 2002).

Our results may well explain the controversy in the literature concerning the early phase of aldosterone action, ATP produces rapid contraction of individual cells and these demonstrate channel activity. Thus aldosterone acting via ATP would appear to increase the open probability of ENaC. However with time there is progressive recruitment of individual cells that parallels the slow increase in electrical conductance. This incremental change in the number of transporting cells would be interpreted as a progressive increase in the apparent apical membrane channel density.

The evolutionary aspects of this system are likely to be important. The transition of the protovertebrates from salt to fresh water required the development of a system that could regulate ion transport so as to maintain the internal milieu in the face of a very variable external environment, hi this context it is of interest that

P2X receptors are apparently restricted to vertebrates (North, 2002). During evolution aldosterone appears to have adopted the same mechanism of action that the cell uses to regulate cell volume in response to a change in osmolality.

Hypotonicity is a potent stimulus for ATP release across the renal epithelia (Jans et al., 2002). We have demonstrated that the application of a hypo-osmolar stimulus to the basolateral side of the A6 monolayer produces a pattern of cell contraction that is very similar to that produced by either aldosterone or ATP. Many studies have confirmed that ATP release is concerned with the control of cell volume (Wang et ah, 1996).

The regulation of extracellular fluid volume and sodium balance are key determinants of blood pressure. In about one third of patients with so-called essential hypertension plasma renin activity is low in association with normal aldosterone levels. The reason for this is unknown. One possibility is that in some individuals there may be a greater response of the renal epithelial cells to aldosterone leading to enhanced sodium reabsorption. It is interesting to speculate in relation to our results that there could well be important variations in this purinergic control system some of which might underlie this and other conditions. It would seem likely that a number of stimuli of sodium transport may also have adopted this very basic cell control and energy system.

5

1.2. Identification of basolateral receptors in the purinergic pathway

Materials and methods

Cell culture 10

A single Xenopus laevis renal epithelial^' A6 cell line (kindly provided by Dr P. DeSmet, Karnolieke Universiteit, Belgium) was used in all the experiments, carried out between 127-134 passages. Cells were routinely maintained in a 25cm² plastic cell culture flask (Corning) at 28⁰C in a humidified incubator with

15 1% CO2 in air as described previously (Sariban-Sohrbay et al., 1984). The culture medium was a mixture of 1 part Ham's F-12 medium with L-Glutamine (Gibco, Parsley, UK) and 1 part L- 15 medium with L-Glutamine (Gibco) modified for amphibian cells at pH 7.4. In addition to these components, the culture medium was also supplemented with-; 10% fetal bovine serum (Gibco), 200 mg/ml

20 streptomycin and 200 - U/ml penicillin (Gibco). The final growth medium osmolality was measured using an automatic Micro-Osmometer (Type 13/13DR- Autocal, CAMLAB₅ Cambridge, UK) and found to be 260 mosmol/kgH₂O. When the cells were 90-95% confluent, they were passaged by removing the growth medium and exposing them to divalent-free (Ca²⁺ and Mg"⁺) medium containing

■25 0.05% trypsin and 0.6mM ethylenediaminetetraacetate (EDTA) (Gibco). After lOmin, the action of the enzyme was inhibited with growth medium containing 10% fetal bovine serum. The cells were then re-suspended and seeded either again in a culture flask at a density of 5XlO³ cells/cm² or on different culture substrates according to the requirements of particular experiments. The cells to be

30 utilized for assessing the intracellular Ca²⁺ were grown on glass coverslips (thickness No. 0 with 13 mm diameter, VWR, UK) at a density of 1x10⁵ cells/cm². The cells that were to be used in investigations of the involvement of P2X4 receptors in sodium transport and topographical observations were all grown on the membrane filter inserts (23 mm diameter, 0.4 _m pore, L6xlO pores/cm², Falcon, Becton Dickinson Labware, Oxford, UK) at a density of 2x10⁵ cells/cm² for 10-12 days until formation of a tight epithelium, which exhibited a stable transepithelial electrical resistance. A6 cells were routinely pretreated with aldosterone using a modified protocol reported by Kemendy et al. 1992. hi brief, this involved the tight A6 cell monolayer being maintained for 24 hours in the growth medium together with 10% fetal calf serum as above, but supplemented with 1.5 mM aldosterone (Sigma, Dorset, UK) added to the basolateral compartments of the cell monolayer. After 24 hours, the cell monolayer was incubated in a serum-free medium, but still in the presence of 1.5μM aldosterone, for a further 24 hours to facilitate the maximal hormonal responsiveness. The cells were then grown in a culture medium which was free of both serum and aldosterone and incubated at 28⁰C in a humidified incubator with 1% CO2 in air for 48 hours.

Prior to being used in experiments, A6 cells were bathed with a modified, isosmotic L- 15 medium (Gibco) with a final osmolality of about 260 mosmol/kgH₂O that was verified with an automatic Micro-Osmometer (Type 13/13DR-Autocal, CAMLAB, UK).

Scanning Ion Conductance Microscope (SICM)

We obtained topographic images of A6 cells using an SICM (Ionoscope Limited, London, UK) as described previously (Korchev et al. 1997a, 1997b). Briefly, the SICM uses a nano-pipette, mounted on a three-axis piezo translation stage and arranged perpendicularly to the cell monolayer as a scanning probe. The SICM feedback control system keeps the ion current through the pipette constant to approach and scan over cells while maintaining a constant separation distance, within approximately nano-pipette tip internal radius from the A6 cell surface. The SICM then produces a 3D topographical image of A6 cell membrane surface.

The SICM nano-pipettes were pulled from borosilicate glass capillaries (Intrafil, 1.0 mm OD x 0.58 mm ID; Intracel Ltd, Hertz, UK), using a laser-based electrode puller (P-2000, Sutter Instrument, Novato, California, USA). To perform the scanning we used a pipette with around 50 run tip internal radius and electrical resistance of about 100 MΩ when submerged in the Ll 5 medium. Measurement of transepithelial electrical resistance (Rf) and calculation of transepithelial electrical conductance (Gt)

A well-developed A6 epithelial cell monolayer has been demonstrated to possess a stable and high transepithelial electrical resistance (Rt) when cultured on the membrane filters (Handler et al. 1981). In our study the Rt was measured using commercial apparatus ENDOHM 24 chamber connected to the EVOM Volt-Ohm- Meter (World Precision Instruments, Hertfordshire, UK) as described previously (Gorelik et al. 2005; Zhang et al. 2005). Briefly, membrane filter inserts with cultured A6 cells were transferred to the ENDOHM 24 chamber for measurement. The Rt measurements were performed in Ll 5 medium at room temperature after the EVOM Volt-Ohm-Meter reading became stable (~30-60 min). The measured resistance output from the EVOM was sampled at IHz with a Digidata 1322A 16- bit data acquisition system and pClamp 8.2 software (Axon Instruments, Inc., Union City, CA, USA). Rt was expressed in Ωcm² and determined by multiplying the measured resistance by the effective membrane area of the A6 cell monolayer, which was calculated to be 4.19 cm2 of the cell culture membrane filter supports. It was essential that the cell monolayer was relatively impermeable during all transepithelial Na⁺ transport and P2X4 assessment experiments.

Only cells with a stable initial Rt of greater than 8,000 Ωcm were chosen for these experiments. Transepithelial electrical conductance (Gt) was then calculated using Gt =1/Rt, and was expressed in Siemens per square centimeter (S/cm²).

Measurements of intracellular calcium concentration

A6 cells grown at low density for one day on glass coverslips were bathed in L15 medium and adapted to a perfusion chamber (RC-25 Chamber, Warner Instruments Inc., U.S.A.). They were incubated with fluo-4 acetoxymetrryl ester (fluo-4 AM; Molecular probes, Eugene, Oregon, U.S.A.) 4 μM for 1 hour at room temperature (24±2 °C) in presence of 0.08 % pluronic F-127 (Sigma). The remaining fluo-4 was washing out with Ll 5 medium followed by 15 min for de- esterification. Intracellular free calcium concentration ([Ca²⁺];) was imaged in individual cells by exciting fluo-4 at 450-480 nm and detecting emitted fluorescence at > 520 nm using an intensified CCD (CoolView IDI camera system, Photonic Science, Sussex, UK) coupled to a Nikon TE-2000 inverted microscope and controlled by Image-Pro Plus software (Media Cybernetics, Woldngham, UK). Images were normally acquired at 2 frames per second with 100 ms individual frame exposure time, whereas for long-term observation of the fluctuations of [Ca²⁺]; images were taken at about 0.16 frame per second with same 100 ms exposure time of every frame to minimum the photo-bleaching. Corresponding time traces show normalized intensity of fluorescence as the ratio of fluorescence to initial fluorescence (FfBO) in the individual cells of interest.

Indirect pharmacological screening for purinergic receptors involved: P2X4 receptor agonists and antagonists

In this study, we were only concerned with the effect of extracellular ATP in the basolateral compartment. Since the inhibitory effect of extracellular ATP on epithelial apical membrane has been previously demonstrated (Cuffe et al. 2000; Leipziger, 2003; Shriley et al. 2005; Wildman et al. 2005), hi order to avoid any. diffusion of ATP agonists from the basolateral side to the apical side, these experiments were performed under continuous perfusion of the apical bath solution (2ml) with Ll 5 medium at the rate of 5 ml/min by gravity feed from the connected reservoirs. The agonists used to screen for P2X4 receptor were ATP analogues including ATPγS, 2-methylthio-ATP (2-MeSATP), α,β-methylene-ATP (αβmeATP) and CTP (Bo et al. 1995; Garcia-Guzman et al. 1997; North et al. 2000; Soto et al. 1996). Because β,γ-methylene-ATP (βγmeATP) is another stable analog of ATP which is more potent at P2X receptors than P2Y receptors (Ralevic et al. 1998), βγmeATP was used too. All agonists were purchased from Sigma and used at final concentration of 5OmM. 10 roM ivermectin (Sigma), which has been proved to potentiate the activity of P2X4 receptors (North et al. 2000; Priel et al. 2004; Ralevic et al. 1998), was also used in our study.

An alternative way to get information about the activation of purinergic receptors is the use of antagonists that block such P2 receptors. As selective P2X4 receptor antagonists are not readily available, we used the generic P2 receptor blockers suramin (100-300 μM) (Jans et al. 2002) used either alone or together with 100- 300 μM P3τidoxal phosphate-6-azo (benzene-2,4-disulfonic) acid (PPADS, nonspecific P2 receptor antagonist) (Shwiebert and Zsembery, 2003; Xia et al. 2004) and added to the A6 cell monolayer hasolateral side. A relatively selective compound used to antagonize P2X4 receptors - 10 μM brilliant blue G (BBG) (Jiang et al. 2000) was added to the basolateral compartment too. PPADS₅ suramin, and BBG were also obtained from Sigma.

Effect of amiloride

To determine whether any observed effect of ATP analogs involved an action via amiloride-sensitive epithelial Na⁺ channels (ENaCs), experiments were repeated with the addition of lOμM amiloride (Sigma) to the apical compartment of the A6 cell monolayer. During experiments, amiloride washout was accomplished by continually perfusing the apical side (2ml) of the A6 cell monolayer with Ll 5 medium at a rate of 6 ml/min by gravity feed from reservoirs connected to the ENDOHM 24 chamber (World Precision Instruments), combined with a suction pump system to remove the solution and maintain the level of the bath stable. Complete removal of amiloride blockade took about 3 min.

Data analysis

All data were expressed as the average of n experiments (mean values ± SEM).

For comparison of the data to assess the statistical significance Student's Mest was used. The P-value and the number of experiments or the numbers of measurements contributing to the mean values reported are given in the figure legends as appropriate. A difference between means at the level of P < 0.05 was considered significant; P < 0.01 was statistically highly significant; P < 0.001 was very highly significant. The statistical analyses were performed using Microcal Origin version 5.0 (Microcal Software Inc., MA U.S.A.).

Results

We have recently found that the basolateral addition of ATPγS (50 μM) induced progressive lateral cell contraction accompanied by apical membrane expansion in an A6 monolayer (Gorelik et al. 2005). In order to further investigate the P2X receptors involved in our findings, we used SICM to observe the changes in membrane structures of A6 cells in response to another non-hydrolysable form of ATP, 2MeSATP₅ which has been demonstrated to be the most potent agonist of recombinant and native P2X4 receptors (Ralevic et al. 1998; Shen et al. 2006). Figure 6A presents six selected time-sequence SICM images of the same area of an A6 cell monolayer taken over a two-hour period. The first two control frames (0:00 to 0:18) show a flat and uniform cell monolayer with well-developed tight junctions between cells and pronounced microvilli covering the cell membrane. After Hie basolateral addition of 2MeSATP (50 mM), the cell morphology changed significantly with progressive lateral cell contraction and apical membrane elevation (frame 0:36 to 1:12). The last frame (1:48) shows gradual recovery of the original morphology of the cells. To highlight these typical changes in cell apical membrane structures, topographical profile changes in a selected A6 cell marked by asterisk in Figure 6 A (frame 0:00) were presented (Fig. 6B). The scanned profiles are across two points of tight-junction-joints "a" and "b", which each sited on the points of apposition of the asterisk marked cell and two other neighbouring cells, represented by the same length of dashed line marked on frames 0:18 and 1:12. Compared with the profile of the control frame (frame 0:18; Fig. 6B upper trace), the profile line scanned after 2MeSATP stimulation (frame 1:12; Fig. 6B lower trace) shows clear lateral contraction (note the arrow marked position change of the tight-junction-joint "b") accompanied by vertical apical membrane elevation. Such 2MeSATP-induced cell morphological changes are very similar to those membrane structure changes that we have previously linked with ENaC activity of A6 cells in response to aldosterone, hypotonic stress or ATPγS (Gorelik et al. 2005).

We further observed that the addition of 50 mM 2MeSATP to the basolateral side ofthe A6 cells, as with our published results of experiments in response to ATPγS - - (Gorelik et al. 2005), produced a rapid rise in cell monolayer transepithelial conductance (Gt) that was inhibited by the epithelial sodium channel (ENaC) inhibitor amiloride (Fig. 7).

The P2X receptor family have a diverse pharmacology, and the agonist potency orders vary significantly between P2X subtypes (North, 2002). The agonist potency profile of the cloned P2X4 receptors is: ATP > 2MeSATP > CTP > αβmeATP (Garcia-Guzman et al. 1997; North et al. 2000; Soto et al. 1996). ATPγS is also an agonist at recombinant P2X4 receptors with a potency generally less than that of ATP (Bo et al. 1995).

To test whether the P2X4 receptor is specifically involved in sodium transport, we tested its agonist sensitivity by measuring the amiloride-sensitive sodium conductance changes (Δ GtENaC) of the A6 monolayer. The basolateral addition of selected P2X agonists (5OmM)₅ including 2MeSATP, ATPγS, βγmeATP, αβmeATP and CTP, all significantly increased the ΔGtENaC of the cell monolayer (Fig. 8). The sensitivity of the involved P2X receptors to 2MeSATP₅ ATPγS and CTP in the A6 monolayer (2MeSATP > ATPγS >CTP) displays a similar agonist potency profile to that of cloned P2X4 receptors.

However, compared with the cloned P2X4 receptors, the A6 cell monolayer had a notably higher sensitivity for the agonist βγmeATP and αβmeATP (2MeSATP > βγmeATP > αβmeATP > CTP) .

P2X4 receptors also have their own characteristic profile with respect to the effects of different ions. Zn²⁺ potentiates the cation conductance induced by ATP at cloned P2X2 and P2X4 receptors, and Cu²⁺ has been reported to increase the ATP-activated current through P2X2 receptors but had no effect at P2X4 receptors (Garcia-Guzman et al. 1997; North et al. 2000; Soto et al. 1996). This ion dependence allows P2X4 receptors to be distinguished from other P2X receptors. In our study, zinc (Fig.9) potentiated the 2MeSATP -induced amiloride-sensitive sodium transport of the A6 monolayer but not Cu²⁺ (data not shown). P2X4 receptors have been demonstrated to be insensitive to the known P2 receptor antagonists suramin and PPADS (Buell et al. 1996; North et al. 2000). Figure 9 indicates that the 2MeSATP-evoked Gt changes of the A6 monolayer cannot be inhibited with suramin and PPADS (100-300 mM). Moreover, the effects of both antagonists on the 2MeSATP-evoked sodium transport of the A6 monolayer was similar to that Townsend-Nicholson et al. (1999) observed at mouse P2X4 receptors and to that found by Bo et al. (1995) with cloned P2X4 receptors, that suramin and PPADS did not reduce but further increased ATP-mediated effects. Ivermectin is widely used in human and veterinary medicine as an antiparasitic agent, and has been proved to potentiate the activity of P2X4 receptors in homomeric and heteromeric configuration, but does not affect extracellular ATP- induced currents in cells expressing other types of purinergic receptors (North, 2002; North et al. 2000; Priel et al. 2004). Compared with the 50 mM 2MeSATP treatment alone, pre-incubating the basolateral side of A6 monolayers with 10 mM ivermectin for 5 min had a significant potentiating action on 2MeSATP- induced_GtENaC of the A6 monolayer (Fig 10A). A relatively selective P2X4 antagonist and a specific P2X7 antagonist brilliant blue G (BBG) (Jiang et al. 2000) was used to further assess the P2X subtypes involved in the 2MeSATP induced GtENaC changes of the A6 monolayer (Fig 10B). Similar to the results published by Jiang et al. (Jiang et al. 2000) using rat and human P2X4 receptors, pre-incubating the A6 monolayer with 10 mM BBG did not totally inhibit the 2MeSATP -induced amiloride-sensitive Gt increase, but significantly reduced the effect of 2MeSATP on ΔGtENaC.

Figure 11 presents two graphs of normalized fluorescence intensity changes in individual fluo-4 loaded A6 cells in response to 2MeSATP. The P2X family has differences in its desensitization kinetics and P2X4 receptors have been classified as slowly desensitizing (North, 2002; Ralevic et al. 1998). The 2MeSATP- induced [Ca²⁺]; elevation in selected eight A6 cells returns to the basal level within about 1-2 min (Fig HA). This slow desensitization processing also indicates that P2X4-like receptors may be involved. In the further experiments, the addition of 100 μM suramin, as expected, did not block the subsequent 50 mM 2MeSATP- evoked [Ca²⁺Jj elevation of 10 selected A6 cells (Fig HB). Nevertheless, the addition of suramin induced [Ca²⁺]; elevation in some A6 cells (Fig 11B), a result similar to that found by Dockrell and co-workers (Dockrell et al. 2001) in cultured rat renal tubular cells.

Discussion

The P2X4 receptor appears to be the most widespread member of the P2X ligand- gated ion channel super family (Bo et al. 2003, 1995; North, 2002). Activation of P2X4 receptors leads to the opening of non-selective cation channel; the recombinant P2X4 receptor is most potently activated by 2MeSATP (Bo et al. 1995; Seguela et al. 1996). In our study, 2MeSATP addition to the basolateral side of the A6 cell monolayer produced progressive cell contraction and apical membrane protrusion. Similar morphological changes, induced with ATPγS and other physiological stimuli, have been shown in our previous experiments to be clearly linked with ENaC activity in A6 cells (Gorelik et al. 2005). The agonist order of potency determined by amiloride-sensitive Gt changes of A6 monolayer to 2MeSATP, ATPγS and CTP (2MeSATP > ATPγS > CTP) displays a similar agonist potency profile to that of those cloned P2X4 receptors but has a notably higher sensitivity for the agonists βγmeATP and αβmeATP. The marked response of the A6 cells to βγmeATP and αβmeATP, which should be inactive or weak ^' agonists at P2X4 receptors (Bo et al. 1995; North, 2002; Ralevic et al. 1998), raises the question as to whether the effect is via any of the currently known P2X receptors. The subtypes that are sensitive to αβmeATP, such as P2X1 or P2X3. receptors, are not likely to be involved, because of their high sensitivity to suramin and PPADS. However, it is known that the ability of added ATP agonists to mimic intercellular signalling molecules also depends on, the rates of ATP- release, hydrolysis by ecto-ATPases, and the rate of ATP synthesis by ectonucleotide diphosphokinases (Joseph et al. 2004). Methylene ATP analogs, such as βγmeATP and αβmeATP, as their name suggests possess a methylene group substituted for the oxygen in the phosphodiester bridge between the phosphate moieties of ATP. Since this renders such anologs relatively resistant to hydrolytic attack by neucleotide phosphohydrolases, they have been used as stable agonists for certain P2 receptors and also as more effective inhibitors of ATP degradation mediated by ecto-ATPases (Joseph et al. 2004). When 50 μM βγmeATP and αβmeATP were utilized as P2X agonists, βγmeATP may more- strongly inhibit ecto- ATPase than αβmeATP in some cell types (Joseph et al. 2004). Moreover, both methylene ATP analogs may also secondarily induce extracellular- ATP synthesis and accumulation of ATP, the most potent agonist for P2X4 receptors (Joseph et al. 2004). It is also clear that βγmeATP can act directly as an agonist at adenosine/Pl receptors (Ralevic et al. 1998). These receptors have also been demonstrated to be involved in the purinergic regulation of Na transport in A6 cells (Gorelik et al. 2005; Ma et al. 1996). Several further distinguishing features of ATP-evoked sodium transport in A6 cells allow us to conclude that it results from the activation of P2X4~lϊke receptors. First is the potentiation by ivermectin and the lack of any blockade by suramin or PPADS βuell et al. 1996; North, 2002). Pre-incubating the basolateral side of the A6 monolayers with 10 IDM ivermectin had a significant potentiating action on 2MeSATP -mediated sodium transport in A6 cells. Neither suramin (100 μM) nor PPADS (lOOμM) inhibited ATPγS- (Gorelik et al. 2005) or 2MeSATPmediated sodium transport, but similiar to the Townsend-Nicholson et al. (Townsend-Nicholson et al. 1999) observation at the mouse P2X4 receptor, the 2MeS ATP-evoked sodium transport are actually potentiated by PPADS and suramin in A6 cells. P2X4 is relatively insensitive to the conventional antagonists suramin and PPADS possibly because of the absence of a lysine residue in the receptor (Buell et al. 1996). Moreover, it has been suggested that the insensitivity of purinergic receptors to suramin is due to its inhibition of the ecto-ATPases which breakdown ATP (Crack et al. 1994). Here we have also demonstrated that suramin can also induce [Ca²⁺]; elevation as specific evidence that it can potentiate the ATP effects on P2 receptors.

In our study, Cu²⁺ had no effect and Zn²⁺ potentiated the 2MeSATP responses again indicating that P2X4 receptors are involved. Pre-incubating the A6 monolayer with a relatively selective P2X4 antagonist and a specific P2X7 antagonist, BBG₃ did not totally inhibit the 2MeSATP -induced conductance increase, but significant reduced the effect of 2MeSATP on sodium conductance, which also suggests the involvement of P2X4-like receptors. Moreover, the kinetics of desensitization of 2MeSATP-induced [Ca²⁺Ji elevation in A6 cells are also in agreement with the slow deactivation of P2X4 receptors (North, 2002).

Studies of the functional properties of heterologous^ expressed P2X receptors, together with examination of their distribution in native tissues, suggests P2X receptors are likely to occur as both homo- and heteromultimers in vivo (Mackenzie et al. 1999; North, 2002). There is some evidence that P2X4 may heteropolymeri.se with P2X6 receptors since they are often found together in native tissues, and can be co-immunoprecipitated (North et al. 2000).

Functionally expressed homomeric P2X6 receptors are sensitive to PPADS and relatively insensitive to suramin, and may contribute αβmeATP sensitivity to heteromeric P2X4/6 receptors (Jones et al. 2004; Khakh et al. 1999).

However, those heteromeric P2X4/6 receptors have been demonstrated to have a higher sensitivity to suramin and PPADS (Le et al. 1998), so these homomeric

P2X6 and heteromeric P2X4/6 receptors are not likely to be involved because of the insensitivity of the A6 cells to suramin and PPADS in our study. However,

P2X4/5 heteromeric can also be formed (Torres et al. 1999), and it is even possible that all three subtypes P2X4/5/6 might co-assemble to form a novel phenotype (Shirley et al. 2005). We cannot rule out the possibility that the widespread P2X4 subunit serves as the principal subunit to form possibly novel heteromers with other P2 and Pl subunits in the distal nephron.

Unwin and Burnstock's group found that only P2X4 and P2X6 receptors were present on the basolateral membrane of the rat distal tubule epithelium (Turner et al. 2003). The fact that our results indicate that there are P2X4-like receptors in

A6 epithelial cell line derived from frog distal tubule is in agreement with their findings. Given our results indicating that ATP release from renal epithelial cells plays a critically important role in the mechanism of action of aldosterone (Gorelik et al. 2005), the precise location of these purinergic receptors is of interest because of their likely role in regulating salt and water balance. We suggest that sodium reabsorption is stimulated by activation of the P2X4 receptors along the tubular basolateral side of the distal nephron, making manipulation of these receptors of interest in conditions with aldosterone excess or enhanced renal responsiveness to mineralocorticoids. Better agonists and antagonists are necessary to investigate the physiology of native P2X4 receptors and results with these may point to new means of treatment.

In summary we have shown the existence of P2X4-like receptors in the A6 cell basolateral membrane. It has been demonstrated that the basolateral P2 receptors facilitate Na+ transport and there is well-documented evidence for apical P2 receptor-mediated inhibition of Na+ transport (Cuffe et al. 2000; Leipziger, 2003: Shirley et al. 2005; Wildman et al. 2005). The complexities of multiple ATP release mechanisms and multiple types of P2 receptors expressed by a given renal epithelial cell along the nephron have also been delineated (Bailey et al. 2000; Bucheimer et al. 2004; Burnstoclς 2006; Insel et al. 2001; Schwiebert et al, 2003; Tuπner et al. 2003). This suggests an elegant reciprocal purinergic system acting both at a basolateral and apical location for control of Na⁺ transport. Such a system would require a mechanism within the cell that resulted in either basolateral or apical release of ATP for the autocrine and/or paracrine regulation of renal tubular function.

EXAMPLE 2 - Preferred pharmaceutical formulations and modes and doses of administration.

The nucleic acids, molecules and pharmaceutical formulations of the present invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

The nucleic acids, molecules and pharmaceutical formulations of the present invention can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for the administration of nucleic acids, molecules and pharmaceutical formulations of the invention. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.

The nucleic acids, molecules and pharmaceutical formulations of the invention can also be delivered by electroincorporation (EI). EI occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. hi EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative method of delivery of the nucleic acids, molecules and pharmaceutical formulations of the invention is the ReGeI injectable system that is thermo-sensitive. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active substance is delivered over time as the biopolymers dissolve.

The nucleic acids, molecules and pharmaceutical formulations of the invention can also be delivered orally. The process employs a natural process for oral uptake of vitamin B₁₂ in the body to co-deliver proteins and peptides. By riding the vitamin B₁₂ uptake system, the nucleic acids, molecules and pharmaceutical formulations of the invention can move through the intestinal wall. Complexes are synthesised between vitamin B ₁₂ analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin Bi₂ portion of the complex and significant bioactivity of the active substance of the complex.

The nucleic acids, molecules and pharmaceutical formulations of the invention can be introduced to cells by "Trojan peptides". These are a class of polypeptides.-- called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al. (1998).

Preferably, the pharmaceutical formulation of the present invention is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the active ingredient.

The nucleic acids, molecules and pharmaceutical formulations of the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the nucleic acids, molecules and pharmaceutical formulations of the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the nucleic acids, molecules and pharmaceutical formulations of the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The nucleic acids, molecules and pharmaceutical formulations of the invention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and., granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcelMose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with- - emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The nucleic acids, molecules and pharmaceutical formulations of the invention can also be administered parenteral^, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques WeIl-IoIO¹WTi to those skilled in the art. 5 ^■

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include

10 suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried- (lyophilised) condition requiring only the addition of the

- . sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile

15 powders, granules and tablets of the kind previously described.

For oral and parenteral administration to human patients, the daily dosage level of the compounds of the invention will usually be administered in single or divided doses. 0

Thus, for example, the tablets or capsules of the compound of the invention may contain an active compound for administration singly or two or more at a time, as appropriate. The physician in any event will determine the actual dosage which will be most suitable for any individual patient and it will vary with the age, 5 weight and response of the particular patient. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited and such are within the scope of this invention.

The nucleic acids and molecules and pharmaceutical formulations of the invention 0 can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1.1,2-tetrafiuoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafiuoropropane (HFA 227EA3), carbon dioxide oi other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

Aerosol or dry powder formulations are preferably arranged so that each metered dose or "puff' contains an effective amount of a compound of the invention for delivery to the patient. It will be appreciated that he overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the nucleic acids, molecules and pharmaceutical formulations of the invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. The nucleic acids, molecules and pharmaceutical formulations of the invention may also be transdermaUy administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthahnic use, the nucleic acids, molecules and pharmaceutical formulations ^{■ ■} of the invention can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the nucleic acids, molecules and pharmaceutical formulations of the invention can be formulated as a suitable ointment containing the active compound suspended ox dissolved in, for example, a mixture with, one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively;, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Generally, in humans, oral or parenteral administration of the nucleic acids, molecules and pharmaceutical formulations of the invention compounds of the invention is the preferred route, being the most convenient.

For veterinary use, the nucleic acids, molecules and pharmaceutical formulations of the invention is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

Conveniently, the formulation is a pharmaceutical formulation.

Advantageously, the formulation is a veterinary formulation. EXAMPLE 3 - Exemplary pharmaceutical formulations

Whilst it is possible for a compound of the^' invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and P3τogen-free.

The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a compound of the invention.

Example A: Tablet

Active ingredient 100 mg

Lactose 200 mg

Starch 50 mg

P olyvinylpyrrolidone 5 mg

Magnesium stearate 4 mg

359 mg

Tablets are prepared from the foregoing ingredients by wet granulation followed by compression. -

Example B: Ophthalmic Solution

Active ingredient 0.5 g

Sodium chloride, analytical grade 0.9 g

Thiomersal 0.001 g

Purified water to 100 ml pH adjusted to 7.5 Example C: Tablet Formulations

The following formulations A and B are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression.

Formulation A me/tablet me/tablet

(a) Active ingredient 250 250

(b) Lactose B.P. 210 26

(c) Povidone B.P. 15 9

(d) Sodium Starch Glycolate 20 12

(e) Magnesium Stearate 5 3

500 SOO

Formulation B me/tablet mε/tablet

(a) Active ingredient 250 250

(b) Lactose 150 -

(c)Avicel PH 101^® 60 26

(d) Povidone B.P. 15 9

(e) Sodium Starch Glycolate 20 12

(f) Magnesium Stearate 5 3

500 300

Formulation C mε/tablet

Active ingredient 100

Lactose 200

Starch 50

Povidone 5

Magnesium stearate 4 359

The following formulations., D and E₅ are prepared by direct compression of the admixed ingredients. The lactose used in formulation E is of the direction compression type.

Formulation D mg/capsule

Active Ingredient 250 Pregelatinised Starch NF15 150

400

Formulation E mg/capsule

Active Ingredient 250 Lactose 150 Avicel ^® 100

500

Formulation F (Controlled Release Formulation^')

The formulation is prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate 'and compression. mg/tablet

(a) Active Ingredient 500 φ) Hydrox}φropylmemylceLl.ulose 112

(Methocel K4M Premium)^® (c) Lactose B.P. 53 (d) Povidone B.P.C. 28

(e) Magnesium Stearate 7

700

Drug release takes place over a period of about 6-8 hours and was complete after 12 hours.

Example D: Capsule Formulations

Formulation A

A capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B mg/capsule

(a) Active ingredient 250

(b) Lactose B.P. 143

(c) Sodium Starch Glycolate 25

(d) Magnesium Stearate 2

420

Formulation C mg/capsule

(a) Active ingredient 250

(b) Macrogol 4000 BP 350 _

600

Capsules are prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule. Formulation D mg/capsule

Active ingredient 250

Lecithin 100 Arachis Oil 100

450

Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate. and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule. mg/capsule

(a) Active ingredient 250 (b) Microcrystalline Cellulose 125

(c) Lactose BP 125

(d) Ethyl Cellulose 13

513

Example E: Injectable Formulation

Active ingredient 0.200 g

Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer (35-40 "C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals. Example F: Intramuscular injection

Active ingredient 0.20 g

Benzyl Alcohol 0.10 g Glucofurol 75^® 1.45 g

Water for Injection q.s. to 3.00 ml

The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).

Example G: Syrup Suspension

Active ingredient 0.2500 g

Sorbitol Solution 1.500O g

Glycerol 2.0000 g

Dispersible Cellulose 0.0750 g

Sodium Benzoate 0.0050 g

Flavour, Peach 17.42.3169 0.0125 ml

Purified Water q.s. to 5.0000 ml

The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.

Example H: Suppository mg/suppository Active ingredient (63 μm)* 250

Hard Fat, BP (Witepsol Hl 5 - Dynamit Nobel) 1770

2020 *The active ingredient is used as a powder wherein at least 90% of the particles are of 63 μm diameter or less.

One fifth of the Witepsol Hl 5 is melted in a steam-jacketed pan at 45⁰C maximum. The active ingredient is sifted through a 200 μm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C, the remaining Witepsol Hl 5 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 μm stainless steel screen and, with continuous stirring, is allowed to cool to 40°C. At a temperature of 38°C to 40°C 2.02 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.

Example I: Pessaries mg/pessary

Active ingredient 250

Anhydrate Dextrose 380

Potato Starch 363

Magnesium Stearate 7

1000

The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

REFERENCES

Alvarez, d. 1. R. & Canessa, C. M. (2003) Am. J. Physiol Cell Physiol 284, C404- C414.

Alvarez, d. 1. R., Canessa, C. M., Fyfe, G. K. & Zhang, P. (2000) Annu. Rev. Physiol 62, 573-594.

Awayda, M. S. & Subramanyam, M. (1998) J Gen. Physiol 111, 97-111.

Bailey MA, Hillman KA and Unwin RJ. P2 receptors in the kidney. JAuton Nerv ■ξμsf 81: 264-270, 2000.

Banderali et al., 1999, J Physiol 519 737-751.

Benos, DJ., L.J.Mandel, and S.A.Simon. (1980) J. Gen. Physiol 76:233-247.

Bo X, Kim M, Nori SL₅ Schoepfer R, Burnstock G and North RA. Tissue distribution of P2X4 receptors studied with an ectodomain antibody. Cell Tissue Res 313: 159-165, 2003.

Bo X, Zhang Y, Nassar M, Burnstock G and Schoepfer R. A P2X purinoceptors cDNA conferring a novel pharmacological profile. FEBS Lett 375: 129-133, 1995.

Brodin, B. and R.Nielsen. (2000) Pflugers Arch. 439:234-239.

Brown, J.J., RJraser, A.F.Lever, and J.I.Robertson. (1972). Br. Med. J2:391-396.

Bucheimer RE and Linden J. Purinergic regulation of epithelial transport. J Physiol 555: 311-321, 2004.

Buell G, Lewis C, Collo G₅ North RA and Surprenant A. An antagonistinsensitive P2X receptor expressed fn epithelia and brain. EMBO J ^' 15: 55-62, 1996.

Burnstock, G. & Williams, M. (2000) J. Pharmacol. Exp. Ther. 295, 862-869.

Burnstock G. Purinergic signalling. Br J Pharmacol 147 Suppl 1: Sl 72-Sl 81, 2006.

Burnstock G and Kennedy C. Is there a basis for distinguishing two types of P2- purinoceptor? Gen Pharmacol 16: 433-440, 1985.

Canessa, CM., L.Schild, G.Buell, B.Thorens, I.Gautschi, J.D.Horisberger, and B.CRossier. (1994) Nature 367:463-467.

Cantiello, H. F., Stow, J. L., Prat, A. G. & Ausiello, D. A. (1991) Am. J Physiol 261, C882-C888. Chen, S. Y., Bhargava, A., Mastroberardino, L., Meijer, O. C₅ Wang, J-, Buse, P., Firestone, G. L., Veixey, F. & Pearce, D. (1999) Proc. Natl. Acad. ScI U. S. A 96, 2514-2519.

Conn JW: Primary aldosteronism: A new clinical syndrome. J Lab Clin Med 45:6-17, 1955.

Conn JW, Cohen EL, Rovner DR: Suppressed plasma renin activity in primary aldosteronism. JAMA 190:213-221, 1964 .

Connell, J.M., R.Fraser, and E.Davies. 2001. Disorders of mineralocorticoid synthesis. Best. Tract. Res. Clin. Endocrinol Metab 15:43-60.

Crabbe, J. (1961) J. Clin. Invest 40, 2103-2110.

Crabbe, J. (1963) Nature 200, 787-788.

Crack BE, Beukers MW, McKechnie KC, IJzerman AP and Leff P. Pharmacological analysis of ecto-ATPase inhibition: evidence for combined enzyme inhibition and receptor antagonism in P2X-purinoceptor ligands. Br J Pharmacol 113: 1432-1438, 1994.

Cuffe JE, Bielfeld-Ackermann A, Thomas J, Leipziger J and Korbmacher C. ATP stimulates Cl- secretion and reduces amiloride-sensitive Na+ absorption in M-I mouse cortical collecting duct cells. J Physiol 524 Pt 1 : 77-90, 2000.

Curtis JJ, Luke RG, Dustan HP, Kashgarian M, Whelchel JD, Jones P, Diethelm AG Remission of essential hypertension after renal transplantation. N Engl J Med 309 1009-1015 1983.

Dockrell ME, Noor MI, James AF and Hendry BM. Heterogeneous calcium responses to extracellular ATP in cultured rat renal tubule cells. Clin Chim Acta 303: 133-138, 2001. Derossi et al. (1998), Trends Cell Biol 8, 84-87.

Eaton, D. C, Malik, B., Saxena, N. C, Al Khalili, O. K. & Yue, G. (2001) J. Membr. Biol 184, 313-319. Edelman, I. S. & Fimognari, G. M. (1968) Recent Prog. Horm. Res. 24, 1-44.

Edelman, I. S., Bogoroch, R. & Porter, G. A. (1963) Proc. Natl. Acad. Sci. U. S. A 50, 1169-1177. Edwards CRW Primary Mineralocorticoid Excess Syndromes in DeGroot Endocrinology 4^th Edition 2001 pp WB Saunders & Co, US .

Fimognari, G. M., Fanestil, D. D. & Edelman, I. S. (1967) Am. J Physiol 213, 954- 962. Eraser, R., J.J.Brown, A.F.Lever₅ J.J.Morton, and J.I.Robertson. 1981. The renin- angiotensin system and aldosterone in hypertension: a brief review of some aspects. Int. J Obes. 5 suppl 1:115-123.

Fronius, M., A.Berk, W.Clauss, and M.Schnizler. 2004. Ion transport across Xenopus alveolar epithelium is regulated by extracellular ATP, UTP and adenosine. Respir. Physiol Neurobiol. 139:133-144.

Garcia-Guzman M, Soto F₃ Gomez-Hernandez JM₅ LundP-E, Stuhmer W Characterisation of recombinant human P2X4 receptor reveals pharmacological differences to the rat homologue Molecular Pharmacology 51 : 109-118 1997.

Garty, H. and L.G.Palmer. 1997. Epithelial sodium channels: function, structure, and regulation. Physiol Rev. 77:359-396. Gorelik, J., Gu, Y., Spohr, H. A., Shevchuk, A. L, Lab, M. J., Harding, S. E., Edwards, C. R., Whitaker, M., Moss, G. W., Benton, D. C. et al. (2002) Biophys J 83, 3296-3303.

Gorelik, J., Zhang, Y., Shevchuk, A. L, Frolenkov, G. L, Sanchez, D., Lab, M. J., Vodyanoy, L, Edwards, C. R., Klenerman, D. & Korchev, Y. E. (2004) MoL Cell Endocrinol. 217, 101 - 108.

Gorelik J, Zhang Y, Sanchez D, Shevchuk A, Frolenkov G, Lab M, Klenerman D, Edwards C and Korchev Y. Aldosterone acts via an ATP autocrine/paracrine system: the Edelman ATP hypothesis revisited. Proc Natl Acad Sd U S A 102: 15000-15005, 2005.

Gross, F. 1958. Renin and hypertension, physiological or pathological agents?. Klin. Wochenschr. 36:693-706.

Gu, Y., Gorelik, J., Spohr, H. A., Shevchuk, A., Lab, M. J., Harding, S. E., Vodyanoy, L, Klenerman, D. & Korchev, Y. E. (2002) FASEB J 16, 748-750.

Handler JS, Perkins FM and Johnson JP. Hormone effects on transport in cultured epithelia with high electrical resistance. Am J Physiol 240: C103-C105, 1981

Hansma, P. K., Drake, B., Marti, O., Gould, S. A. & Prater, C. B. (1989) Science 243, 641-643.

Helman, S. L, Liu, X., Baldwin, K., Blazer- Yost, B. L. & Els, W. J. (1998) Am. J Physiol 274, C947-C957.

Insel PA, Ostrom RS, Zambon AC, Hughes RJ, Balboa MA, Shehnaz D, Gregorian C, Torres B, Firestein BL, Xing M and Post SR. P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides. Clin Exp Pharmacol Physiol 28: 351-354, 2001. Jans, D., Srinivas, S. P., Waelkens, E., Segal, A., Laxiviere, E., Simaels, J. & Van Driessche, W. (2002) J Physiol 545, 543-555.

Jans D, De Weer P₅ Srinivas SP₅ Lariviere E₅ Simaels J and Van Driessche W. Mg(2+)-sensitive non-capacitative basolateral Ca(2+) entry secondary to cell swelling in the polarized renal A6 epithelium. J Physiol 541 : 91-101 , 2002.

Jiang LH₅ Mackenzie AB₅ North RA and Surprenant A. Brilliant blue G selectively blocks ATP-gated rat P2X(7) receptors. MoI Pharmacol 58: 82-88, 2000.

Jones CA, Vial C₅ Sellers LA₅ Humphrey PP₅ Evans RJ and Chessell JJP. Functional regulation of P2X6 receptors by N-linked glycosylation: identification of a novel alpha beta-methylene ATP-sensitive phenotype. MoI Pharmacol 65:979-985, 2004.

Joseph SM, Pifer MA₅ Przybylski RJ and Dubyak GR. Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation. Br J Pharmacol 142: 1002-1014, 2004.

Kemendy, A. E., Kleyman, T. R. & Eaton, D. C. (1992) Am J Physiol 263, C825- C837.

Khakh BS, Proctor WR, Dunwiddie TV₅ Labarca C and Lester HA. Allosteric control of gating and kinetics at P2X(4) receptor channels. J Neurosci 19: 7289- 7299, 1999.

Kleyman, T. R., Coupaye-Gerard, B. & Ernst, S. A. (1992) J. Biol. Chem. 267, 9622-9628.

Korchev YE, Bashford CL, Milovanovic M, Vodyanoy I and Lab MJ. Scanning ion conductance microscopy of living cells. Biophys J 73 : 653-658, 1997a.

Korchev, Y. E., Milovanovic, M., Bashford, C. L., Bennett, D. C, Sviderskaya, E. V., Vodyanoy, I. & Lab, M. J. (1997b) JMicrosc 188, 17-23.

Kunzelmann, K., R.Schreiber, and ABoucherot. 2001. Mechanisms of the inhibition of epithelial Na(+) channels by CFTR and purinergic stimulation. Kidney Int. 60:455-461.

Le KT₅ Babinski K and Seguela P. Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18: 7152-7159, 1998.

Leipziger, J., D.Kerstan₅ R.Nitschke, and R.Greger. 1997. ATP increases [Ca2+]i and ion secretion via a basolateral P2Y-receptor in rat distal colonic mucosa. P fingers Arch. 434:77-83. Leipziger J. Control of epithelial transport via luminal P2 receptors. Am J Physiol Renal Physiol 284: F419-F432, 2003.

Lifton, R.P., A.G.Gharavi, and .D.S.Geller. 2001. Molecular mechanisms of human hypertension. Cell 104:545-556.

Lim PO, Rodgers P₅ Cardale K₅ et al: Potentially high prevalence of primary aldosteronism in a primary-care population [letter] [see comments]. Lancet 353:40 1999. Ma H and Ling BN. Luminal adenosine receptors regulate amiloride-sensitive Na+ channels in A6 distal nephron cells. Am J Physiol 270: F798-F805, 1996.

Macala, L. J. & Hayslett, J. P. (2002) Am. J Physiol Renal Physiol 283, F1216- F1225.

Mackenzie AB, Surprenant A and North RA. Functional and molecular diversity ofpurinergic ion channel receptors. Ann N YAcadSci 868: 716-729, 1999.

McCoy, D.E., A.L.Taylor, BA.Kudlow, KXarlson, MJ.Slattery, L.M.Schwiebert, E.M.Schwiebert, and B.A.Stanton. 1999. Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X and P2Y purinergic receptors. Am. J Physiol 277:F552-F559.

Menenton P, Jeunemaitre X, de Wardener HE, MacGxegor GA. Links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases. Physiol Rev. 2005 Apr;85(2):679-715. Review.

Nakano, H., Furuya, K., Furuya, S. & Yamagishi, S. (1997) Pflugers Arch. 435, 1- 8. North RA, Surprenant A Pharmacology of cloned P2X receptors Annual Review of Pharmacology and Toxicology 40:563-580 2000.

North, R. A. (2002) Physiol Rev. 82, 1013-1067. Padfield PL, Brown JI, Davies D, et al: The myth of idiopathic hyperaldosteronism. Lancet 2:83-84, 1981.

Palmer, L. G. 1982. Ion selectivity of the apical membrane Na channel in the toad urinary bladder. J. Membr. Biol. 67:91-98.

Palmer, L. G. and G.Frindt. 1986a. Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proc. Natl. Acad. Sci. U. S. A 83:2767-2770.

Palmer, L.G. and G.Frindt. 1986b. Epithelial sodium channels: characterization bjr using the υatch-clamp technique. Fed. Proc. 45:2708-2712. Pearce, D., Verrey, F., Chen, S. Y., Mastroberaxdino, L., Meijer, O. C₅ Wang, J. & Bhargava, A. (2000) Kidney Int. 57, 1283-1289.

Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A₅ Perez A₅ Palensky J.WittesJ The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med. 1999 Sep 2;341(10):709-17.

Priel A and Silberberg SD. Mechanism of ivermectin facilitation of human P2X4 receptor channels. J Gen Physiol 123: 281-293, 2004.

Rafferty, K.A., Jr. and R.W.Sherwin. 1969. The length of secondary chromosomal constrictions in normal individuals and in a nucleolar mutant of Xenopus laevis. Cytogenetics. 8:427-438.

Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413-492, 1998.

Rozansky, D. J., Wang, J., Doan, N., Purdy, T., Faulk, T., Bhargava, A., Dawson, K. & Pearce, D. (2002) Am. J Physiol Renal Physiol 283, F105-F113.

Sariban-Sohraby S, Burg MB and Turner RJ. Aldosterone-stimulated sodium uptake by apical membrane vesicles from A6 cells. J Biol Chem 259: 11221- 11225, 1984.

Schrier,R.W. Renal and Electrolyte Disorders. Sixth. 2003. USA₅ LIPPINCOTT WILLIAMS & WILKINS.

Schwiebert, E. M. & Kishore, B. K. (2001) Am. J Physiol Renal Physiol 280, F945-F963.

Schwiebert EM. ATP release mechanisms, ATP receptors and purrnergic signalling along the nephron. Clin Exp Pharmacol Physiol 28: 340-350, 2001. Schwiebert EM and Zsembery A. Extracellular ATP as a signaling molecule for epithelial cells. Bio chim Biophys Acta 1615: 7-32, 2003.

Seguela P, Haghighi A₅ Soghomonian JJ and Cooper E. A novel neuronal P2x ATP receptor ion channel with widespread distribution in the brain. JNeurosci 16: 448-455, 1996.

Shen JB, Pappano AJ and Liang BT. Extracellular ATP-stimulated current in wild-type and P2X4 receptor transgenic mouse ventricular myocytes: implications for a cardiac physiologic role of P2X4 receptors. FASEB J20: 277-284, 2006.

Shirley DG₅ Bailey MA and Unwin RJ. In vivo stimulation of apical P2 receptors in collecting ducts: evidence for inhibition of sodium reabsorption. Am J Physiol Renal Physiol 288: F1243-F1248, 2005. Simpson, S. A., Tait, J. F., Wettstein, A., Neher, R., Von Euw, J., Schindler, O. & Reichstein, T. (1954) Experientia 1O₅ 132-133.

Snyder, P. M. (2002) Endocr. Rev. 23, 258-275.

Soto F, Garcia-Guzman M, Gomez-Hernandez JM₅ Hollmann M₅ Karschin C and Stuhmer W. P2X4: an ATP-activated ionotropic receptor cloned from rat brain. Proc Natl Acad Sci USA 93: 3684-3688, 1996. Taylor, C.T., D.C.Winter, M.M.Skelly, D.P.O'Donoghue, G.C.O'Sullivan, B. J.Harvey, and A.W.Baird. 1999. Berberine inhibits ion transport in human colonic epithelia. Eur. J. Pharmacol. 368:111-118.

Tong, Q., Booth, R. E., Worrell, R. T. & Stockand, J. D. (2004) Am. J Physiol Renal Physiol 286, F1232-F1238.

Torres GE, Egan TM and Voigt MM. Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. J Biol Chem 21 A: 6653-6659, 1999. Townsend-Nicholson A, King BF₅ Wϊldman SS and Burnstock G. Molecular cloning, functional characterization and possible cooperativity between the murine ^• P2X4 and P2X4a receptors. Brain Res MoI Brain Res 64 : 246-254, 1999.

Turner CM, Vonend O, Chan C, Burnstock G and Unwin RJ. The pattern of distribution of selected ATP-sensitive P2 receptor subtypes in normal rat kidney: an immunobistological study. Cells Tissues Organs 175: 105-117, 2003.

Turner et al., 2004, Cells Tissues Organs 178 168-79. Ussing, H. H. & Zerahn, K. (1951) Acta Physiol Scand. 23, 110-127.

Wang, Y., Roman, R., Lidofsky, S. D. & Fitz, J. G. (1996) Proc. Natl. Acad. Sci. U. S A 93, 12020-12025. Watlington, CO., F.M.Perkins, PJ.Munson, and J.S.Handler. 1982. Aldosterone and corticosterone binding and effects on Na+ transport in cultured kidney cells. " Am. J Physiol 242.-F610-F619.

Wildman SS, Marks J₅ Churchill LJ₅ Peppiatt CM, Chraibi A₅ Shirley DG₅ Horisberger JD₅ King BF and Unwin RJ. Regulatory Interdependence of Cloned Epithelial Na+ Channels and P2X Receptors. JAm Soc Nephrol 2005.

Witzgall H, Lorenz R, Von Werder K, et al: Dopamine reduces aldosterone and

18-hydroxycorticosterone response to angiotensin II in patients with essential low- renin hypertension and idiopathic hyperaldosteronism. Clin.Sci 68:291-299, 1985.

Wolff et al., 2005, Am J Physiol 288 624-632. WuIS₅ P., Vallon, V., Huang, D. Y., VoUd, H., Yu₃ F., Richter, K, Jansen, M., Schlunz, M., Klingel, K., Loffing, J. eϊ al (2002) JCUn. Invest 110, 1263-1268.

Xia SL₅ Wang L, Cash MN₅ Teng X, Schwalbe RA and Wingo CS. Extracellular ATP-induced calcium signaling in mIMCD-3 cells requires both P2X and P2Y purinoceptors. Am J Physiol Renal Physiol 287: F204-F214, 2004.

Yamamoto, T. & Suzuki, Y. (2002) Am. J Physiol Gastrointest. Liver Physiol 283, G300-G308.

Zhang Y₅ Gorelik J₅ Sanchez D₅ Shevchuk A₅ Lab M, Vodyanoy I₅ Klenerman D₅ Edwards C and Korchev Y. Scanning ion conductance microscopy reveals how a functional renal epithelial monolayer maintains its integrity. Kidney Int 68: 1071- 1077, 2005.

Claims

1. Use of a compound for preventing and/or alleviating a condition in an individual associated with abnormal sodium homeostasis wherein the compound is capable of modulating the activity of a component of the purinergic signalling pathway.

2. The use according to Claim 1 wherein the condition in an individual is selected from the group comprising: primary aldosteronism; secondary aldosteronism; heightened sensitivity to aldosterone and/or essential hypertension.

3. Use of a compound for inhibiting and/or reducing sodium import into one or more cell, wherein the compound is capable of modulating the activity of a component of the purinergic signalling pathway.

4. The use according to any one of Claims 1 to 3 wherein the compound is capable of inhibiting and/or reducing the activity of a component of the purinergic signalling pathway.

5. The use according to any one of Claims 1 to 3 wherein the compound is capable of activating and/or increasing the activity of a component of the purinergic signalling pathway.

6. The use according to any one of Claims 1 to 4 wherein the component of the purinergic signalling pathway is a purinergic receptor.

7. The use according to Claim 6 wherein the purinergic receptor is a monomeric purinergic receptor.

8. The use according to Claim 6 or 7 wherein the purinergic receptor is selected from the group comprising: the Pl purinergic receptor and the P2 purinergic receptor.

9. The use according to any one of Claims 6 to 8 wherein the purinergic receptor is the P2X4 purinergic receptor.

10. The use according to Claim 6 wherein the purinergic receptor is a multimeric purinergic receptor.

11. The use according to Claim 6 or 10 wherein the purinergic receptor is a dimeric purinergic receptor or a trimeric purinergic receptor.

12. The use according to any one of Claims 6, 10 or 11 wherein the purinergic receptor comprises the Pl purinergic receptor and/or the P2 purinergic receptor.

13. The use according to Claim 12 wherein the P2 purinergic receptor is the P2X4 purinergic receptor.

14. The use according to Claim 12 or 13 wherein the purinergic receptor is selected from the group comprising:

iv) a dimer comprising the P2X4 purinergic receptor and the

P2X5 purinergic receptor; v) a trimer comprising the P2X4 purinergic receptor and the

P2X5 purinergic receptor and the P2X6 receptor; vi) a multimer comprising the P2X4 receptor and a Pl purinergic receptor.

15. The use according to any of Claims 6 to 14 wherein the compound capable of reducing and/or inhibiting the activity of the purinergic receptor is an antagonist of the purinergic receptor.

16. The use according to Claim 15 wherein the antagonist is an antagonist of the Pl purinergic receptor.

17. The use according to Claim 16 wherein the compound is 8-cyclopentyl- 1,3-dipropyl-xanthine.

18. The use according to Claim 15 wherein the antagonist is an antagonist of the P2 purinergic receptor.

19. The use according to Claim 18 wherein the antagonist is selected from the group comprising: pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) and suramin.

20. The use according to Claim 15 wherein the antagonist is an antagonist of the P2X4 purinergic receptor.

21. The use according to Claim 20 wherein the antagonist is selected from the group comprising: cibacron blue; bromophenol blue; brilliant blue G.

22. The use according to any one of Claims 1 to 4 wherein the component of the purinergic signalling pathway is phosphatidylinositol-3 -kinase.

23. The use according to Claim 22 wherein the compound capable of reducing and/or inhibiting the activity of phosphatidylinositol-3 -kinase is an antagonist of phosphatidylinositol-3-kinase.

24. The use according to Claim 22 or 23 wherein the compound is 2-(4- _. . morpholinyl)-8-phenil-l(4H)-benzopyrari-4-one hydrocnloride.-

25. The use according to any one of Claims 1 to 4 wherein the component of the purinergic signalling pathway is a calcium channel.

26. The use according to Claim 25 wherein the compound capable of reducing and/or inhibiting the activity of the calcium channel is a calcium channel blocker.

27. The use according to Claim 25 or 26 wherein the compound selected from the group consisting of: nifedipine, nicardipine and amlodipine.

28. The use according to any one of Claims 1 to 4 wherein the component of the purinergic signalling pathway is a sodium channel selected from the group comprising: the epithelial sodium channel (ENaC).

29. The use according to Claim 28 wherein the compound capable of reducing and/or inhibiting the activity of the sodium channel is a sodium channel antagonist.

30. The use according to Claim 28 or 29 wherein the compound is selected from the group consisting of: amiloride.

31. The use according to any one of Claims 1 to 3 or 5 wherein the component of the purinergic signalling pathway is an apical P2Y receptor.

32. The use according to Claim 31 wherein the compound capable of activating and/or increasing the activity of the apical P2Y receptor is an antagonist of the apical P2Y receptor.

33. The use according to Claim 32 wherein the compound is selected from the group consisting of: suramin and/or pyridoxal phosphate-6-azo(benzene- 2,4-disulfonic acid).

34. The use according to any one of Claims 3 or 4 to 33 wherein the cell is a kidney cell or a cell in which there is a rnmeralocorticoid receptor.

35. The use according to Claim 34 wherein the kidney cell is a basolateral epithelial cell.

36. Use of a compound capable of modulating the activity of a component of the purinergic signalling pathway in the manufacture of a medicament for preventing and/or alleviating a condition in an individual associated with abnormal sodium homeostasis.

37. A method of inhibiting and/or reducing sodium import into one or more 5 cell in vitro, wherein the method comprises treating one or more cell with a compound capable of modulating the activity of a component of the purinergic signalling pathway.

38. A method of preventing and/or alleviating a condition in an individual 10 associated with abnormal sodium homeostasis, wherein the method comprises administering an effective amount of a compound capable of modulating the activity of a component of the purinergic signalling pathway to an individual in need thereof.

15 39. The use according to Claim 36 or the method according to Claim 38 wherein the condition is selected from the group comprising: primary aldosteronism; secondary aldosteronism; heightened sensitivity to aldosterone and/or essential hypertension.

20 40. A method of identifying a compound capable of modulating the activity of a component of the purinergic signalling pathway comprising the steps of:

(i) providing a compound to be tested;

(ii) providing a component of the purinergic signalling pathway;

- 25 (iii) treating the component of the purinergic signalling pathway with . . . the compound and measuring and/or determining the activity of the component of the purinergic signalling pathway;

(iv) identifying the compound as capable of modulating the activity of a component in the event that the activity of the component is 30 altered.

41. The method according to Claim 40 wherein the compound is capable of inhibiting and/or reducing the activity of a component of the purinergic signalling pathway.

42. The method according to Claim 40 or 41 wherein the component of the purinergic signalling pathway is a purinergic receptor as defined in any one of Claims 6 to 14.

5

43. The method according to any one of Claims 40 to 42 wherein the purinergic receptor comprises or consists of the P2X4 purinergic receptor.

.

44. The method according to Claim 40 or 41 wherein the component of the 10 purinergic signalling pathway is phosphatidyls sitol-3 -kinase as defined in Claim 22 or a calcium channel as defined in Claim 25 or the epithelial sodium channel (ENaC) as defined in Claim 28.

45. The method according to Claim 40 wherein the compound is capable of 15 activating and/or increasing the activity of a component of the purinergic signalling pathway.

46. The method according to Claim 40 or 45 wherein the component of the purinergic signalling pathway is the apical P2Y receptor.

20

47. The method according to any one of Claims 40 to 46 further comprising the step:

(v) making a compound identifiable by the method of any one of

Claims 40 to 46. 25. .

48. The method according to any one of Claims 40 to 47 further comprising the step:

(vi) formulating the compound with a pharmaceutically-acceptable carrier to form a pharmaceutical composition. 30

49. A pharmaceutical composition comprising a compound capable of modulating the activity of a component of the purinergic signalling pathway and a pharmaceutically-acceptable carrier.

50. A use, method or pharmaceutical composition substantially as described herein.