AU1500800A - Regulation of nitric oxide synthase activity - Google Patents

Description

WO 00/28076 PCT/AU99/00968 1 REGULATION OF NITRIC OXIDE SYNTHASE ACTIVITY 2 This invention relates to the regulation of the 3 activity of the enzyme nitric oxide synthase, and in 4 particular to regulation of activity of endothelial and 5 neuronal nitric oxide synthases. We have found that the 6 phosphorylation of endothelial and neuronal nitric oxide 7 synthases by several protein kinases, including protein 8 kinase C and the AMP-activated protein kinase, regulates 9 their activity 10 11 BACKGROUND OF THE INVENTION 12 Nitric oxide (NO) has recently been recognised as 13 an important mediator of a very wide variety of cellular 14 functions, and is present in most if not all mammalian 15 cells (Moncada, S. and Higgs, A., 1993). It is implicated 16 in a range of disorders, hypertension, 17 hypocholesterolaemia, diabetes, heart failure, aging, 18 inflammation, and the effects of cigarette smoking, and is 19 especially important in vascular biology. It regulates 20 systemic blood pressure as well as vascular remodelling 21 (Rudic et al., 1998) and angiogenesis in response to tissue 22 ischaemia (Murohara et al., 1998). NO is synthesised from 23 the amino acid L-arginine by the enzyme nitric oxide 24 synthase (NOS). 25 Three isoforms of NOS have been identified: 26 neuronal NOS (nNOS), which is found in neuronal tissues and 27 skeletal muscle (nNOSp isoform); inducible NOS (iNOS), 28 found in a very wide variety of mammalian tissues including 29 activated macrophages, cardiac myocytes, glial cells and 30 vascular smooth muscle cells; and endothelial NOS (eNOS), 31 found in vascular endothelium, cardiac myocytes and blood 32 platelets. Endothelial cells produce NO in response to WO 00/28076 PCT/AU99/00968 -2 1 shear stress generated by the streaming of blood on the 2 endothelial layer. 3 The three isoforms of NO synthase have an amino 4 acid sequence identity of approximately 55%, with strong 5 sequence conservation in regions involved in catalysis. 6 For all three isoforms, the mechanism of NO synthesis 7 involves binding of the ubiquitous calcium regulatory 8 protein calmodulin (CaM) to the enzyme. However, the 9 conditions under which CaM is bound appear to be different 10 for iNOS, at least insofar as calcium concentration is 11 concerned. These three NOS enzymes have been intensively 12 studied, and the field has been recently reviewed; see for 13 example Michel and Feron (1997); Harrison (1997); and Mayer 14 and Hellens (1997). Although it was known from earlier 15 studies that eNOS could be multiply phosphorylated, the 16 mechanism of these phosphorylation events, including the 17 enzyme responsible for phosphorylation, and the role of 18 phosphorylation in modulation of eNOS function was not 19 known. 20 AMP-activated protein kinase (AMPK) is a 21 metabolic stress-sensing protein kinase which is known to 22 play an important role in the regulation of acetyl-CoA 23 carboxylase, leading to the acceleration of fatty acid 24 oxidation during vigorous exercise or ischaemia. AMPK is 25 well known as a regulator of lipid metabolism, and in 26 particular is known to have a role in cholesterol 27 synthesis, as reviewed in Hardie and Carling (1997). The 28 AMPK is also considered to play an important role in 29 exercise-enhanced glucose transport (Hayashi et al., (1998) 30 which is distinct from the insulin-mediated glucose uptake 31 mechanism. AMPK has mainly been studied in the liver, 32 heart and skeletal muscle. AMPK has been purified, and the 33 genes encoding the enzyme subunits were cloned (See 34 International Patent Applications WO 00/28076 PCT/AU99/00968 -3 1 numbersPCT/GB94/01093and PCT/US97/00270 and publication 2 W097/25341). 3 The mammalian AMPK (Mitchelhill et al, 1994) is 4 related to the Saccharomyces cereviseae SNFl protein 5 kinase. It is required for the expression of glucose 6 repressed genes in response to nutritional stress which 7 requires growth on alternative carbon sources (Celenza and 8 Carlson, 1986); both the mammalian and yeast kinases are 9 activated by upstream kinases (Hardie and Carling, 1997). 10 The AMPK is involved in metabolic stress responses through 11 phosphorylation at Ser-79 and concomitant inhibition of 12 acetyl-CoA carboxylase and HMG-CoA reductase (Hardie and 13 Carling, 1997). Multiple AMPK isoforms occur. They 14 comprise apy heterotrimers consisting of either al or a2 15 catalytic sub-units (Stapleton et al, 1996; Stapleton et 16 al, 1997a), together with the non-catalytic subunits P and 17 y (Mitchelhill et al, 1994; Carling et al, 1994; Stapleton 18 et al, 1994), which are related to the yeast siplp and 19 snf4p respectively. 20 The AMPK a2 sub-unit gene is on chromosome 1 21 (Beri et al., 1994), the al sub-unit gene is on 22 chromosome 5, the 01 and 71 sub-unit genes are on 23 chromosome 12, the f2 sub-unit gene is on chromosome 1, and 24 the y2 sub-unit gene is localised on chromosome 7 25 (Stapleton et al, 1997). Ay3 gene has been detected using 26 an expressed sequence tag (EST) generated by genome 27 sequencing (Accession No AA178898). 28 One of the genes encoding eNOS is on 29 chromosome 7, close to the gene for the 72 sub-unit of 30 AMPK. Another gene encoding nNOS is found on 31 chromosome 12. (The human gene map; SEE 32 http://www.ncbi.nlm.nih.gov/cgi- WO 00/28076 PCT/AU99/00968 -4 1 bin/SCIENCE96/tsrch?QTEXT=nitric+oxide+synthase) 2 Recent work has shown that the AMPK in cardiac 3 and skeletal muscle is activated by vigorous exercise or by 4 ischaemic stress (Winder and Hardie, 1996; Vavvas et al, 5 1997; Kudo et al, 1995). This led us to investigate the 6 localization of the AMPK isoforms in these tissues. The 7 AMPK-Q2 isoform is present in capillary endothelial cells 8 in cardiac and skeletal muscle, and the AMPK-ol isoform 9 occurs in cardiac myocytes and vessels. The presence of 10 AMPK in endothelial cells led us to test bacterially 11 expressed eNOS as a substrate, and we found that it is 12 readily phosphorylated by either AMPK-tl or AMPK-x2. 13 We have now surprisingly found that the 14 AMP-activated protein kinase phosphorylates and regulates 15 endothelial NO synthase. We find that the AMPK 16 phosphorylates eNOS at two sites. In the presence of 17 calcium and calmodulin, Ser-1177 in the human sequence, and 18 Ser-1179 for the bovine sequence is phosphorylated in the 19 COOH-terminal tail of the enzyme, causing activation of 20 eNOS by shifting the calmodulin-dose dependence. In the 21 absence of added calcium and calmodulin, phosphorylation 22 also occurs at Thr-495 in the eNOS calmodulin-binding 23 sequence, and inhibits the enzyme. Ischaemia of the heart 24 causes activation of the AMPK and of eNOS, mimicking the 25 effects of phosphorylation at Ser-1177. Phosphopeptide 26 specific antibodies to phosphorylated Ser-1177 were used to 27 confirm that this site was phosphorylated during ischaemia. 28 Our results are of special interest because they identify a 29 link between metabolic stress, which reduces ATP and 30 increases AMP, and signalling through eNOS to control 31 nutrient availability (via arterial vasodilation) as well 32 as suppressing myocardial contraction. This couples the 33 metabolic status of endothelial cells and myocytes with the WO 00/28076 PCT/AU99/00968 -5 1 vascular supply and mechanical demands. Our results 2 provide a new insight into the post-translational 3 regulation of eNOS which is of particular significance for 4 the cardiovascular and skeletal muscle field. In addition, 5 similarities in structure and behaviour between eNOS and 6 nNOS have been identified, enabling us to identify 7 modulators of the activity of both these enzymes. 8 9 SUMMARY OF THE INVENTION 10 According to a first aspect, the invention 11 provides a method of identifying modulators of AMPK 12 mediated activation of a nitric oxide synthase enzyme 13 selected from the group consisting of eNOS, nNOS and nNOSg, 14 comprising the step of testing the ability of putative 15 modulators to increase or decrease phosphorylation of the 16 enzyme; said increase or decrease depending on the 17 calmodulin and calcium ion concentrations. 18 Preferably the specific phosphorylation of 19 Ser-1177 is assessed in the presence of calcium and 20 calmodulin. 21 In an alternative aspect, the invention provides 22 a method of identifying modulators of AMPK-mediated 23 inhibition of eNOS, comprising the step of testing a 24 putative modulator for its ability to decrease or increase 25 AMPK-mediated phosphorylation of eNOS in the presence of 26 limiting calcium ions. Preferably specific phosphorylation 27 of Thr-495 is assessed. 28 Compounds able to increase phosphorylation of 29 Ser-1177 or decrease phosphorylation of Thr-495 are 30 referred to herein as activators, and compounds able to 31 decrease phosphorylation of Ser-1177 or increase 32 phosphorylation of Thr-495 are referred to as inhibitors.

WO 00/28076 PCT/AU99/00968 -6 1 In both aspects of the invention, one or more of 2 the following activities may optionally be additionally 3 assessed for each putative activator or inhibitor 4 identified by the method of the invention: 5 (a) Effect on smooth muscle contraction; 6 (b) Effect on inotropic activity of the 7 heart; 8 (c) Effect on chronotropic activity of the 9 heart; and 10 (d) Effect on platelet function. 11 It is expected that because the phosphorylation 12 site equivalent to Thr-495 in the eNOS calmodulin-binding 13 site is absent from the neuronal form of NOS, inhibitors 14 and activators identified by the method of the invention 15 will have at least some degree of tissue specificity. 16 Compounds that activate the AMP-activated protein 17 kinase are expected to be useful in ischaemic heart disease 18 by promoting both glucose and fatty acid metabolism, as 19 well as by increasing NOS activity to improve nutrient and 20 oxygen supply to the myocytes and to reduce mechanical 21 activity. These compounds would also have utility in 22 pulmonary hypertension and in obstructive airways disease. 23 For the purposes of this specification it will be 24 clearly understood that the word "comprising" means 25 "including but not limited to", and that the word 26 "comprises" has a corresponding meaning. 27 28 BRIEF DESCRIPTION OF THE FIGURES 29 Figure 1 shows ilmunofluorescence localization of 30 AMPK-X2 in the heart and in the tibialis anterior muscle.

WO 00/28076 PCT/AU99/00968 -7 1 Panel A shows a negative control section of rat 2 heart stained with control rabbit IgG and control mouse 3 IgG, together with anti-rabbit-FITC and anti-mouse-Texas 4 Red. 5 Panel B shows a section of rat heart stained with 6 affinity-purified rabbit polyclonal antibody against 7 AMPK-oJ2 (491-514) and anti-rabbit-FITC. 8 Panel C shows the same section as Panel B, 9 stained with a monoclonal antibody against rat endothelium 10 recA-1 and anti-mouse-Texas Red. 11 Panel D shows the overlay of Panels B and C. 12 Colocalization can be seen by the coincidence of staining. 13 The arrows highlight specific endothelial cells that are 14 stained by both antibodies. 15 Panel E shows a negative control section of rat 16 tibialis anterior muscle stained with control rabbit IgG 17 and control mouse-IgG, together with anti-rabbit-FITC and 18 anti-mouse-Texas Red. 19 Panel F shows a section stained with affinity 20 purified rabbit polyclonal antibody against AMPK-a2 21 (491-514) and anti-rabbit-FITC. 22 Panel G shows the same section as in Panel B, 23 stained with a monoclonal antibody against rat endothelium 24 recA-1 and anti-mouse-Texas Red. 25 Panel H shows the overlay of Panels E and F. 26 Colocalization can be seen by the coincidence of staining. 27 Figure 2 illustrates phosphorylation of 28 recombinant eNOS by AMPK. 29 Top panel: eNOS was incubated with rat liver 30 AMPK-xl and [y- 3 2 P] ATP. 31 Lane 1: Coomassie-stained SDS-PAGE; WO 00/28076 PCT/AU99/00968 -8 1 Lane 2: Autoradiograph. 2 Lower panel: 32 P-tryptic phosphopeptide map of 3 eNOS. 4 Figure 3 shows the effect of phosphorylation of 5 eNOS by the AMPK with or without added Ca 2 +-CaM. Rat 6 heart eNOS purified by 2',5'-ADP-Sepharose affinity 7 chromatography was phosphorylated by AMPK in the presence 8 of 0.8 pM CaM/3.2 [1M Ca 2 (closed circles), in the absence 9 of Ca -CaM (closed triangles) and without AMPK (open 10 squares). After phosphorylation, samples were diluted and 11 eNOS activity was measured. The lower panels show 12 phosphopeptide maps for rat heart eNOS phosphorylated in 13 the presence and absence of added Ca -CaM. 14 Figure 4 shows the effect of ischaemia on the 15 activities of AMPK-al, AMPK-a2 and eNOS. 16 Panel A shows the results of immunoprecipitation 17 using antibody specific for AMPK-al and AMPK-2, assayed 18 using the SAMS peptide substrate. Results shown are mean 19 SEM for n=5. 20 Panel B shows eNOS activity measured at 500 nM 21 CaM. 22 Panel C shows eNOS activities with full CaM-dose 23 responses for a representative experiment. Ischaemia time 24 points: 0 min (open squares), 1 min (closed diamonds), 25 10 min (closed circles) and 20 min (open triangles). The 26 results of 4 replicates were the same, except that in one 27 case the 20 min ischaemia eNOS CaM-dependence remained the 28 same as for 10 min. 29 Figure 5 shows a comparison of NOS sequences. 30 Phosphorylation site sequences for eNOS and nNOS are 31 indicated in a schematic model of NOS. Sequences from the 32 CaM-binding region (around the Thr-495 phosphorylation site WO 00/28076 PCT/AU99/00968 -9 I in eNOS) and for the COOH-terminal tail (around the 2 Ser-1177 phosphorylation site in eNOS) are shown. 3 Figure 6 shows the effect of treatment of bovine 4 aortic endothelial cells with phorbol ester (PMA) and 5 okadaic acid on eNOS activity (upper pane) and the 6 phosphorylation at Ser-1177 and Thr-495 (lower panel). 7 Figure 7 shows the effect of treatment of bovine 8 aortic endothelial cells with 3-isobutyl-1-methylxanthine 9 (IBMX) and calyculin A on the phosphorylation at Ser-1177 10 and Thr-495. 11 Figure 8 shows a summary illustration of the 12 regulation of eNOS by phosphorylation at Thr-495 and Ser 13 1177, mediated by protein kinases PKC, AMPK and Akt. 14 Reversal of the phosphorylation at these sites is mediated 15 by protein phosphatases PP1 and PP2A in response to 16 treating the cells with IBMX and PMA respectively. 17 Figure 9 shows the effect of a 30 second bicycle 18 sprint exercise on nNOS phosphorylation in human muscle. 19 The nNOS was extracted from biopsy material and probed for 20 phosphorylation at Ser-1417 using an anti-phosphopeptide 21 antibody. The left panel shows an immunoblot, and the 22 right panel shows quantitative analysis of 5 individuals. 23 24 DETAILED DESCRIPTION OF THE INVENTION 25 The invention will now be described in detail by 26 way of reference only to the following non-limiting 27 examples and to the figures. 28 We have surprisingly found that in the presence 29 of Ca -calmodulin (CaM) eNOS is phosphorylated by AMPK at 30 Ser-1177, resulting in activation, whereas phosphorylation 31 of eNOS in the absence of Ca occurs predominantly at 32 Thr-495, a site in the CaM-binding sequence, resulting in WO 00/28076 PCT/AU99/00968 - 10 I inhibition. It had previously been considered that 2 phosphorylation was solely inhibitory. We have also found 3 that ischaemia of the heart leads to rapid activation of 4 both isoforms of the metabolic stress-sensing enzyme AMPK 5 and eNOS. These data suggest that the AMPK may operate an 6 "inside-out" signalling pathway that leads to arterial 7 vasodilation and reduced myocardial contraction, so 8 coupling the metabolic status of endothelial cells and 9 myocytes with the vascular supply and mechanical activity. 10 11 Example 1 Immunofluorescence Localisation of AMPK-x2 12 in Heart and Skeletal Muscle 13 Confocal immunofluorescence microscopy using 14 affinity-purified rabbit polyclonal antibody directed 15 against AMPK-X2 (antibody 491-414. Staining with 16 fluorescence-labelled anti-rabbit antibody showed that the 17 a2 isoform is found predominantly in capillary endothelial 18 cells in both cardiac muscle and skeletal muscle, while 19 cardiac myocytes and blood vessels showed intense but 20 diffuse staining for the al AMPK isoform. In skeletal 21 muscle, the a2 isoform was found in endothelial cells of 22 capillaries, and in fast-twitch muscle fibres, whereas the 23 al isoform was found in Type I aerobic fibres. 24 Localisation of AMPK-a2 in capillary endothelial cells in 25 both cardiac and skeletal muscle is illustrated in 26 Figure 1. 27 28 Example 2 AMPK Phosphorylates Recombinant -eNOS 29 Bacterially expressed eNOS, coexpressed with CaM 30 by the method of Rodriguez-Crespo et al (1996), was 31 phosphorylated by either AMPK-al, as shown in Figure 2 top WO 00/28076 PCT/AU99/00968 - 11 I panel, or AMPK-02. Recombinant eNOS phosphorylation by 2 immunoprecipitated AMPK-L2 was detected. Since we have 3 been unable to purify high specific activity AMPK-U2, no 4 further characterisation of eNOS regulation or the sites of 5 phosphorylation by the a2 isoform was undertaken. Analysis 6 of the phosphorylation sites in eNOS following tryptic 7 digestion revealed four phosphopeptides generated from 8 three separate sites (Figure 2 bottom panel, A, A', B, C). 9 Identification of phosphorylation sites by mass 10 spectrometry and Edman sequencing, using the modified 11 method described by (Mitchelhill and Kemp, 1999), revealed 12 that Ser-1177 was the most prominent phosphorylation site, 13 as shown in Figure 2 bottom panel, A, A', and that its 14 phosphorylation was dependent on the presence of Ca 2 +-CaM. 15 Phosphopeptide isolation from in-gel tryptic 16 digests was carried out as described by Mitchelhill et al 17 (1997a). Greater than 98% of the radioactivity was 18 recovered from the gel. Peptides isolated and characterized 19 by mass spectrometry and Edman sequencing are set out in 20 Table 1.

WO 00/28076 PCT/AU99/00968 -12 0 HHHH H H zi n > .nci rc H w. m~ w 0 -) (d m ) r) ) 0 ci) 04 0)o Q j41 4~ 4 0 1 0 0)4 04 a 0 0 0)0~ EIZ -H 014 H4 0 QU) H4WJr 0 ci) 0 >10 -H 04 0 0) -1 o4 roW 0 -H 4-J a) > 0 n 0 0 c0 :ii Lfl w 04 00 a) WO 00/28076 PCT/AU99/00968 - 13 The location of the phosphorylation site in 2 peptide A, TQXFSLQER, was identified by 32 P-phosphate 3 release sequencing (Mitchelhill et al, 1997a). eNOS 4 phosphorylated by the AMPK-cXl was no longer recognized by 5 the antibody to the eNOS COOH-terminal tail; nor was it 6 eluted from the ADP-Sepharose affinity column by 7 100 mM NADPH. These properties prevented the direct 8 confirmation of Ser-1177 phosphorylation in situ. This is 9 illustrated in Venema et al, 1996. 10 A second site, Thr-495, was phosphorylated in the 11 absence of Ca 2 -CaM or when EGTA was present. This is 12 illustrated in Figure 2 bottom panel, B. This residue is 13 located in the CaM-binding sequence, 14 TRKKT 495 FKEVANAVKISASLM, 15 between the oxidase and reductase domains of eNOS (Venema 16 et al, 1996). Ser-101 in the N-terminal region of eNOS was 17 identified as a minor site of phosphorylation (Figure 2 18 bottom panel, C). 19 Synthetic peptides containing Thr-495 or Ser-1177 20 were readily phosphorylated by AMPK, with similar kinetic 21 values to the SAMS peptide substrate. The peptide 22 containing Thr-495, GTGITRKKTFKEVANAVK, was phosphorylated 23 with a Km of 39 ± 10 4M and a Vmax of 24 6.7 ± 0.6 pmol/min/mg, whereas the peptide containing 25 Ser-1177, RIRTQSFSLQERQLRG was phosphorylated with a Km of 26 54 ± 6 JIM and a Vmax of 5.8 ± 0.3 pmol/min/mg. These are 27 comparable to results obtained using the well-characterized 28 SAMS peptide substrate, which has a Km 33 ± 3 pM and a Vmax 29 of 8.1 ± 1.5 pmol/min/mg (Michell et al, 1996). The in 30 vitro phosphorylation of the peptides confirms the 31 identification sites of phosphorylation. 32 WO 00/28076 PCT/AU99/00968 - 14 I Example 3 Effect of Ca 2+-CaM on Phosphorylation of 2 eNOS by AMPK 3 The eNOS activity was determined by measuring 4 L-[ 3 H]-citrulline production, using the method of Balligand 5 et al, 1995. The recombinant eNOS was coexpressed with 6 CaM, as described by Rodriguez-Crespo and Ortiz de 7 Montellano, 1996. Partially purified rat heart eNOS 8 contained some Ca 2 -CaM. In the absence of added EGTA, CaM 9 dependence was observed at .0-100 nM added CaM. In order to 10 investigate the changes in NOS activity with 11 phosphorylation in the absence and presence of Ca 2 -CaM, 12 EGTA buffering was used to achieve CaM dose response curves 13 in the range 0-1 pM. Routinely, 7-15 pM EGTA was added to 14 make eNOS activity dependent upon added CaM. Where 15 Ca -CaM was used in the phosphorylation reaction prior to 16 eNOS assay, the samples were either diluted so that the 17 extra Ca 2+-CaM was negligible, or the indicated 18 concentrations represent total final concentrations of 19 added Ca 2 +-CaM. 20 Cardiac eNOS was partially purified as follows. 21 Twenty rat hearts were homogenised in 80 ml of ice-cold 22 buffer A [50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 23 1 mM DTT, 50 mM NaF, 5 mM Na Pyrophosphate, 24 10 gg/ml Trypsin inhibitor, 2 pg/ml Aprotinin, 25 1 mM Benzamidine, 1 mM PMSF, 10% Glycerol, 1% Triton-X 26 1001. The homogenate was put on ice for 30 min and 27 centrifuged at 16,000 x g for 30 min. The supernatant was 28 incubated with 2 ml of 2',5'-ADP-Sepharose (Bredt and 29 Snyder, 1990). The suspension was incubated for one hour 30 before washing in a fritted column, with 20 ml of buffer A 31 and 20 ml of buffer A containing 0.5 M NaCl, and then with 32 20 ml of buffer B [50 mM Tris-HCl, pH 7.5, 1 mM DTT, 33 10% Glycerol, 0.1% Triton-X-100]. eNOS was eluted with 34 buffer B containing 2 mM NADPH, then subjected to WO 00/28076 PCT/AU99/00968 - 15 I centrifugal filtration (ULTRAFREE-MC MILLIPORE) to remove 2 NADPH. Immunoblotting was used for selective detection of 3 eNOS rather than nNOS. 4 Phosphorylation of eNOS by AMPK in the presence 5 of Ca2+ -CaM resulted in activation, but CaM-dependence was 6 retained, as shown in Figure 3 top panel. Activation 7 shifted the dose response curve for CaM to the left. 8 Phosphopeptide mapping revealed that activation of eNOS was 9 correlated with phosphorylation of Ser-1177 but not of Thr 10 495, as shown in Figure 3 lower panel. Phosphorylation 11 without added Ca 2 +-CaM enhanced Thr-495 phosphorylation, 12 suppressed Ser-1177 phosphorylation, and inhibited eNOS 13 activity (Figure 3 top panel). The inhibition of eNOS 14 activity by Thr-495 phosphorylation is consistent with 15 earlier reports that phosphorylation of synthetic peptides 16 corresponding to this region by protein kinase C inhibits 17 CaM-binding (Matsubara et al, 1996). Similar results have 18 been reported for nNOS (Loche et al, 1997). 19 20 Example 4 Effect of Ischaemia on Activities of 21 AMPK-cl, AMPK-2 and eNOS 22 Langendorf preparations of isolated perfused rat 23 heart were subjected to ischaemia according to the method 24 of Kudo et al (1995). AMPK-Xl and AMPK-a2 isoforms were 25 immunoprecipitated using a2 (490-516) or al (231-251) 26 antibodies, and assayed using the SAMS peptide substrate 27 (Michell et al, 1996; Hardie and Carling, 1997). eNOS 28 activity was measured as described in Example 3. The 29 results are shown in Figure 4. Both al and a2 isoforms are 30 activated, as shown in Figure 4A, indicating that AMPK is 31 activated in both capillary endothelial cells, which have 32 predominantly the a2 isoform, and in cardiac myocytes, WO 00/28076 PCT/AU99/00968 - 16 I which have predominantly the al isoform. AMPK activation 2 during ischaemia is also accompanied by eNOS activation and 3 changes in the CaM dependence, as shown in Figures 4B and 4 4C, mimicking the effect of eNOS phosphorylation by AMPK in 5 vitro, as shown in Figure 3. 6 Polyclonal antibodies were raised against 7 synthetic phosphopeptides based on the eNOS sequence: 8 RIRTQSpFSLQER and GITRKKTpFKEVANCV. Rabbits were immunized 9 with phosphopeptides coupled to keyhole limpet haemocyanin 10 and then emulsified in Freund's complete adjuvant, using 11 conventional methods. The antibodies were purified using 12 the corresponding phosphopeptide affinity columns after 13 thorough preclearing with dephosphopeptide affinity 14 columns. The specificity of the purified antibodies was 15 confirmed using both EIA and immunoblotting, confirming 16 that they did not recognize recombinant dephospho-eNOS. 17 Using the anti-phosphopeptide antibodies to Ser 18 1177 and Thr-495 phosphorylation sites we observed that 19 phosphorylation of Ser-1177 was increased approximately 3 20 fold by ischaemia, but that there was no detectable change 21 in the Thr-495 phosphorylation under these conditions. 22 Heart muscle contains eNOS in both capillary endothelial 23 cells and cardiac myocytes (Balligand et al, 1995), with 24 low levels of the nNOS g isoform (Silvagno et al, 1996). 25 The sequences of the three types of NOS are 26 compared in Figure 5, which shows the CaM-binding region 27 and the C-terminal tail. In nNOS Ser-1417 corresponds to 28 eNOS Ser-1177, whereas iNOS is truncated, and has a Glu in 29 this region. Both iNOS and nNOS lack a phosphorylatable 30 residue equivalent to Thr-495 in the CaM-binding region.

WO 00/28076 PCT/AU99/00968 - 17 11 2 3 Example 5 Effect of Stimulation of Protein Kinase C on 4 eNOS Phosphorylation 5 Bovine aortic endothelial cells cultured in 0.1% 6 foetal calf serum for 20 hours (serum starved) were 7 subjected to treatment with the protein kinase C activator 8 0.1 pM phorbol-12-myristate-13-acetate (PMA) for 5 min. 9 PMA treatment increased the phosphorylation of eNOS at Thr 10 495 and decreased the phosphorylation at Ser-1177, as 11 measured using anti-phosphopeptide specific antibodies. 12 The antibodies used were the same as those described in 13 Example 4. The results are shown in Figure 6. In cells 14 cultured in medium without calcium we observed a 4-fold 15 decrease in Ser-1177 phosphorylation. Furthermore, when 16 cells were incubated in standard medium containing calcium 17 addition of the calcium ionophore A23187 (10gM for 90 18 seconds) increased Ser-1177 phosphorylation by a further 7 19 fold. Preincubation of the cells with 0.5 pM okadaic acid 20 prevented the dephosphorylation of Ser-1177 by PMA 21 treatment, and greatly augmented the phosphorylation of 22 Thr-495 (Results mean ± SEM, n =6). Since okadaic acid 23 inhibits protein phosphatase PP2A, the results indicate 24 that PP2A is responsible for dephosphorylation of Ser-1177. 25 The changes observed in Thr-495 and Ser-1177 26 phosphorylation in response to treatment with PMA and 27 okadaic acid were reflected in the activity of eNOS. 28 Increased phosphorylation of Thr-495 with PMA or PMA plus 29 okadaic acid was associated with reduced eNOS activity. 30 Okadaic acid alone increased Ser-1177 phosphorylation 31 without altering Thr-495 phosphorylation, and was 32 associated with increased eNOS activity (Figure 6 upper 33 panel).

WO 00/28076 PCT/AU99/00968 - 18 2 3 Example 6 Effect of Inhibition of Phosphodiesterase 4 and Phosphatase on the Phosphorylation of eNOS 5 The experimental details were similar to those 6 for Example 5. Bovine aortic endothelial cells were 7 preincubated with or without 10 nM of the phosphatase 8 inhibitor calyculin A for 10 min, and then incubated with 9 or without 0.5 mM of the phosphodiesterase inhibitor, 3 10 isobutyl-1-methylxanthine (IBMX) for 5 min. As shown in 11 Figure 7, IBMX treatment caused enhanced phosphorylation of 12 Ser-1177 and dephosphorylation of Thr-495. Preincubation 13 with calyculin A prevented the dephosphorylation of Thr 14 495. (Results mean ± SEM, n =6). Since calyculin A 15 inhibits protein phosphatase PP1, the results indicate that 16 PP1 is responsible for dephosphorylation of Thr-495. 17 18 DISCUSSION 19 Since the identification of the Ser-1177 20 phosphorylation site by the present inventors, it has been 21 recognized that other protein kinases phosphorylate at this 22 site. In particular, the protein kinase Akt (also named 23 PKB) phosphorylates Ser-1177 in response to stimulation of 24 endothelial cells by vascular endothelial growth factor 25 (VEGF ) (Fulton et al.1999; Michell et al.,1999) or to fluid 26 shear stress (Dimmeler et al., 1999; Gallis et al., 1999). 27 In the study by Gallis et al. (1999) it was reported that 28 fluid shear stress stimulated the phosphorylation of Ser 29 116 in the sequence KLQTRPSPGPPPA. Neither the kinase 30 responsible nor the functional effects of phosphorylation 31 of this site on eNOS has yet been identified. This 32 phosphorylation site is present in the oxidase domain.

WO 00/28076 PCT/AU99/00968 - 19 1 We have found that phosphorylation of eNOS at 2 Thr-495 by protein kinase C occurs in endothelial cells 3 that have been serum starved and incubated in calcium-free 4 medium in the presence of the phorbol ester PMA. There is 5 a reciprocal relationship between phosphorylation at Ser 6 1177 and Thr-495 in endothelial cells. Protein kinase C 7 phosphorylates both sites in vitro, but stimulation of 8 protein kinase C in endothelial cells with phorbol ester 9 causes enhanced Thr-495phosphorylation but marked 10 phosphorylation of Ser-1177. The dephosphorylation of Ser 11 1177 is prevented by okadaic acid but not by calyculin A, 12 indicating that phosphatase PP2A is responsible. Okadaic 13 acid also greatly enhances the phosphorylation of Thr-495 14 in response to phorbol ester. Thrombin, which also acts 15 via protein kinase C, stimulates phosphorylation of Thr-495 16 and dephosphorylation of Ser-1177. 17 In contrast, treatment of endothelial cells with 18 the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine 19 (IBMX) causes a pronounced dephosphorylation of eNOS at 20 Thr-495 and enhanced Ser-1177 phosphorylation. 21 Dephosphorylation of Thr-495 in response to IBMX is blocked 22 by treatment with calyculin A, suggesting that phosphatase 23 PP1 is responsible for Thr-495 dephosphorylation. 24 These relationships are summarised in Figure 8. 25 We find that exercise of skeletal muscle results in the 26 phosphorylation of nNOSg at Ser-1417, the site 27 corresponding to Ser-1177 in eNOS (see Figure 5). 28 Electrical stimulation of rat extensor digitorum longus 29 (EDL) muscle was found to activate the AMPK, to 30 phosphorylate acetyl CoA carboxylase at Ser-79 (the 31 inhibitory site), and to-phosphorylate nNOSg at Ser 1417. 32 Similarly, in biopsies of human skeletal muscle following 33 vigorous exercise, such as a 30-second bicycle sprint, 34 there is a 10-fold increase in phosphorylation on Ser-79 in WO 00/28076 PCT/AU99/00968 - 20 1 acetyl CoA carboxylase and a 7.5-fold increase in nNOSR 2 phosphorylation at Ser-1417 (see Figure 9). 3 Endothelially-derived NO has a critical role in 4 preventing premature platelet adhesion and aggregation that 5 leads to thrombus formation (Radomski and Moncada, 1993). 6 There is evidence that the protective effects of elevated 7 high-density lipoprotein (HDL) on the cardiovascular system 8 may be mediated via increased platelet NO production. 9 Apolipoprotein E, a component of HDL, acts on a receptor 10 (apoER2) present in platelets to stimulate the NO signal 11 transduction pathway (Riddell et al., 1997; Riddell and 12 Owen, 1999). 13 Activation of eNOS by phosphorylation of its 14 COOH-terminal tail gives new insight into eNOS 15 autoinhibition. The increased activity and shift in the 16 CaM-dose dependence with phosphorylation at Ser-1177 17 suggest that in eNOS, and perhaps nNOS, the COOH-terminal 18 tails act as partial autoregulatory sequences analogous to 19 those in the CaM-dependent protein kinases (Kemp and 20 Pearson, 1991; Kobe et al, 1996). 21 The COOH-terminal tail of eNOS is only fully 22 accessible to the AMPK when Ca 2 +-CaM is bound, consistent 23 with this region being buried in the absence of CaM. As 24 can be seen from Figure 5, there is a high level of 25 similarity between eNOS and nNOS in their COOH-terminal 26 tails, whereas iNOS is distinct. It is known that the iNOS 27 CaM-binding, which is characterised by a low 28 Ca -dependence, requires both the canonical CaM-binding 29 sequence and distal residues in the COOH-terminus that 30 cannot be satisfied by nNOS chimeras (Ruan et al, 1996). 31 Without wishing to be boufnd by any proposed mechanism, we 32 believe that eNOS and nNOS are autoinhibited by their 33 COOH-terminal tails, requiring a two-stage activation WO 00/28076 PCT/AU99/00968 - 21 1 process for full activity with both CaM-binding and 2 phosphorylation in the tail, whereas iNOS requires only CaM 3 binding. Recently Salerno et al (1997) proposed that an 4 insert sequence in the FMN-binding domain may also be 5 important in autoregulation. 6 Previous studies have shown that eNOS may be 7 phosphorylated both in vitro and in vivo, but the precise 8 sites of phosphorylation and the function of the 9 phosphorylation events have not hitherto been fully 10 characterized (reviewed in Michel and Feron, 1997). eNOS 11 is the first example of an enzyme activated by AMPK to be 12 identified, and is also unusual because phosphorylation can 13 lead to either activation or inhibition, depending on the 14 availability of Ca 2 -CaM. Other enzymes, notably the 15 cyclin-dependent protein kinases, are activated or 16 inhibited by phosphorylation, but this is catalysed by 17 different protein kinases. Protein kinase C phosphorylates 18 Thr-495 in eNOS, demonstrating intersecting regulatory 19 pathways acting on eNOS by phosphorylation of Thr-495 or 20 Ser-1177. It is also possible that persistent activation 21 of protein kinase C, for example in response to 22 hyperglycaemia induced by diabetes, could chronically 23 suppress phosphorylation of eNOS at Ser-1177, and thereby 24 reduce its activity. 25 The regulation of eNOS by AMPK extends the 26 conceptual relationship between the yeast snflp kinase and 27 the AMPK. Snflp kinase modulates the supply of glucose from 28 the environment by secreting invertase whereas the 29 mammalian AMPK integrates metabolic stress signalling with 30 the control of the circulatory system. Thus intracellular 31 metabolic stress signals _within endothelial cells and 32 myocytes can elicit improved nutrient supply and suppress 33 mechanical activity of the muscle.

WO 00/28076 PCT/AU99/00968 - 22 1 It will be apparent to the person skilled in the 2 art that while the invention has been described in some 3 detail for the purposes of clarity and understanding, 4 various modifications and alterations to the embodiments 5 and methods described herein may be made without departing 6 from the scope of the inventive concept disclosed in this 7 specification. 8 References cited herein are listed on the 9 following pages, and are incorporated herein by this 10 reference.

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Claims (13)

1. A method of identifying modulators of AMPK-mediated activation of a nitric oxide synthase enzyme selected from the group consisting of eNOS, nNOS and nNOS1, comprising 5 the step of testing putative modulators for their ability to increase or decrease phosphorylation of the enzyme, said increase or decrease depending on the calmodulin and calcium ion concentrations.

2. A method according to claim 1, in which the specific 10 phosphorylation of Ser-1177 is assessed in the presence of calcium and calmodulin.

3. A method of identifying modulators of AMPK-mediated inhibition of eNOS, comprising the step of testing a putative modulator for its ability to decrease or increase 15 AMPK-mediated phosphorylation of eNOS in the presence of limiting calcium ions.

4. A method according to claim 3, in which the specific phosphorylation of Thr-495 is assessed.

5. A method according to any one of Claims 1 to 4, in 20 which one or more of the following activities is additionally assessed: (a) Effect on smooth muscle contraction; (b) Effect on inotropic activity of the heart; (c) Effect on chronotropic activity of the heart; or 25 (d) Effect on platelet function.

6. A method according to any one of Claims 1 to 5, in which the modulator is an activator, as herein defined.

7. A method according to Claim 6, in which the activator promotes both glucose metabolism and fatty acid metabolism. WO 00/28076 PCT/AU99/00968 - 29

8. A method according to any one of Claims 1 to 5, in which the modulator is an inhibitor, as herein defined.

9. A method according to any one of Claims 3 to 8, in which the modulator acts preferentially on non-neuronal 5 cells.

10. A method according to Claim 1 or Claim 2, in which the modulator promotes the dephosphorylation of Ser-1177 and inhibits eNOS activity.

11. A method according to Claim 3, in which the modulator 10 promotes the dephosphorylation of Thr-495 and stimulates eNOS activity.

12. A method according to Claim 1 or Claim 2 in which the modulator promotes phosphorylation of nNOS or nNOSg at Ser

1417. 15

13. A method according to Claim 1 or Claim 2 in which the modulator promotes dephosphorylation of nNOS or nNOSg at Ser-1417.