EA036081B1 - Modified radioligand method for quantitative assessment of beta-adrenoreceptor binding activity on human t-lymphocyte surface - Google Patents

Abstract

The invention relates to the field of medicine and provides a modified radioligand method for quantitative assessment of -adrenoreceptor (-AR) binding activity on human T-lymphocyte surface which ensures reliable assessment of specific binding with 1- and 2-adrenoreceptors in low concentrations, and can be used for testing during clinical studies. The method includes pre-incubation of a medium containing T-lymphocytes with a ligand specific to 1- and 2-AR with subsequent interaction with [I]cyanopindolol. The technical result is significant improvement of assay sensitivity in respect of 1-AR binding activity analysis, and the possibility of identification of typical characteristics variation patterns for -AR unit influenced by pre-incubation with specific ligands.

Description

The technical field to which the invention relates

The invention relates to the field of medicine. More specifically, the invention provides a modified radioligand method for quantifying the binding activity of β-adrenergic receptors (β-AR) on the surface of human T-lymphocytes, allowing for reliable determination of specific binding to β1- and β2-adrenergic receptors at their low concentrations, on the basis of which assays can be performed for clinical research.

Prior art

In the structure of non-infectious pathology, cardiovascular and broncho-pulmonary pathologies are of the greatest importance. Diseases of the cardiovascular system are in the first place among the causes of morbidity and mortality, both in Russia and in other countries of the world [Chazova I.E., Chuchalin A.G., Zykov K.A., Ratova L.G. / / Systemic hypertension. 2013.10 (1) p. 5-35].

Among broncho-pulmonary pathologies, broncho-obstructive diseases require special attention. [Global Initiative for Asthma (GINA) (updated 2014); Global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease (GOLD). Update 2014.] Many patients with these pathologies need to be prescribed drugs that act on β-adrenergic receptors.

By their structure, adrenergic receptors belong to a group of receptors connected to guanine nucleotide binding proteins (G-protein). Anatomically, β-adrenergic receptors are subdivided into β1-, β2- and β3-adrenergic receptors, among which the following are most significant for the diagnosis and treatment of cardiovascular and bronchopulmonary diseases:

1) β1-adrenergic receptors, the maximum number of which is found in the right atrium;

2) β2-adrenergic receptors, which are present mainly in the lungs [Sano M, Yoshimasa T., Yagura T., Yamamoto I. Life Sci. 1993. v. 52 (12). P.1063-70].

In lung diseases, therapy is often based on β-adrenergic agonists, and in cardiovascular diseases, β-blockers, which are ligands for the corresponding receptors. Changes in the expression and affinity of receptors under the influence of appropriate drugs often lead to a decrease in the effectiveness of the prescribed therapy and in some cases to the development of serious side effects, such as an increase in heart rate, prolongation of the QT interval, a decrease in potassium levels, bronchospasm, etc.

The ligand-receptor interaction depends on the type of ligand (agonist or inverse agonist) and is manifested in a change in cellular activity. Agonists, binding to the receptor, activate the G-protein and increase the intracellular content of cyclic adenosine monophosphate (cAMP). Simultaneously with the appearance of the clinical effect, these changes in the cell also cause a change in the activity of the receptor. Often the appointment of agonists leads to a decrease in sensitivity to the ligand and desensitization of receptors [Mickey J., Tate R., Lefkowitz R.J.J. Biol: Chem. 1975. No. 250. P. 5727-5729; Perkins J.P., Waldo G.L., Harden T.K. Federation Proc. 1982. V. 41. P. 1327], which in some cases causes an unfavorable change in the clinical course of the disease in the form of worsening bronchial obstruction and lack of response to the therapy. The use of antagonists, on the contrary, leads to an increase in the density of β-adrenergic receptors [Parfenova EF, Krasnikova T.L., Aripova N.A. and others. Ger. Archive. 1993.65 (4). from. 49-52] on the surface of mammalian cells and can affect their affinity.

Changes in receptor activity are noted not only under the influence of pharmacological drugs. It was noted that in diseases of the cardiovascular and bronchopulmonary systems, there is already an initially altered background of receptor activity. In patients with ventricular premature beats, the number of β2-adrenoreceptors on peripheral blood lymphocytes is statistically higher than in the normal group [Krasnikova T.L., Yurkova V.B., Kuzmina M.M. and other Cardiology. 1989. No. 7. S.25-29].

Patients with arterial hypertension also have a statistically significantly increased expression of β2-adrenoreceptors in relation to the norm, which, in addition, correlates with the severity of the disease. At the same time, the presence of heart failure in the latter leads to a decrease in the indicator of maximum binding of β2-ligands. [Qing F., Rahman S.U., Rhodes C.G. et al., Am. J. Respir. Crit. Care Med. 1997.155 (3). P. 1130-1134].

When examining patients with obstructive pulmonary pathology, conflicting data are noted: some sources report an initially low density of β2-receptors on the surface of blood cells, while others do not note the relationship between changes in the density of receptors on the surface of blood cells with the presence of bronchial asthma [P.G. Stanley, L. Duriseti, Sh. Underwood, S. Allred, P.A. Insel. J. Clin. Invest. 1980. V. 65 (3). P.577-585].

In recent decades, a number of both direct and indirect methods have been developed to determine receptor activity: assessment of changes in affinity, expression of receptors, as well as their activity due to changes in secondary messengers.

Methods of binding to radioactive ligands allow us to assess the parameters of interest for receptors both initially and during the use of drugs that affect β-adrenorecetors, and also allow us to determine their affinity [Blankesteijn W. M, Graafsma S. J., Hectors M. P. Y. et al. Eur.

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J. Clin. Pharmacol. 1992.42 (6). P. 613-8]. The density of surface β-adrenergic receptors is determined by the method of specific binding using a specific ligand labeled with a radioactive isotope ¹²⁵ I or ³ N. Non-specific binding is determined by adding a significant excess of unlabeled ligand. The specific binding of the ligand to the receptor is calculated from the difference between total and nonspecific binding, the receptor density and the dissociation constant are determined in Scatchard coordinates using the linear regression coefficient. From the curves of displacement of the labeled ligand calculate the dissociation constant of the receptor-ligand according to the Cheng-Prussov formula [Williams L. T., Snyderman R., Lefkowitz RJJ Clin. Invest. 1976, v. 57 (1). Pp. 149-55; Krasnikova T.L., Korichneva I.L., Radyukhin V.A. Biochemistry. 1989.54 (2), p. 235-243].

However, the described traditional radioligand method for determining the amount and affinity of β-adrenergic receptors is laborious, expensive and requires a large amount of blood to be drawn, especially when studying the effect of drugs. The latter is most important in assessing the dynamics of the effect of drugs acting on β-adrenergic receptors, which makes this method of little use in real clinical practice.

The closest, in fact, technical solution is a method for the selective quantitative assessment of the binding activity of β-adrenergic receptors on the surface of human leukocytes, disclosed in patent publication EA 026837 (publ. 05/31/2017), in the implementation of which to assess the binding activity of β1-adrenergic receptors for specific binding [ ²⁵ 1] cyanopindolol with cells determine the difference between the binding of [ ¹²⁵ 1] cyanopindolol by cells in the presence of 1.5-2.0 μM non-radioactive cyanopindolol and binding of [ ²⁵ 1] cyanopindolol by cells in the presence of 0.20-0.28 μM β1-specific ligand CGP- 20712 (chemical name - [2 - ((3-carbamoyl-4hydroxy) phenoxy) ethylamino] -3- [4- (1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy] -2-propanol dihydrochloride), for which the reaction mixture containing an aqueous solution of [ ¹²⁵ 1] cyanopindolol, phosphate buffered saline (PBS), a solution of unlabeled ligand (cyanopindolol or CGP-20712) and the cell suspension are incubated for 45-60 min at 37 ° C with caution With proper stirring, the process is stopped by adding ice water (cold water with ice), the cells are centrifuged at 1800-2000g for 10-12 minutes, the supernatant is discarded, the precipitate is carefully suspended in cold (+ 4 ° C) PBS and centrifuged again under the same conditions , the supernatant is discarded and the sediment is carefully suspended in cold PBS and centrifuged at 10000-12000g for 2-3 minutes, the supernatant is discarded, and the sediment is counted on a γ-counter, measuring the amount of radioactive material in each sample, after which the binding activity of β1-adrenergic receptors is calculated by the difference between the total binding and β-specific binding of cyanopindolol, per 1 million cells.

For a quantitative selective assessment of the binding activity of β2-adrenergic receptors, the difference in binding [ ¹²⁵ 1] cyanopindolol by cells in the presence of 1.5-2.0 μM non-radioactive cyanopindolol and binding of [ ²⁵ 1] cyanopindolol by cells in the presence of 0.14-0.18 μM β2-specific ligand is determined ICI 118551 (chemical name (±) -erythro hydrochloride - ^ * ^ *) - 1- [2,3 (dihydro-7-methyl-1H-inden-4-yl) oxy] -3 - [(1-methylethyl) amino] -2-butanol), for which the reaction mixture containing an aqueous solution of [ ¹²⁵ 1] cyanopindolol, phosphate buffered saline (PBS), a solution of unlabeled ligand (cyanopindolol or ICI 118551) and the cell suspension are incubated for 45-60 min at 37 ° C with gentle stirring, the process is stopped by adding ice water (cold water with ice), the cells are centrifuged at 1800-2000g for 10-12 minutes, the supernatant is discarded, the precipitate is carefully suspended in cold (+ 4 ° C) PBS and centrifuged again at the same conditions, the supernatant is discarded and the sediment is carefully pendate in cold PBS and centrifuged at 10000-12000g for 2-3 minutes, the supernatant is discarded, and the precipitate is counted on a γ-counter, measuring the amount of radioactive material in each sample, after which the binding activity of β2-adrenergic receptors is calculated from the difference in total binding and β -specific binding of cyanopindolol, per 1 million cells.

The disadvantage of the closest analogue is its low sensitivity in relation to β1-adrenergic receptors. Thus, there is a need to develop an improved radioligand assay method that substantially overcomes the disadvantage of the prior art solution.

In accordance with the invention, there is provided a method for radioligand determination of the binding activity of β1- and β2-adrenergic receptors of human T-lymphocytes, for which

a) taking blood from the object from a peripheral vein;

b) the allocation of T-lymphocytes in a conventional way;

c) setting up four parallel analyzes with the addition of 100 μl [ ¹²⁵ I] cyanopindolol in each of them;

d) incubation for 25-30 minutes at 37 ° C and a shaker speed of 100 rpm;

e) stopping the reaction by adding cold water;

f) centrifugation of the reaction mass at 2000 g for 10-12 minutes with discarding the supernatant;

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g) suspending the pellet in cold (+ 4 ° C) PBS buffer, followed by centrifugation under the same conditions, discarding the supernatant;

h) suspending the pellet in cold (+ 4 ° C) PBS buffer, followed by centrifugation at 10000-12000 g for 2-3 min with discarding the supernatant;

i) counting the precipitate on a γ-counter to measure the amount of radioactive material in each sample, and

l) calculation of the binding activity of β1-adrenergic receptors by the difference between the total binding and β-specific binding of cyanopindolol per 1 million cells, characterized in that when four parallel analyzes are performed in the first of them, water is added to the medium containing T-lymphocytes, in the second, in cold cyanopindolol is added to the excess, in the third add e1-AP ligand CGP 20712, in the fourth add b2-AP ligand ICI 118551 and pre-incubation of each analyzed sample is carried out for 25-30 min at 37 ° C and a shaker speed of 100 rpm;

m) calculation of the binding activity of β2-adrenergic receptors by the difference between the total binding and the beta-specific binding of cyanopindolol per 1 million cells, characterized in that when four parallel analyzes are performed in the first of them, water is added to the medium containing T-lymphocytes, in the second, in cold cyanopindolol is added to the excess, in the third, b1-AP ligand CGP 20712 is added, in the fourth, b2-AR ligand ICI 118551 is added, and each analyzed sample is preincubated for 25-30 minutes at 37 ° C and a shaker speed of 100 rpm.

List of drawings

FIG. 1 shows histograms of the binding activity of β-adrenergic receptors of T-lymphocytes of healthy volunteers and volunteers suffering from diseases.

FIG. 2 is a graph showing the dependence of the percent binding of [^^ cyanopindolol by ADL-7A cells in the presence of CGP-20712 on the concentration of the latter.

FIG. 3 shows a graph of the dependence of the percentage of binding of [^ -cyanopindolol by A2R9 cells in the presence of ICI 118551 on the concentration of the latter.

FIG. 4 shows histograms of the binding activity of e1-AP and B2-AP of T-lymphocytes in cases of pre-incubation and without pre-incubation.

The essence of the invention

As the closest analogue, the authors of the present invention proposed a method for assessing the binding activity of β-1- and β-2-adrenergic receptors, based on the displacement of iodine-125 labeled cyanopindolol from the cellular receptors, nonspecifically binding to several types of receptors, ligands with high specificity for adrenergic receptors: CGP-20712 and ICI 118551. There has been an association of β2-AR with other clinical and laboratory parameters of proven clinical significance. At the same time, the binding activity of e1-AP lymphocytes was at an extremely low level, approximately 10-20 times lower than the binding activity of β2-AP of the same cells. Therefore, the previously obtained results on the binding activity of e1-AP T-lymphocytes have not been comprehensively analyzed due to their low reliability.

However, in the course of ongoing studies with T-lymphocytes of persons suffering from combined cardiorespiratory pathology, the authors found that some types of cardiac pathologies are characterized by extremely high binding activity of β1-adrenergic receptors of T-lymphocytes. This was the basis for re-examining the issue of specificity and reliable selectivity of the previously proposed analysis method, and also necessitated the development of a modified method in order to increase the reliability of the results obtained on the binding activity of β1-adrenergic receptors of T-lymphocytes.

In the context of the present invention, the binding activity of cellular β-AR is defined as the specific binding of a labeled ligand under certain standardized conditions. Since the amount of radioactive ligand bound to the receptor is the main measured parameter for determining the binding activity, it is believed that it is more convenient to use the value of cpm-million cells for quantitative assessment. This allows for quantitative comparisons of changes in binding activity both under the influence of various external factors and in dynamics.

Unfortunately, (N-cyanopindolol does not have high specificity for various β-ARs. According to some reports, the binding constants for β1- and β2-adrenergic receptors from different sources vary in the range of 7-40 pM (Engel G., Hoer D., Berthold R ., Wagner H. // Naunyn Schmiedebergs Arch. Pharmacol. 1981. Vol. 317 (4). Pp. 277-285) This led to the idea of using specific ligands CGP-20712 and ICI for the selective determination of β1- and β2-adrenergic receptors 118551, first published in 1989 (Tsuchihashi H., Nagatomo T, Imai S. // J. Pharmacobiodyn. 1989 Vol. 12 (9). Pp. 509-516).

However, this approach is difficult to use to determine the binding activity of e1-AP lymphocytes. The amount of βΤ-AP on the surface of lymphocytes is too small, and the specific β1-ligand CGP20712 cannot specifically displace [ ² d] cyanopindolol from e1-AP simply because their amount is too small. In healthy volunteers (Fig.1A, samples 1, 2, 3), the binding activity of e1-AP

- 3 036081 is very low and is at a level not exceeding 0.1 fmol (10- ¹⁶ mol) receptors on Tlimfotsitov 1 million, or about 60 receptors per cell. In patients with broncho-pulmonary pathology (Fig. 1A, samples 4, 5, 6), this level is significantly higher. In patients with idiopathic cardiac arrhythmias (Fig. 1B), the binding activity of β1-adrenergic receptors is significantly higher, at the level of 1-1.5 fmol per 1 million cells, and is comparable to the binding activity of β2-AR. This fact determines the importance of developing a highly sensitive method for determining the binding activity of β1-AR as a clinical indicator.

To confirm the specificity of the previously proposed method, experiments were carried out to displace the labeled ['Ccyanopindolol from the cell receptors of the ADL-7A line by the ligand CGP20712 at various concentrations (Fig. 2). A similar series of experiments on the displacement of labeled [I-Ccyanopindolol by the ligand ICI 118551 at various concentrations was carried out for the A2R9 line (Fig. 3).

The authors of the present invention have unexpectedly found that changing the order of addition of reagents to the incubation mixture dramatically increases the binding activity of β 1 adrenoreceptors, and also makes it possible to identify characteristic patterns of changes in the characteristics of the β-AR unit under the influence of preliminary incubation with specific ligands. In the context of the present invention, such a procedure is called pre-incubation. It was proposed to first incubate cells with specific ligands CGP-20712 or ICI 118551, and then add labeled ['^ Ccyanopindolol. The results of these experiments on ADL-7A and A2R9 cells are presented in table. 1.

Table 1

Cells The amount of bound radioactivity, imp / min mln cells % binding of the maximum Ligand free with CGP-20712 with ICI 118551 Without PI PI Without PI PI Without PI PI ADL-7A 53392 - - - - 100 - ADL-7A - 17059 11846 - - 32 22 ADL-7A - - - 41335 52391 78 98 A2R9 55978 - - - - 100 - A2R9 - 54296 52391 - - 97 92 A2R9 - - - 7293 12445 thirteen 22

PI - pre-incubation

Interestingly, pre-incubation of cells with CGP-20712 (b1-specific ligand) activates cellular e1-AP, but at the same time, pre-incubation with ICI 118551 (b2-specific ligand) does not significantly change the binding activity of b2-AR.

From the data given in table. 1, it follows that CGP-20712, even at a very high concentration (0.44 μM), practically does not displace [^ Ccyanopindolol from cellular β2-AR: the displacement is about 3%. This confirms the specificity of the proposed modified analysis method, since the selected concentration of CGP-20712 will have a minimal effect on the binding activity of the radioactive ligand β2-AP by T lymphocytes. At the same time, the ligand ICI 118551 in high concentration at the level of 0.32 μM partially (about 20%) displaces [Ig Ccyanopindolol from cellular lymphocytic β1-AR. However, for the selective determination of the binding activity of β 1-AR T-lymphocytes, this distortion of the quantitative assessment will not exceed 10%, even for individuals with extremely high binding activity of β 1-AR T-lymphocytes, which is observed in rare pathologies.

Thus, the proposed modified method for determining the binding activity of β1 and β2-adrenergic receptors of human T-lymphocytes makes it possible to unexpectedly overcome the drawback of the prior art with the achievement of the technical result of the invention - a significant increase in the sensitivity of determining the binding activity of β1-AR.

Information confirming the possibility of carrying out the invention

Materials.

For the analysis, specific ligands are used: CGP-20712 (β 1-blocker), ICI 118551 (β2-blocker) and cyanopindolol obtained from Sigma (USA). The radioactive isotope [ ¹²⁵ I] in the form of sodium iodide with a molar activity of more than 2000 Ci / mmol was obtained from V / O Isotope (Russian Federation). The rest of the reagents used in the studies were of no less than analytical grade.

Patient characteristics.

All included volunteers sign an informed letter before being included in the study.

- 4 036081 consent in accordance with all the rules of ethical provision of the Declaration of Helsinki and the National Standard of the Russian Federation Good Clinical Practice GOST R 52379-2005.

The study included 11 volunteers: 3 volunteers without cardiovascular and bronchopulmonary pathologies; 3 patients with moderately severe bronchial asthma of a controlled course; 5 patients with cardiac arrhythmias (idiopathic arrhythmia).

All volunteers are over 18 years of age and do not take drugs that affect β-adrenergic receptors. Participants undergo a medical examination, examination using standard methods for assessing the state of the cardiovascular and bronchopulmonary systems before blood sampling. Patients with bronchial asthma do not take long-acting β2-agonists and Mholinolytic drugs for 48 hours, and short-acting ones - for 12 hours before the study.

The study of β1-adrenergic receptors is carried out in 3 healthy volunteers and 3 patients with bronchopulmonary pathology. The determination of the binding activity of β2-adrenergic receptors is carried out in 5 volunteers with combined cardiorespiratory pathology.

Obtaining [ ¹² 5 [| cyanopindolol.

The introduction of the radioactive isotope ¹²⁵ I into the cyanopindolol molecule is carried out by a modified method described in the article Greenwood FC, Hunter WM The preparation of labelled human growth hormone of high specific radioactivity. Biochem. J., 1963, 89 (1). P. 114-123.

The reaction mixture for radioiodination contains 1 μg of cyanopindolol, 0.2 M potassium phosphate buffer (pH 7.0), 1 mCi Na ^l25 I in a volume of 50 μl. The reaction is initiated by adding 10 ml of a solution of chloramine T (10 mg / ml). The incubation time at room temperature is 1 min. The reaction is stopped by the addition of 10 μl sodium thiosulfate (20 mg / ml) and after 5 min incubation at room temperature 1 μl of cold sodium iodide solution (6 mg / ml) is added.

The target product is isolated by HPLC (chromatograph manufactured by Gilson, France) on a Nucleosil 100 C-18 column (5 μm, 150 mm) in ion-pair mode. Buffer A: 10% acetic acid, buffer B: 100% acetonitrile. Elution is carried out with an acetonitrile concentration gradient from 0 to 100% in 20 min at a flow rate of 0.5 ml / min. The eluate is detected by UV absorption at 280 nm and by a flow-through radioactivity detector. The fractions containing [ ¹²⁵ 1] cyanopindolol are combined, evaporated to dryness, dissolved in 70% ethanol, and stored at -20 ° C.

Blood sampling and cell isolation.

The patient's blood from the peripheral vein is taken into 2 S-Monovette EDTA KE vacuum tubes (Sarstedt, Germany) with a volume of 9 ml. The blood is allowed to stand for 60 minutes at room temperature until the erythrocytes are deposited. Next, leukocyte-rich plasma is used.

Plasma from two tubes (18 ml) was taken and carefully layered onto 15 ml of Histopaque-1077 (Sigma, USA; cat. # H8889) contained in a 50 ml tube. For work, use sterile tubes, sterile pipettes or pipettes with a dispenser. Centrifugation is carried out at room temperature for 25 min in a centrifuge with a horizontal rotor at 400 g. After centrifugation, the interphase ring containing mononuclear cells (lymphocytes) is collected in clean 50 ml tubes.

The lymphocyte suspension is washed once with 30 ml of PBS buffer (phosphate-buffered saline, pH 7.2, 2 mM EDTA; autoMACS Rinsing solurion, Miltenyi Biotec, Germany, cat. No. 130-091-222) and centrifuged for 10 min at 400 g. The supernatant is removed and the pellet is resuspended in 500 μl PBS and transferred to 1.5 ml Eppendorf microcentrifuge tubes using an automatic pipette and filter tips. Centrifuged for 10 min at 400 g at room temperature. The supernatant is carefully discarded.

The sediment from the test tubes is used for the isolation of T-lymphocytes using the Pan T cell isolation kit (human) (Miltenyi Biotec, Germany, cat. No. 130-096-535), according to the instructions for the kit. For this, the pellet in a test tube is resuspended in 40 μl of PBS-BSA buffer and 10 μl of a mixture of antibodies (Pan T Cell Biotin Antibody Cocktail) is added, mixed well and incubated for 7 min in a refrigerator at 2-8 ° C. To the resulting mixture, 30 μl of PBS-BSA buffer and 20 μl of magnetic particles (Pan T Cell MicroBeard Cocktail) are added to the test tube, gently mixed and incubated for 15 min in a refrigerator at 2-8 ° C. Next, 500 μl of PBS-BSA buffer is added to the test tube, mixed and the resulting suspensions are passed through magnetic columns previously washed with PBS-BSA buffer. Magnetic separation is carried out using an OctoMACS separator (Miltenyi Biotec, Germany, cat. No. 130-042-201) according to the manufacturer's instructions. The flowing solution (T cells) is collected in a test tube.

Transfer 500 μl of the solution from the test tube to Eppendorf, add 500 μl of nutrient medium 199, mix well. From the volume obtained (1 ml), 10 μl is taken into a separate eppendorf for cell counting (approximately 106 cells / ml). The resulting cells are stored in a refrigerator at 4 ° C for no more than 24 hours prior to use in radioligand analysis.

Pre-incubation with specific ligands.

Four separate Eppendorfs are placed in (1) 10 μl of water, (2) 10 μl of an aqueous solution of cold cyanopindolol with a concentration in the range of 10 ^-6 -10 ^-8 M, containing an excess of cold

- 5 036081 cyanopindolol, (3) 10 μl 0.20-0.28 μM βΐ-specific ligand CGP-20712 (chemical name dihydrochloride [2 - ((3-carbamoyl-4-hydroxy) phenoxy) ethylamino] -3- [ 4- (1-methyl-4-trifluoromethyl-2imidazolyl) phenoxy] -2-propanol) and (4) 10 μl 0.14-0.18 μM β2-specific ligand ICI 118551 (chemical name - (±) -erito hydrochloride - (A *> *) - 1- | 2.3- (dihydro-7-methyl-1H-inden-4-yl) oxy | -3 [(1-methylethyl) amino] -2-butanol), phosphate buffer solution ( PBS) and 100 μl of a cell suspension with a concentration of 5-10 million / ml. The mixtures are preincubated for 25-30 minutes at 37 ° C and gentle shaking at 100 rpm. The process is stopped by adding ice water (cold water with ice), the cells are centrifuged at 1800-2000 g for 10-12 minutes, the supernatant is discarded, the precipitate is carefully suspended in cold (+ 4 ° C) PBS and centrifuged again under the same conditions. The supernatant is discarded and the pellet is carefully suspended in cold PBS, centrifuged at 10,000-12,000 g for 2-3 minutes, and the supernatant is discarded.

Radioligand analysis of β-adrenergic receptor binding activity.

For experiments with pre-incubation, each of the pellets (1) - (4) after centrifugation is diluted with PBS buffer with 0.1% BSA to a concentration of 10 mln / ml and 100 μl of the cell suspension are incubated for 25-30 min at 37 ° C and gentle stirring on a shaker with a speed of 100 rpm. Then, 100 µl of a solution of [ ¹²⁵ 1] cyanopindolol in PBS buffer with a concentration of 1000 cpm-µl is added to the reaction mixture and incubated for another 25-30 min. The process is stopped by adding 400 μl of ice water (cold water with ice) to each sample. To remove unbound radioactivity, the cells are centrifuged at 2000 g for 10 min, the pellet is washed three times by suspension in 200 μL of cold PBS buffer and centrifugation under the same conditions, and then counted on a Wallac Wizard 1470 γ counter (PerkinElmer, USA). The binding activity of β1- and β2-adrenergic receptors is calculated from the difference between the total binding and beta-specific binding of cyanopindolol, per 1 million cells.

Claims (1)

CLAIM The method of radioligand determination of the binding activity of β1-adrenergic receptors of human T-lymphocytes, for the implementation of which is carried out a) taking blood from the object from a peripheral vein; b) the allocation of T-lymphocytes in a conventional way; c) setting up four parallel analyzes, while in the first of them water is added to the medium containing T-lymphocytes, in the second cold cyanopindolol is added in excess, in the third β1-ΑΡ ligand CGP 20712 is added, in the fourth β2-ΑΡ ligand ICI 118551 is added; d) incubation for 25-30 minutes at 37 ° C and a shaker speed of 100 rpm; e) adding 100 μl [ ¹²⁵ I] cyanopindolol to all four pre-incubated reaction mixtures; f) stopping the reaction by adding cold water; g) centrifugation of the reaction mass at 2000 g for 10-12 minutes, discarding the supernatant; h) suspending the pellet in cold (+ 4 ° C) PBS buffer, followed by centrifugation under the same conditions, discarding the supernatant; i) suspending the pellet in cold (+ 4 ° C) PBS buffer, followed by centrifugation at 10000-12000 g for 2-3 minutes, discarding the supernatant; j) counting the precipitate on a γ-counter to measure the amount of radioactive material in each sample, and k) Calculation of the binding activity of β1-αdrenoreceptors by the difference between the total binding and beta-specific binding of cyanopindolol, per 1 million cells.