ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 5, pp. 429–435. © Pleiades Publishing, Inc., 2006. Original Russian Text © V.I. Vanina, Yu.A. Kovalitskaya, A.A. Kolobov, E.A. Kampe-Nemm, Yu.A. Zolotarev, V.V. Yurovskii, V.M. Lipkin, E.V. Navolotskaya, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 5, pp. 477–484.
Stress-Protective Effect of the Synthetic ACTH-Like Peptide Leucocorticotropin V. I. Vaninaa, Yu. A. Kovalitskayaa, A. A. Kolobova, E. A. Kampe-Nemmb, Yu. A. Zolotarevc, V. V. Yurovskiid, V. M. Lipkina, and E. V. Navolotskayaa, 1 a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Pushchino Branch), Russian Academy of Sciences, pr. Nauki 6, Pushchino, Moscow oblast, 142290 Russia b State Research Center Institute of Highly Pure Biopreparations, Federal Agency for Healthcare and Social Development, Pudozhskaya ul. 7, St. Petersburg, 197110 Russia c Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, Moscow, 123182 Russia d Department of Medicine, University of Maryland, Baltimore, MD, 21201 USA Received November 14, 2005; in final form, December 12, 2005
Abstract—We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81–88 sequence of the precursor of human interleukin-1α ([3H]GKVLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (Kd 2.2 ± 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10–20 µg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 µg/animal, three times) or significantly decreases (at a dose of 10 µg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor. Key words: adrenal cortex, adrenocorticotropic hormone, interleukin-1, peptides, receptors DOI: 10.1134/S1068162006050050
INTRODUCTION We have earlier found that more than 80% homology exists between the GK-VLKKRR fragment corresponding to the 81–88 sequence of human interleukin 1α precursor and the GKPVGKKRR fragment corresponding to the 10–18 sequence of ACTH [1].2 We synthesized octapeptide GKVLKKRR (it was called leucocorticotropin) and demonstrated its high affinity and binding specificity for the ACTH receptor on murine splenocytes and macrophages. The main ACTH function is the stimulation of synthesis and secretion of glucocorticoids by the cells of adrenal zona fasciculata and by the reticularis cells. Sensitivity of adrenal cells to ACTH depends on expression and function of the G-protein-bound recep1
Corresponding author; phone: (27) 73-6668; fax: (27) 33-0527; e-mail:
[email protected]. 2 Abbreviations: ACTH, adrenocorticotropic hormone; CRH, corticotrophin-releasing hormone; HPAA, hypothalamus–pituitary– adrenal axis; pIL-1α, precursor of interleukin-1α; LCT, leucocorticotropin; MC2R, melanocortin-2 receptor; 11-HOCS, 11hydroxycorticosteroids; PAM, phenacylamidomethyl; POMC, proopiomyelocortin; and SEM, standard mean error.
tor (melanocortin-2 receptor), which belongs to the melanocotin receptor subfamily [2–5]. The hormone binding to MC2R results in an increase in the adenylate cyclase activity and, as a result, activation of protein kinase A [6–8]. Kapas et al. studied binding of ACTH fragments to the recombinant murine ACTH receptor expressed in the HeLa human cells [9]. They demonstrated that the ACTH-(11-24) fragment effectively competed with 125I-labeled ACTH for the binding (I50 of approximately 1 nM), but, unlike the full-size hormone, cannot activate the adenylate cyclase. This means that this fragment can be regarded as an ACTH antagonist. We have already demonstrated that LCT has a high affinity for the ACTH receptor on immunocompetent cells [1] and now plan to investigate its binding to the ACTH receptor of the adrenal cortex and its effect on the adenylate cyclase activity and content of glucocorticoids in adrenal cortex of experimental animals. The goal of this study is the obtaining of [3H]LCT and the investigation of its binding to adrenal cortex membranes of rats in norm and after cold and heat shock.
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Fig. 1. A comparison of amino acid sequences of human ACTH, LCT, and the fragments of precursors of human (HproIL-1α), mouse (MproIL-1α), and rat (RproIL-1α) interleukin-1α. The coinciding amino acid residues are printed in boldface.
RESULTS AND DISCUSSION HPAA plays a key role in the regulation of stressrealizing systems of organism [10]. Any stress leads to an increase in the synthesis and secretion of CRH [11, 12]. CRH passes to the adrenohypophysis (anterior part of hypophysis) trough the portal vascular system, where it binds to the specific receptors (CRHR1 and CRHR2) of corticotrophic cells (the cells of promyelocortin precursor of ACTH). Expression of these receptors was shown to increase at stress [13]. CRH is a main stimulator of synthesis and secretion of POMC [14]. ACTH released in bloodstream achieves adrenal glands, is bound to the specific G protein–bound receptor of the cells of zona fasciculata and the reticularis cells [4, 5], and induces synthesis and secretion of glucocorticoids, which inhibit the synthesis and secretion of CRH and ACTH (POMC) by the principle of negative feedback [15, 16]. Recently, mRNA of the ACTH receptor (MC2R) has been found in human hypothalamus, and the proposal was made that ACTH can regulate its own secretion through an ultra-short feedback loop hypophysis– hypothalamus [17]. Antistress effect of glucocorticoids is directed to mobilization of host defense (release of catecholamines, activation of enzymes of glucogenesis, increase in lipolysis, inhibition of protein synthesis, increase in the glucagon secretion, decrease in the insulin secretion, etc) [18]. A comparison of amino acid sequences of human ACTH and fragments of the human, murine, and rat pIL-1α (Fig. 1) demonstrates that the highest degree of homology exists between the fragment corresponding to the 81–88 sequence of human pIL-1α (the LCT peptide) and the ACTH-(10–18) fragment. LCT differs from the corresponding pIL-1α fragments of mouse and rat by one equivalent substitution in position 83 (Ile for Val). Thus, pIL-1α involves the highly conservative ACTH-like 81–88 sequence. Peptide corresponding to this sequence or involving it can be formed during pIL-1α processing along with IL-1α. We prepared [3H]LCT and studied its binding to the membranes isolated from rat adrenal cortex. The experiments demonstrated that [3H]LCT was specifically
bound to the membranes of rat adrenal cortex (Fig. 2) under the conditions we chose (see the Experimental section). The dynamic equilibrium was established in [3H]LCT binding, cpm 30000
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Fig. 2. (a) Dependence of (1) total, (2) specific, and (3) nonspecific binding of [3H]LCT to the membranes of rat adrenal cortex on the incubation time. (b) Analysis of the specific binding of (1) [3H]LCT and (2) 125I-labeled ACTH-(11–24) to the membranes of rat adrenal cortex in the Skatchard coordinates (B and F are the molar concentrations of the bound labeled peptide and the free labeled peptide, respectively).
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the system of the labeled peptide and receptor at 4°C after 1 h and was maintained for no less than 2 h (Fig. 2a). Therefore, we determined the equilibrium dissociation constant (Kd) 1 h after the beginning of the reaction of [3H]LCT binding to the membranes. The nonspecific binding of [3H]LCT was 8.4 ± 1.6% from its total binding under these conditions. The Scatchard curve (curve 1) given in Fig. 2b demonstrates that [3H]LCT is bound to one type of the highly affinity receptors on the membranes of adrenal cortex (Kd 2.1 ± 0.2 nM). The unlabeled ACTH-(4–10), ACTH-(1–24), ACTH-(11–24), somatostatin, β-endorphin, and [Met5]-enkephalin were tested as potential competitors for the characteristics of the binding specificity of [3H]LCT. The experimental results (Table 1) demonstrated that only ACTH-(1–24) and ACTH-(11– 24) inhibited the specific binding of [3H]LCT to the membranes of adrenal cortex (Ki 1.7 ± 0.1 and 1.9 ± 0.2, respectively). The inhibiting activity of other peptides was very low (Ki > 10 µM). Kapas et al. [9] reported that the ACTH-(11–24) fragment is an antagonist of the ACTH receptor: it effectively competes with 125I-labeled ACTH for the binding to the cloned receptor of this hormone (IC50 ~ 1 nM), but, unlike ACTH, did not activate adenylate cyclase. We obtained 125I-labeled ACTH-(11–24) and found that it is bound to the ACTH receptor on the membranes of rat adrenal cortex with a high affinity (Kd – 1.8 ± 0.1 nM, see curve 2 in Fig. 2b). We also demonstrated that only LCT and ACTH-(1–24) displaced 125I-labeled ACTH-(11–24) from its complex with the receptor (Ki 2.0 ± 0.1 and 1.9 ± 0.1 nM, respectively) (Table 2). Other unlabeled peptides (somatostatin, β-endorphin, and [Met5]enkephalin) were tested as potential competitors of ACTH-(4–10) and proved to be inactive (Ki > 10 µM). We should note that practically the same values of Bmax – 1.064 ± 0.128 and 1.056 ± 0.116 pmol/mg protein, respectively) were obtained for two different test systems using [3H]LCT and [125I]ACTH-(11–24) as labeled ligands. This result indicates that these kinetic characteristics of binding are close to true. Thus, LCT and ACTH-(11–24) are bound to the ACTH receptor on the membranes of the rat adrenal cortex. The experiments demonstrated that the LCT binding to the membranes of rat adrenal cortex had no effect on the adenylate cyclase activity (Table 3). Hence, both ACTH-(11–24) and LCT are antagonists of the ACTH receptor. The IL-1 anti-inflammatory cytokine [19–21] and its receptors [20, 22, 23] are continuously expressed by the immune system cells and neurons and glia cells of the human and mammalian brains. Along with other anti-inflammatory cytokines (IL-6 and TNF-α), IL-1 activates HPAA by stimulating the CRH secretion in hypothalamus [24–27]. The data on the IL-1 effect on HPAA on the level of hypophysis and adrenal glands were mainly obtained using in vitro system. IL-1 was shown to increase expression of the POMC gene and RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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Table 1. Inhibition of specific binding of [3H]LCT to the membranes of rat adrenal cortex by the unlabeled peptides Peptide ACTH-(1–24) ACTH-(11–24) ACTH-(4–10) Somatostatin β-Endorphin [Met5]enkephalin
I50,* nM
Ki,* nM
5.6 ± 0.2 6.2 ± 0.3 >10000 >10000 >10000 >10000
1.7 ± 0.1 1.9 ± 0.2 >10000 >10000 >10000 >10000
* ±, Standard deviation.
Table 2. Inhibition of specific binding of 125I-labeled ACTH-(11–24) to the membranes of rat adrenal cortex by the unlabeled peptides Peptide LCT ACTH-(1–24) ACTH-(4–10) Somatostatin β−Endorphin; [Met5]enkephalin
I50,* nM
Ki,* nM
7.6 ± 0.2 7.2 ± 0.2 >10000 >10000 >10000 >10000
2.0 ± 0.1 1.9 ± 0.1 >10000 >10000 >10000 >10000
* ±, Standard deviation.
Table 3. Effect of LCT, ACTH-(1–24) and ACTH-(11–24) on the adenylate cyclase activity of the membranes of rat adrenal cortex Adenylate cyclase activity*
Peptide concentration, nM
LCT
ACTH-(1–24) ACTH-(11–24)
0
1.43 ± 0.12 1.43 ± 0.12
1.43 ± 0.12
0.1
1.46 ± 0.13 1.44 ± 0.15
1.48 ± 0.12
1
1.46 ± 0.15 1.82 ± 0.17
1.54 ± 0.18
10
1.43 ± 0.18 2.23 ± 0.19
1.50 ± 0.14
100
1.52 ± 0.16 2.38 ± 0.21
1.43 ± 0.12
1000
1.41 ± 0.18 2.36 ± 0.17
1.49 ± 0.12
* cAMP nmol/mg of protein ± standard deviation.
the ACTH release by corticotropic cells of the frontal part of hypophysis [28, 29] and to stimulate the secretion of glucorticoids by the cells of adrenal cortex [20] despite the fact that the IL-1 receptors were not found in the adrenal glands [30, 31]. At the same time, van der Meer et al. [32] reported that the HPAA activation by the anti-inflammatory cytokines (IL-1, IL-6, and TNF-α) is not mediated by the direct effect of these cytokines on hypophysis and/or adrenal glands. In any
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case, the results of this work allow a proposal that the ACTH-like peptide antagonist of the ACTH receptor capable of the HPAA inhibition arises by the pIL-1α processing along with the HPAA activator (IL-1). The results of studies of the LCT effect on the level of 11HOCS in adrenal glands and blood plasma of rats in vivo confirm this point of view. The 11-HOCS content in the rat adrenal glands in the absence of temperature action was 115 µg/g of tissue in average (see Figs. 3, 4). This content increased 2.4- (Fig. 3) and threefold (Fig. 4) in the rats subjected to the cold and hot shocks, respectively. LCT has practically no effect on the 11-HOCS level in the adrenal glands of rats at rest (Figs. 3, 4) after its three times intranasal administration at the dose of 10–20 µg/rat. At the same time, LCT abolished (at the dose of 20 µg/rat, three times) or significantly decreases (at the dose of 10 µg/rat, three times) the sharp increase in the 11-HOCS content in adrenal glands induced by the temperature shock (Figs. 3, 4). EXPERIMENTAL We used in this study ACTH-(4–10) and ACTH-(1– 24) peptides, somatostatin, β-endorphin, and [Met5]enkephalin (Sigma, United States); 1,3,4,6-tetrachloro-3α,6α-diphenylglucouryl (Iodogen), saccharose, BSA, EDTA, EGTA, Tris, PMSF, and NaN3 (Serva, Germany); N-methylpyrrolidone, diisopropylcarbodiimide, 1-hydroxybenzotriazole, and thioanisole (Merck, Germany); and Unisolv 100 scintillator (Amersham, UK). The rest of reagents were of the os. ch. quality (special purity grade). Distilled water was additionally purified on a Mono-Q system (Millipore, United States). Mature male rats of the SD line (180–210 g of body mass) were purchased from the breeding nursery of the Pushchino Branch of the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry. LCT (GKPVGKKRR) and ACTH-(11–24) (KPVGKKRRPVKVYP) were synthesized on an Applied Biosystems 430A automatic synthesizer (United States) using Boc/Bzl-strategy of peptide chain elongation. The syntheses were carried out on phenacylamidomethyl (PAM) polymer according to the in situ procedure proposed earlier [33]. Both peptides were purified to homogeneity by the preparative rpHPLC on a Gilson chromatograph (France) equipped with a Delta Pack C18 column (39 × 150 mm, 100 Å, 5 µm) eluted with a gradient of acetonitrile in 0.1% trifluoroacetic acid (from 10 to 40% for 30 min) at a flow rate of 10 ml/min. The purity of the peptides was higher than 97%. Molecular masses of the peptides were determined by mass spectrometry on a Finnigan mass spectrometer (United States). The amino acid analysis was carried out on a LKB 4151 Alpha Plus amino acid analyzer (Sweden) after their acidic hydrolysis in 6 M HCl at 110°ë for 22 h. We obtained the following results:
Gly 1.22, Val 1.00, Lys 2.92, and Arg 2.10 for LCT and Pro 3.40, Gly 1.00, Tyr 1.00, Val 2.78, Lys 4.62, and Arg 1.40 for ACTH-(11–24). [3H]LCT was prepared using the reaction of hightemperature solid phase catalytic isotope exchange (HTSPCIE) [34, 35]. Aluminum oxide (50 mg) was added to a solution of LCT (2.0 mg) in water (0.5 ml), and the mixture was evaporated on a rotary evaporator. The dry residue was mixed with catalyst (5% Rh/Al2O3) (10 mg), placed into an ampoule of 10 ml volume. The ampoule was evacuated, filled with gaseous tritium to 250 mm of Hg, heated to 170°C, and kept for 20 min at this temperature. The ampoule was cooled, evacuated, blown out with hydrogen, and evacuated again. The labeled peptide was extracted from the solid reaction mixture with 50% aqueous ethanol (2−3 ml) and evaporated. This procedure was repeated two times for removal of labile tritium. The [3H]LCT was purified by HPLC on a Kromasil column (4 × 150 mm, 5 µm) at 20°C with a gradient elution with methanol in 0.1% trifluoroacetic acid (from 42 to 70% for 20 min) at a flow rate of 3 ml/min with detection on a Beckman spectrophotometer at 254 and 280 nm. The tritium incorporation into the peptide was calculated on the basis of liquid scintillation counting. Introduction of 125I into ACTH-(11–24) (10 µg) was achieved using Na125I (1 mCi) and iodogen [36]. The iodinated peptide was isolated from the reaction mixture on a column (0.9 × 10 cm) with Sephadex G-10 by elution with 50 mM phosphate buffer (pH 7.4) at a flow rate of 5 ml/h. Retention time of the labeled peptide was determined in a control experiment with unlabeled peptide. Radioactivity of the fractions was measured on a Mini-Gamma Counter (LKB, Sweden). The fractions with maximal radioactivity corresponding to those with the unlabeled peptide in control were combined, and the total and specific activities of the resulting peptide were determined. The purity of the iodinated peptide was determined by TLC on glass plates with aluminum oxide in 4 : 1 : 1 butan-1-ol–acetic acid–water. The peptide was detected on the plate by autoradiography. The membranes were isolated from the rat adrenal cortex according to the procedure [37]. The protein concentration was determined by the Lowry method [38] using BSA as a standard. Reaction of the [3H]LCT binding to the membranes. The labeled peptide (100 µl, 10–10–10–7 M) was placed into glass siliconized tubes. Three parallel samples were taken for each concentration. Tris-HCl buffer (50 mM, containing 0.6 mg/ml PMSF, pH 7.5, 100 µl), for total binding) or 10–3 M solution of the unlabeled peptide in the buffer (for nonspecific binding) and a suspension of the freshly isolated membranes (800 µl, 0.5 mg of protein) were added. The tubes were incubated for 1 h at 4°C. The reaction mixture was filtered through GF/B fiberglass filters (Whatman, UK), and the filters were three times washed with ice-cooled buffer
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Fig. 4. Effect of LCT on the level of 11-HOCS in the rat adrenal glands in the absence of temperature treatment and at the hot shock. The peptide was intranasally administered at a dose of (a) 20 and (b) 10 µg/animal three times 3, 2, and 1 day before the shock.
LCT
Cold shock
Fig. 3. Effect of LCT on the level of 11-HOCS in the rat adrenal glands in the absence of temperature treatment and at the cold shock. The peptide was intranasally administered at a dose of (a) 20 and (b) 10 µg/animal three times 3, 2, and 1 day before the shock.
solution. The radioactivity was counted on the filters using LS 5801 scintillation counter (Beckman, United States). The specific binding of [3H]LCT to the membranes was determined from the difference between its total and its specific binding. The equilibrium dissociation constant (Kd) of the [3H]LCT binding with the membranes and the receptor density (Bmax is the maximum binding ability per mg of protein) were determined from the curve of dependence of ratio of the molar concentrations of the bound (B) and free (F) labeled peptide on the molar concentration of the bound labeled peptide (B) (Scatchard plot) [39]. Inhibition of the [3H]LCT specific binding by the unlabeled ACTH-(4–10), ACTH-(11–24), ACTH-(1– 24), somatostatin, and b-endorphin. The membrane suspension (0.5 mg of protein) was incubated with the labeled LCT (5 nM) and one of the potential inhibitors (at the concentration range from 10–10 to 10–4 M, three repetitions for each concentration) as described above. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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The inhibition constant (Ki) was determined according to the equation: Ki = [I]50/(1 + [L]/Kd), where [L] is the molar concentration of [3H]LCT; Kd. the equilibrium constant of the complex dissociation; and [I]50, the concentration of unlabeled ligand causing 50% inhibition of specific binding of the labeled LCT [40]. The value of [I]50 was determined from the curve of dependence of the inhibition (%) on the molar concentration of inhibitor. The Kd value was preliminarily determined as described above. Binding of 125I-labeled ACTH-(11–24) to the membranes was carried out according to the scheme described above for [3H]LCT. The radioactivity on filters was counted on a Mini-Gamma counter (LKB, Sweden). The value of specific binding of the 125Ilabeled ACTH-(11–24) to the membranes was determined as a difference between its total and its nonspecific binding. The results of three independent experiments were analyzed by the method [39]. Inhibition of the specific binding of 125I-labeled ACTH-(11–24) by the unlabeled LCT, ACTH-(4– 10), ACTH-(1–24), somatostatin, and b-endorphin. The membranes (0.5 mg of protein) were incubated
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with the labeled peptide (5 nM) and with one of the unlabeled peptides taken at the concentration range from 10–10 to 10–4 M (three repetitions for each concentration) as described above. The results of three independent experiments were analyzed by the method [40]. The inhibition constant (Ki) was determined by the equation given above. Adenylate cyclase activity was determined using α[32P]ATP by the method proposed earlier [41]. The reaction was carried out in the following medium: 40 mM Tris-HCl (pH 7.4) containing 50 µM ATP, 4 mM cAMP, 12 mM phosphoenol pyruvate, and pyruvate kinase (2 µg/ml). α[32P]ATP (200000–500000 cpm) in the medium (50 µl) and a suspension (50 µl) of membranes of the rat adrenal cortex (experiment) or the same volume of the buffer (control) were placed into glass silanized tubes cooled in an ice bath. The tubes were transferred into a thermostat and kept for 45 min at 34°ë. The reaction was stopped by the addition of 0.5 M HCl. The tubes were placed in a boiling water bath for 15 min, transferred into an ice bath, and 1.5 M imidazole (100 µl) was added in each tube. The content of each tube was applied onto a separate column with aluminum oxide (1 cm3, activity II by Brockmann) and washed with distilled water (5 ml). The enzyme activity was determined by the substrate decrease and expressed in nmol of cAMP formed for 10 min per mg of protein of adrenal cortex membrane. The model of cold shock was created by subjection of rats to free swimming for 3 min in a cuvette filled with water cooled to 4°ë. The model of heat shock. Rats were kept in an aerated thermochamber at 40°ë for 1 h. An LCT solution in isotonic solution (20 µl) at doses of 10, 20, or 50 µg/animal was intranasally introduced three, two, or one day before the temperature treatment. Rats of the control group were not subjected to the shock. The values of biochemical characteristics of this group were taken to be 100%. Content of the 11-HOCS glucocorticoids in blood and adrenal glands was determined as described in [42–44]. This method is based on the ability of 11-HOCS to fluoresce after a treatment with a mixture of sulfuric acid and ethanol (3 : 1 v/v). The 11-HOCS levels were separately determined in blood and adrenal glands for each rat of the experimental and control groups. The contents of fluorescing products were measured at λ530 nm on a Hitachi 850 fluorimeter (Japan) (λext 470 nm). The amount of 11-HOCS was determined from a calibration curve and expressed in µg/g of tissue. An LCT solution in isotonic solution (20 µl) at doses of 10, 20, and 50 µg/animal was intranasally introduced one day before the decapitation. The reliability of differences between the experimental and control results was determined by the Student t-criterion.
ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research, project no. 05-04-48060, by the program Leading Scientific Schools, project no. NSh-312.2003.4, program of Molecular and Cellular Biology and Naukogrady, project no. 04-04-97200, and by International Scientific and Technical Center, project no. 2615. REFERENCES 1. Zav’yalov, V.P., Maioro, V.A., Safonova, N.G., Navolotskaya, E.V., Volodina, E.Y., and Abramov, V.M., Immunol. Lett., 1995, vol. 46, pp. 125–128. 2. Cone, R.D., Mountjoy, K.G., Robbins, L.S., Nadeau, J.H., Johnson, K.R., Roselli-Rehfuss, L., and Mortrud, M.T., Ann. N. Y. Acad. Sci., 1993, vol. 680, pp. 342–363. 3. Clark, A.J.L., The Melanocotrin Receptors, Totowa, New Jersey: Humana, 2000, pp. 361–384. 4. Beuschlein, F., Fassnacht, M., Klink, A., Allolio, B., and Reincke, M., Eur. J. Endocrinol., 2001, vol. 144, pp. 199–206. 5. Clark, A.J.L., Noon, L., Swords, F.M., Hunyady, L., and King, P., Ann. N. Y. Acad. Sci., 2003, vol. 994, pp. 111– 117. 6. Haynes, R.C., Jr., Koritz, S.B., and Peron, F.G., J. Biol. Chem., 1959, vol. 234, pp. 1421–1423. 7. Grahame-Smith, D.G., Butcher, R.W., Ney, R.L., and Sutherland, E.W., J. Biol. Chem., 1967, vol. 242, pp. 5535–5541. 8. Côté, M., Paye, M.D., Rousseau, E., Guillon, G., and Gallo-Payet, N., Endocrinol, 1999, vol. 140, pp. 3594– 3601. 9. Kapas, S., Cammas, F.M., Hinson, J.P., and Clark, A.J., Endocrinology, 1996, vol. 137, pp. 3291–3294. 10. Dallman, M.F., Akana, S.F., Cascio, C.S., Darlington, D.N., Jacobson, L., and Levin, N., Recent Prog. Horm. Res., 1987, vol. 43, pp. 113–173. 11. Vale, W., Spiess, J., Rivier, C., and Rivier, J., Science, 1981, vol. 213, pp. 1394–1397. 12. Spiess, J., Rivier, J., Rivier, C., and Vale, W., Proc. Natl. Acad. Sci. USA, 1981, vol. 78, pp. 6517–6321. 13. Wang, T.Y., Chen, X.Q., Du, J.Z., Xu, N.Y., Wei, C.B., and Vale, W.W., Neuroscience, 2004, vol. 128, pp. 111– 119. 14. Antoni, F.A., Endocr. Rev., 1986, vol. 7, pp. 351–378. 15. Keller-Wood, M.E. and Dallman, M.F., Endocr. Rev., 1984, vol. 5, pp. 1–24. 16. Dallman, M.F., Akana, S.F., Jacobson, L., Levin, N., Cascio, C.S., and Shinsako, J., Ann. N. Y. Acad. Sci., 1987, vol. 512, pp. 402–414. 17. Morris, D.G., Kola, B., Borboli, N., Kaltsas, G.A., Gueorguiev, M., McNicol, A.-M., Ferrier, R., Jones, T.H., Baldeweg, S., Powell, M., Czirják, S., Hanzély, Z., Johansson, J.O., Márta Korbonits M., and Grossman, A.B, J. Clin. Endocrinol. Metab., 2003, vol. 88, pp. 6080– 6087. 18. Riad, M., Mogos, M., Thangathurai, D., and Lumb, P.D., Curr. Opin. Crit. Care, 2002, vol. 8, pp. 281–284.
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