[The effect of hypoxia on cholinesterase activity in rat sensorimotor cortex].

This study reports the dynamics of changes in postnatal ontogenesis of the activity of soluble and membrane-bound forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in sensorimotor cortex of rats as well as the pattern of their changes after prenatal hypoxia (E14, 7% O2, 3 h) or acute hypoxia in adult animals (4 months, 7% O2, 3 h). In normally developing rats the activity of the membrane-bound AChE form in the sensorimotor cortex gradually increased up to the end of the first month after birth and remained at this high level during all further postnatal ontogenesis, while the activity of the soluble form of AChE reached its maximum on the 10th day after birth and decreased significantly by the end of the first month. In animals exposed to prenatal hypoxia the activity both of the soluble and membrane bound forms of AChE during the first two weeks after birth was 20-25% lower, as compared to controls but increased by the end of the first month and even exceeded the control values remaining increased up to old age (1.5 years). The activity of both BChE forms in rat sensorimotor cortex at all stages of postnatal ontogenesis was significantly lower than of AChE, although the dynamics of their changes was similar to that of AChE. Prenatal hypoxia led to a decrease in the activity of the membrane-bound form of BChE, as compared to controls, practically at all developmental stages studied, but was higher at the end of the first month after birth. At the same time, the activity of the soluble form of BChE was decreased only on the 20th day of development, as compared to the control, but increased from the end of the first month of life onwards. Acute hypoxia in adult rats also led to a decrease in the activity of both forms of AChE and BChE in the sensorimotor cortex but the dynamics of these changes was different for each enzyme. Thus, insufficient oxygen supply to the nervous tissue at different stages of ontogenesis has a significant effect on the activity and ratio of various forms of cholinesterases exhibiting either growth factor or signaling properties. This may lead to changes in brain development and formation of behavioural reactions, including learning and memory, and also increase the risk of development of the sporadic form of Alzheimer's disease (AD)--one of the most common neurodegenerative diseases of advanced age. This study expands our knowledge of the properties of brain cholinesterases under normal and pathological conditions and may be useful for developing new approaches towards prevention and treatment of AD.

[Attenuation of inhibitory influence of hormones on adenylyl cyclase systems in the myocardium and brain of rats with obesity and type 2 diabetes mellitus and effect of intranasal insulin on it].

The functional state of the adenylyl cyclase signaling system (ACSS) and its regulation by hormones, the inhibitors of adenylyl cyclase (AC)--somatostatin (SST) in the brain and myocardium and 5-nonyloxytryptamine (5-NOT) in the brain of rats of different ages (5- and 7-month-old) with experimental obesity and a combination of obesity and type 2 diabetes mellitus (DM2), and the effect of long-term treatment of animals with intranasally administered insulin (II) on ACSS were studied. It was shown that the basal AC activity in rats with obesity and DM2 was increased in the myocardium, and to the lesser extent in the brain, the treatment with II reducing this parameter. The AC stimulating effects of forskolin are decreased in the myocardium, but not in the brain, of rats with obesity and DM2. The treatment with II restored the AC action of forskolin in the 7-month-old animals, but has little effect on it in the 5-month-old rats. In obesity the basal AC activity and its stimulation by forskolin varied insignificantly and weakly changed in treatment of animals with II. The AC inhibitory effects of SST and 5-NOT in the investigated pathology are essentially attenuated, the effect of SST to the greatest extent, which we believe to be associated with a reduction in the functional activity of Gi-proteins. The II treatment of animals with obesity and with a combination of obesity and DM2 restored completely or partially the AC inhibiting effects of hormones, to the greatest extent in the brain. Since impaired functioning of ACSS is one of the causes of the metabolic syndrome and DM2, their elimination by treatments with II can be an effective approach to treat these diseases and their CNS and cardiovascular system complications.

[Regulation of adenylyl cyclase signaling system by insulin, biogenic amines, and glucagon at their separate and combined action in the muscle membranes of the mollusc Anodonta cygnea].

In smooth muscles of mollusc Anodonta cygnea, hormones produce regulatory effects on the adenylyl cyclase (AC) signaling system via receptors of the serpentine (biogenic amine, isoproterenol, glucagon) and of tyrosine kinase (insulin) types. Intracellular mechanisms of their action are interconnected. Use of hormones, their antagonists, and pertussis toxin at the combined action of insulin and biogenic amines or of glucagon on the AC activity allows revealing possible intersection points in mechanisms of their action. The combined effect of insulin and serotonin or of glucagon leads to a decrease of stimulation of AC by these hormones, whereas at action of insulin and isoproterenol the AC-stimulatory effect of insulin is blocked, while the AC-inhibitory effect of isoproterenol is preserved both in the presence and in the absence of the non-hydrolyzed GTP analog - guanylylimidodiphosphate (GppNHp). Specific blocking of the AC-stimulatory serotonin effect by cyproheptadine - an antagonist of serotonin receptors - did not affect stimulation of AC by insulin. Beta-adrenoblockers (propranolol and alprenolol) interfered with inhibition of the AC activity by isoproterenol, but did not change the AC stimulation by insulin. Pertussis toxin blocked the AC-inhibitory effect of isoproterenol and attenuated the AC-stimulatory effect of insulin. Thus, in muscles of the mollusc Anodonta cygnea there have been revealed negative interrelations between the AC system, which are realized at the combined effect of insulin and serotonin or of glucagon, probably at the level of receptor of the serpentine type (serotonin, glucagon), while at action of insulin and isoproterenol - at the level of interaction of G1 protein and AC.

[Study of hormone responsiveness of the adenylyl cyclase signaling system in erythrocyte membranes of patients with type 2 diabetes mellitus].

Peptides of the insulin superfamily (insulin, insulin-like growth factor, relaxin), epidermal.growth factor (ECF) and biogenic amines (isoproterenol, adrenalin, noradrenalin, serotonin) stimulate the adenylyl cyclase signaling system (ACSS). In erythrocyte membranes from a control group of patients, the hormone activating affect on ACSS was potentiated in the presence of guanylylimidinodiphosphate (CppNHp). In erythrocyte membranes from patients of various severity of type 2 diabetes mellitus (DM2, early, medium and severe), the basal activity of AC was higher than in the control group and its responsiveness to hormones was different. It was reduced in patients with early and severe forms of DM2 both in the presence and absence of CppNHp. In patients with the medium severity of the disease, the stimulating effect of biogenic amines was not changed but there was no potentiating effect of CppNHp. The insulin superfamily peptides and ECF stimulated AC in the erythrocyte membranes of patients with the medium severity of DM2 to the same extent as in the control while, at the early and severe stages of the disease, the AC sensitivity to these hormones was significantly reduced. These data suggest that DM2 results in disturbances of the hormone stimulating properties of ACSS by insulin superfamily peptides, ECF and biogenic amines. In erythrocyte membranes, DM2 disturbs ACSS functions at the level of the catalytic component and its responsiveness to hormone action at the level of interactions between CG, and AC.

[Changes in the activity of amyloid-degrading metallopeptidases leads to disruption of memory in rats].

In old male Wistar rats (older than 12 months), or adult males (3-4 months) subjected to prenatal hypoxia (7% 02, 3 h, E14), a disruption of short-term memory was observed. The prenatal hypoxia also led to a decrease in the brain cortex expression of metallopeptidases neprilysin (NEP) and endothelin-converting enzyme (ECE-1) which regulate some neuropeptides and are the main beta-amyloid-degrading enzymes. Moreover, a significant decrease (by 2.7 times) in NEP activity in the sensorimotor cortex of old and adult rats subjected to prenatal hypoxia (by 1.7 times) was observed. To confirm possible involvement of these enzymes in memory, the analysis of the effect of microinjections of phosphoramidon (an inhibitor of NEP and ECE-1), and thiorphan (an inhibitor of NEP) into the rat sensorimotor cortex was carried out. In a two-level radial maze test, a disruption of short-term memory was observed 60 and 120 min after i.c. injection ofphosphoramidon (5.9 microg/microl) and 30 and 60 min after i.c. injection of thiorphan (2.5 microg/microl). The involvement of NEP and ECE-1 in short-term memory suggests that a decrease in the level of expression and activity of metallopeptidases involved in metabolism of beta-amyloid peptide (Abeta) and other neuropeptides is one of the main factors in disruption of cognitive functions after prenatal hypoxia or in the process of ageing.

[Adenylyl cyclase signaling mechanisms of the insulin superfamily peptide action and their impairment in myometrium of pregnant women with type 2 diabetes].

For the first time we found in myometrium of the women and pregnant women that adenylyl cyclase (AC) stimulating effects of relaxin, insulin and insulin growth factor 1 are realized via six-component AC signaling mechanisms involving the following signaling chain: receptor-tyrosine kinase ==> Gi protein (beta gamma dimmer) ==> phosphatidylinositol 3-kinase ==> protein kinase C (zeta) ==> Gs protein ==> adenylyl cyclase (AC), which are similar to the discovered adenylyl cyclase signaling mechanisms of insulin and relaxin action in vertebrates (rat) and invertebrates (mollusk). The effect of relaxin is more pronounced as compared with other peptides (relaxin > insulin > insulin-like growth factor-1) in myometrium of pregnant women. It is connected with the specific role ofrelaxin as main regulator of reproductive functions. For the first time we revealed the functional defects in distal parts of adenylyl cyclase signaling mechanisms of the insulin superfamily peptides action in the condition type-2 diabetes (the increase of the basal adenylyl cyclase activity and decrease of the peptide-stimulated AX activity in presence of guanilylimidodiphosphate). The defects are localized on the level of Gs protein, adenylyl cyclase and their functional coupling. The data obtained confirm our conception about molecular defects in hormoneregulated adenylyl cyclase system as a key reason of type-2 diabetes.

[Structural and functional organization of the adenylyl cyclase signaling mechanism of insulin-like growth factor 1 revealed in the muscle tissue in vertabrates and invertebrates].

Based on the earlier discovered by the authors adenylyl cyclase signaling mechanisms (ACSM) of action of insulin and relaxin, the study was performed of the presence a similar action mechanism of another representative of the insulin superfamily--the insulin-like growth factor 1 (IGF-1) in the muscle tissues of vertebrates (rat) and invertebrates (mollusc). For the first time there was detected participation of ACSM in the IGF-1 action, including the six component signaling cascade: receptor tyrosine kinase --> G(i)-protein (betagamma-dimer) --> phosphatidylinositol-3-kinase (PI-3-K) --> protein kinase Czeta (PKCzeta) --> G(-)protein --> adenylyl cyclase. By this mechanism structural-functional organization at postreceptor stages, in coincides completely with the mechanism of insulin and relaxin, which we revealed in rat skeletal muscle. In smooth muscle of the mollusc Anodonta cygnea this ACSM of action of IGF-1 has only one difference--the protein kinase C included in this mechanism is represented not by PKCzeta isoform, but by another isoform close to PKCepsilon of the vertabrate brain. Earlier we revealed the same differences in muscle of this mollusc in the ACSM of action of insulin and relaxin.

[Structural and functional characteristics of the adenylyl cyclase signaling system regulated by biogenic amines and peptide hormones in the muscle of a worm Lumbricus terrestris].

It has been shown for the first time that biogenic amines (catecholamines and tryptophane derivatives) stimulate dose-dependently activity of adenylyl cyclase (AC) and GTP-binding of G-proteins in muscle of the cutaneous-muscle bag of the earthworm Lumbricus terrestris. By efficiency of their stimulating action on the AC activity, biogenic amines can be arranged in the following sequence: octopamine > tyramine > tryptamine = serotonin > dopamine > isoproterenol = adrenalin. The sequence of efficiency of their action on GTP-binding is somewhat different: serotonin > tryptamine > octopamine > dopamine = tyramine > adrenaline > isoproterenol. Sensitivity of AC and G-proteins in the worm muscle to biogenic amines is similar with that in smooth muscle of the molluse Anodonta cygnea (invertebrates), but differs markedly by this parameter from the rat myocardium (vertebrates). It has also been revealed that AC in the worm muscle is regulated by peptide hormones relaxin and somatostatin whose action is comparable with that in the mollusk muscle, but much weaker that the action of these hormones on the rat myocardium AC activity. Use of C-terminal peptides of alpha-subunits of G-proteins of the stimulatory (385-394 Galpha(s)) and inhibitory (346-355 Galpha(i2)) types that disrupt selectively the hormonal signal transduction realized via G(s)- and G(i)-proteins, respectively, allowed establishing that the AC-stimulating effects of relaxin, octopamine, tyramine, and dopamine in the worm muscle are realized via the receptors coupled functionally with G(s)-protein; the AC-inhibiting effect of somatostatin is realized via the receptor coupled with G(i)-protein, whereas serotonin and tryptamine activate both types of G-proteins.

Variations in functional activity of the hormone-sensitive adenylate cyclase system in tissues of gastropod mollusks with streptozotocin-induced diabetes.

Treatment of gastropod mollusks of pond snail Lymnaea stagnalis and orb snail Coretus corneus with streptozotocin was followed by an increase in hexose content in the hemolymph and development of the diabetic state (day 1 after treatment). Functional activity of the hormone-sensitive adenylate cyclase system significantly decreased in the muscles and hepatopancreas of mollusks with diabetes. We revealed a decrease in the regulatory effects of biogenic amines and peptide hormones that were realized via stimulatory (octopamine, dopamine, serotonin, tryptamine, and relaxin) and inhibitory G proteins (somatostatin). Disturbances in the hepatopancreas were more pronounced than in the muscle. The severity of disorders in the adenylate cyclase system reached maximum 1 day after streptozotocin treatment. The sensitivity of this system to hormonal and nonhormonal agents was partially restored on days 3 and 5. Hexose content in the hemolymph was elevated after streptozotocin treatment, but returned to normal on day 3. Our results indicate that hyperglycemia is one of the key factors for dysfunction of the adenylate cyclase system in mollusks with the diabetic state.

[The disturbance of the transduction of adenylyl cyclase inhibiting hormonal signal in myocardium and brain of rats with experimental type II diabetes].

At present, the data obtained by us and other authors give evidence that disturbances in hormonal signaling systems are the main causes of development of pathological changes and complications under the diabetes. However, the molecular mechanisms of these disturbances remain obscure, especially in the case of insulin-independent type II diabetes. Using neonatal streptozotocin model of 80- and 180-days type II diabetes the changes in functional activity of hormone-regulated adenylyl cyclase (AC) signaling systems components in the myocardium and the brain striatum of diabetic rats in comparison with the control animals were found. The transduction of AC inhibitory hormonal signal meditated through Gi proteins was shown to by disturbed under diabetes. This was manifested in both the decrease of hormone inhibitory effect on AC activity and weakening of hormone stimulation of G-protein GTP-binding activity. In the case of noradrenaline (myocardium) the inhibitory pathway of AC regulation by the hormone was vanished and the stimulation pathway, in contrary, was protected. Prolongation of diabetes from 80 up to 180 days led to some weakening of Gi-protein-mediated hormonal signal transduction. Stimulating effect of biogenic amines and relaxin on the AC activity and GTP-binding in the myocardium and brain of diabetic rats were weakly changed in the case of both 80- and 180-days diabetes. To sum up, the experimental type II diabetes caused disturbances mainly in Gi-coupled signaling cascades participating in hormone inhibition of AC activity.

Studies of the molecular mechanisms of action of relaxin on the adenylyl cyclase signaling system using synthetic peptides derived from the LGR7 relaxin receptor.

The peptide hormone relaxin produces dose-dependent stimulation of adenylyl cyclase activity in rat tissues (striatum, cardiac and skeletal muscle) and the muscle tissues of invertebrates, i.e., the bivalve mollusk Anodonta cygnea and the earthworm Lumbricus terrestris, adenylyl cyclase stimulation being more marked in the rat striatum and cardiac muscle. Our studies of the type of relaxin receptor involved in mediating these actions of relaxin involved the first synthesis of peptides 619-629, 619-629-Lys(Palm), and 615-629, which are derivatives of the primary structure of the C-terminal part of the third cytoplasmic loop of the type 1 relaxin receptor (LGR7). Peptides 619-629-Lys(Palm) and 615-629 showed competitive inhibition of adenylyl cyclase stimulation by relaxin in rat striatum and cardiac muscle but had no effect on the action of relaxin in rat skeletal muscle or invertebrate muscle, which is evidence for the tissue and species specificity of their actions. On the one hand, this indicates involvement of the LGR7 receptor in mediating the adenylyl cyclase-stimulating action of relaxin in rat striatum and cardiac muscle and, on the other, demonstrates the existence of other adenylyl cyclase signal mechanisms for the actions of relaxin in rat skeletal muscle and invertebrate muscle, not involving LGR7 receptors. The adenylyl cyclase-stimulating effect of relaxin in the striatum and cardiac muscles was found to be decreased in the presence of C-terminal peptide 385-394 of the alpha(s) subunit of the mammalian G protein and to be blocked by treatment of membranes with cholera toxin. These data provide evidence that in the striatum and cardiac muscle, relaxin stimulates adenylyl cyclase via the LGR7 receptor, this being functionally linked with G(s) protein. It is also demonstrated that linkage of relaxin-activated LGR7 receptor with the G(s) protein is mediated by interaction of the C-terminal half of the third cytoplasmic loop of the receptor with the C-terminal segment of the alpha(s) subunit of the G protein.

[Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptus anser].

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca2+ was studied and it was found that calcium cations at concentrations of 0.5-10 microM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 microM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca2+. Calmodulin antagonists--chlorpromazine, W-7, and W-5--caused a dose-dependent decrease of the enzyme activity stimulated by 5 microM Ca2+ with IC50 values of 35, 137, and 174 microM, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 microM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 microM calcium ions, but were markedly inhibited by 100 microM Ca2+. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca2+, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or were restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity does not D. depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.

[Reactivity of the adenylyl cyclase system in rat tissues to biogenic amines and peptide hormones under starvation condition].

Under starvation condition, sensitivity of the adenylyl cyclase system to regulatory action of biogenic amines and peptide hormones in rat tissues are changed. In the myocardium and skeletal muscles, after 2 and 4 days of starvation, the regulatory effects of isoproterenol and relaxin acting via G,-proteins on the adenylyl cyclase activity and the G-protein GTP-binding are significantly increased compared with control. At the same time, regulatory effects ofsomatostatin which are realized via Gi-proteins, on adenylyl cyclase system in the myocardium are decreased. Under prolonged starvation consisting of two consecutive 4-days periods, the effects of hormones acting via Gs-proteins on the adenylyl cyclase activity in muscle tissues are decreased to control value levels. The effects of hormones acting via Gi-proteins are largely reduced. In the brain, intensification of adenylyl cyclase stimulating hormonal effects was late and only observed after a 4-day starvation. Unlike muscle tissues, the increase of adenylyl cyclase stimulating effects in the brain is preserved after two-period starvation. The weakening of adenylyl cyclase inhibiting hormonal signals both in the brain and muscles is observed after a 2-day starvation and then the weakening is intensified. Possible role of glucose level and basal adenylyl cyclase activity in determination of the sensitivity of the adenylyl cyclase system to hormones under study is discussed. It is suggested that one of the key causes of physiological changes in animal organism under starvation involves alteration of hormonal signalling systems sensitivity, in particular that of the adenylyl cyclase system, to hormone regulatory action.

Functional coupling of hormone receptors with G proteins in the adenylate cyclase system of the rat muscle tissues and brain under conditions of short-term hyperglycemia.

The sensitivity of components of the adenylate cyclase signaling system (heterotrimer G proteins and adenylate cyclase enzyme) to the regulatory effects of hormones mediated through G proteins (stimulatory effect of isoproterenol and relaxin and inhibitory effects of somatostatin) was decreased in the myocardium of hyperglycemic rats under conditions of transitory hyperglycemia caused by intravenous glucose and in hyperglycemia associated with insulin insufficiency in 24-h type 1 streptozotocin-induced diabetes mellitus. Changes in hormone sensitivity of the adenylate cyclase system were tissue-specific: clearly manifest in the myocardium, minor in skeletal muscles, and virtually absent in the brain of hyperglycemic rats. The main disorders of this system in the myocardium were observed at the stage of hormone receptor coupling with G proteins, which was seen from reduced stimulatory effect of GppNHp on adenylate cyclase activity and attenuation of the regulatory effect of hormones on adenylate cyclase enzyme and G proteins functionally coupled with it.

Changed sensitivity of adenylate cyclase signaling system to biogenic amines and peptide hormones in tissues of starving rats.

In the myocardium and skeletal muscles of rats deprived of food for 2 days, basal activity of adenylate cyclase decreased, while the sensitivity of adenylate cyclase signaling system to the stimulating effects of non-hormonal agents (guanine nucleotides and NaF) and beta-agonist isoproterinol modulating adenylate cyclase through stimulating G proteins increased. In starving organism, the regulatory effects of hormones realizing their effects through inhibitory G proteins (somatostatin in the myocardium and bromocryptin in the brain) weakened. Their inhibitory effects on forskolin-stimulated adenylate cyclase activity and stimulating effects on binding of guanosine triphosphate decreased. In the brain of starving rats, the differences in the sensitivity of the adenylate cyclase signaling system to hormones and nonhormonal agents were less pronounced than in the muscle tissues, which attested to tissue-specific changes in the functional state of this system under conditions of 2-day starvation.

[Streptozocin model of diabetes mellitus in the mollusc Anodonta cygnea: functional state of the adenylyl cyclase mechanisms of action of peptides of the insulin superfamily and their effect on carbohydrate metabolism enzymes].

In terms of development of evolutionary biomedicine using invertebrate animals as models for study of molecular grounds of various human diseases, for the first time the streptozocin (ST) model of insulin-dependent diabetes in the mollusc Anodonta cygnea has been developed. This model is based on the following authors' data: (1) redetection of insulin-related peptides (IRP) in mollusk tissues: (2) discovery of the adenylyl cyclase signal mechanism (ACSM) of action of insulin and other peptides of the insulin superfamily in tissues of mammals, human, and mollusc. A. cygnea; (3) concept of molecular defects in hormonal signal systems as causes of endocrine diseases. Studies on the ST model have revealed in mollusc smooth muscle on the background of hyperglycemia at the 2nd, 4th, and 8th day after the ST administration a decrease of the ACSM response to activating action of insulin, IGF-1, and relaxin. These functional disturbances were the most pronounced at the 2nd day of development and rather less marked at the 4th and 8th day. Analysis of data on effect of hormonal and non-hormonal (NaF, GIDP, and forskolin) ACSM activators has shown that the causes of impair of signal-transducing function of this mechanism are (1) a hyperglycemia-induced increase of the basal AC activity and as a consequence--a decrease of the enzyme catalytic potentials in response to hormone; (2) a decrease of functions of Gs-protein and of its coupling with AC. Besides, administration of ST produced in the mollusc muscles an attenuation of regulation by insulin of carbohydrate metabolism enzyme (glucose-6-phosphate dehydrogenase, glycogensynthase). The pattern of disturbances in the studied parameters in the mollusc is very similar to that revealed by the authors in rat and human muscle tissues in type 1 diabetes.

[Comparative study of molecular mechanisms of natural and synthetic polycationic peptides action on the activity of the adenylyl cyclase signaling system].

The molecular mechanisms of action of natural and synthetic polycationic peptides, forming amphiphilic helices, on the heterotrimeric G-proteins and enzyme adenylyl cyclase (AC), components of hormone-sensitive AC system, were studied. It is shown that synthetic peptides C-epsilonAhx-WKK(C10)-KKK(C10)-KKKK(C10)-YKK(C10)-KK (peptide I) and (GRGDSGRKKRRQRRRPPQ)2-K-epsilonAhx-C(Acm)(peptide II) in dose-dependent manner stimulate the basal AC activity, inhibit forskolin-stimulated AC activity and decrease both stimulating and inhibiting AC effects of the hormones in the tissues (brain striatum, heart muscle) of rat and in smooth muscles of the mollusc Anodonta cygnea. AC effects of these peptides are decreased after membrane treatment by cholera and pertussis toxins and are inhibited in the presence of the peptides, corresponding to C-terminal regions 385-394 alphas- and 346-355 alphai2-subunits of G-proteins. These data give evidence that the peptides I and II act on the signaling pathways which are realized through Gs- and Gi-proteins. At the same time, natural polycationic peptide mastoparan acts on AC system through Gi-proteins and blocks hormonal signals mediated via Gi-proteins only. Consequently, the action of mastoparan on G-proteins is selective and differs from the action of the synthetic peptides. It is also shown that peptide II, with branched structure, directly interacts not only with G-proteins (less effective in comparison with peptide I with hydrophobic radicals and mastoparan), but also with enzyme AC, the catalytic component of AC system. On the basis of data obtained the following conclusions were made: 1) the formation of amphiphilic helices is not enough for selective activation of G-protein by polycationic peptides, and 2) the primary structure of the peptides, the distribution of positive charged amino acids and hydrophobic radicals in them are very important for selective interaction between polycationic peptides and G-proteins.

[Study of molecular mechanisms of the relaxin action on adenylyl cyclase signaling system using synthetic peptides derived from the relaxin receptor LGR7].

The peptide hormone relaxin in dose-dependent manner stimulates adenylyl cyclase activity in the rat tissues (brain striatum, heart and skeletal muscles) and the muscle tissues of invertebrates--bivalve mollusk Anodonta cygnea and earthworm Lumbricus terrestris. Adenylyl cyclase stimulating effect of the hormone is most expressed in striatum and heart muscles of rats. For identification of the type ofrelaxin receptors, participating in the realization of this effect of the hormone, the peptides 619-629, 619-629-Lys(Palm) and 615-629 derived from the primary structure of C-terminal region of the third intracellular loop of the relaxin receptor of type 1 (LGR7), were synthesized by us for the first time. It is shown that peptide: 619-629-Lys(Palm) and 615-629 in competitive manner inhibit the stimulation of the adenylyl cyclase by relaxin in brain striatum and heart muscle of rats. At the same time, these peptides do not change stimulating effect of the hormone in the skeletal muscles of rat and in the muscles of invertebrates. Thus, the peptide action on adenylyl cyclase effect of relaxin is tissue- and species-specific. These data, on the one hand, demonstrate participation of receptor LGR7 in realization of adenylyl cyclase stimulating effect of relaxin in striatum and heart muscle of rats and, on the other, give evidence for existence of another adenylyl cyclase signaling mechanisms of relaxin action in the skeletal muscles and the muscle of invertebrates, which do not involve LGR7 receptor. The adenylyl cyclase stimulating effect of relaxin in striatum and heart muscle was decreased in the presence of C-terminal peptides 385-394 of alpha(s)-subunit of mammalian G protein and was blocked by treatment of the membranes with cholera toxin. On the basis of data obtained the following conclusions were made: (i) in striatum and heart muscle the relaxin stimulates adenylyl cyclase through LGR7 receptors functionally coupled with Gs protein, and (ii) the coupling between hormoneactivated relaxin receptor LGR7 and Gs protein is realized via the interaction of C-terminal part of receptor third intracellular loop and C-terminal segment of Gs protein alpha-subunit.