A. O. Shpakov

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 αs 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 Gs protein. It is also demonstrated that linkage of relaxin-activated LGR7 receptor with the Gs 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 αs subunit of the G protein.

Molecular mechanisms for the effect of mastoparan on G proteins in tissues of vertebrates and invertebrates

The peptide toxin mastoparan increased GTP-binding activity of heterotrimeric G proteins in tissues of vertebrate and invertebrate animals, the effect of mastoparan in mussel tissues being less pronounced. The stimulatory effect of mastoparan on GTP binding was not observed after treatment of membranes with pertussis toxin that selectively modulates function of Gi proteins. Activity of mastoparan decreased in the presence of C-terminal peptide 346–355 from the Gi protein αi2-subunit. Mastoparan dose-dependently decreased the stimulatory effect of hormones on GTP binding in tissues of rats and mussels. The influence of these hormones on the cell is realized via Gi proteins. However, mastoparan did not modulate the effect of Gs protein-activating hormones.

Decrease in functional activity of G-proteins hormone-sensitive adenylate cyclase signaling system, during experimental type II diabetes mellitus.

The development of experimental type II diabetes mellitus in rats was accompanied by dysfunction of inhibitory and stimulatory heterotrimeric G-proteins, components of hormone-sensitive adenylate cyclase signal system. The function of inhibitory G-proteins decreased most significantly under these conditions, which is seen from weakened regulatory effects of somatostatin (in the myocardium) and bromocriptine (in the brain striatum) realized via inhibitory G-proteins in diabetic rats compared to controls. These hormones produce less pronounced inhibitory effect on forskolin-induced activation of adenylate cyclase. In the myocardium of diabetic rats, the stimulatory effects of isoproterenol and relaxin on adenylate cyclase realized via stimulatory G-proteins were decreased to a lesser extent. In the striatum of diabetic rats the stimulatory effect of serotonin and relaxin did not differ from the control. Therefore, dysfunction of stimulatory G-proteins during type II diabetes mellitus is characterized by tissue specificity. Synthetic peptides corresponding to functionally important regions in α-subunits of G-proteins and relaxin receptor LGR7 less effectively inhibited hormone signal transduction via the adenylate cyclase system in rats with type II diabetes. These changes reflect abnormal coupling between receptors and G-proteins in tissues of diabetic rats.

Androgen Deficiency in Male Rats with Prolonged Neonatal Streptozotocin Diabetes

We studied the diurnal dynamics of testosterone concentration in male rats with 240-day neonatal streptozotocin-induced diabetes mellitus, which is similar to human type 2 diabetes mellitus. We also studied the effects of intranasal administration of luliberin on testosterone level and the regulation of activities of adenylate cyclase and stimulatory G-proteins in the testicles of diabetic and intact animals by human chorionic gonadotropin. In rats with neonatal diabetes, a decrease in the mean diurnal level of testosterone and its morning rise were observed. The increase in testosterone level 30 min after luliberin administration was significantly reduced in diabetic animals, but no differences in the response to luliberin were observed in intact and diabetic rats 2-6 h after the treatment. The stimulatory effects of human chorionic gonadotropin on adenylate cyclase activity and binding of guanosine triphosphate by stimulatory G-proteins were reduced in the plasma membranes from the testicles of rats with neonatal diabetes in comparison with control specimens. Therefore, rats with neonatal diabetes were characterized by androgen deficiency, which can be related to the impairment of hypothalamic–pituitary–gonadal axis and reduced sensitivity of the adenylate cyclase system in the testicles of diabetic rats to human chorionic gonadotropin.

Molecular Mechanisms of Modified Sensitivity of the Adenylate Cyclase Signaling System to Biogenic Amines during Streptozotocin-Induced Diabetes

We demonstrated changes in the sensitivity of the adenylate cyclase signaling system to biogenic amines (adrenoceptor agonists and serotonin) underwent a change in skeletal muscles of rats with 30-day streptozotocin-induced diabetes. Isoproterenol had a less significant stimulatory effect on adenylate cyclase in diabetic rats. Hormonal signals via Gi proteins were suppressed in animals with diabetes, which determined a greater stimulatory effect of norepinephrine and serotonin on adenylate cyclase. Hormones less significantly increased guanosine triphosphate-binding activity of G proteins in diabetic rats, which reflects the impairment of their functional coupling with receptors.

Role of phosphatidylinositol-3-kinase and protein kinase Cζ in the adenylate cyclase signal mechanism of action of relaxin in muscle tissues of rats and mollusks

We showed that phosphatidylinositol-3-kinase and protein kinase Cζ are involved in the adenylate cyclase signal mechanism of relaxin action. A selective inhibitor of phosphatidylinositol-3-kinase wortmannin blocked the stimulatory effect of relaxin on adenylate cyclase in rat skeletal muscles and Anodonta cygnea smooth muscles. Antibodies against protein kinase Cζ abolished the relaxin-induced stimulation of adenylate cyclase in rat muscles, but not in mollusk muscles. Our results indicate that phosphatidylinositol-3-kinase and protein kinase Cζ play a role in the adenylate cyclase signal mechanism of relaxin action.

The Leptin, Dopamine and Serotonin Receptors in Hypothalamic POMC-Neurons of Normal and Obese Rodents

The pro-opiomelanocortin (POMC)-expressing neurons of the hypothalamic arcuate nucleus (ARC) are involved in the control of food intake and metabolic processes. It is assumed that, in addition to leptin, the activity of these neurons is regulated by serotonin and dopamine, but only subtype 2C serotonin receptors (5-HT2CR) was identified earlier on the POMC-neurons. The aim of this work was a comparative study of the localization and number of leptin receptors (LepR), types 1 and 2 dopamine receptors (D1R, D2R), 5-HT1BR and 5-HT2CR on the POMC-neurons and the expression of the genes encoding them in the ARC of the normal and diet-induced obese (DIO) rodents and the agouti mice (Ay/a) with the melanocortin obesity. As shown by immunohistochemistry (IHC), all the studied receptors were located on the POMC-immunopositive neurons, and their IHC-content was in agreement with the expression of their genes. In DIO rats the number of D1R and D2R in the POMC-neurons and their expression in the ARC were reduced. In DIO mice the number of D1R and D2R did not change, while the number of LepR and 5-HT2CR was increased, although to a small extent. In the POMC-neurons of agouti mice the number of LepR, D2R, 5-HT1BR and 5-HT2CR was increased, and the D1R number was reduced. Thus, our data demonstrates for the first time the localization of different types of the serotonin and dopamine receptors on the POMC-neurons and a specific pattern of the changes of their number and expression in the DIO and melanocortin obesity.

Peptides Derived from the Third Cytoplasmic Loop of the Serotonin Subtype 1B Receptor Selectively Inhibit Transmission of Serotoninergic Signals via Their Homologous Receptors

A leading role in the interactions of most serotonin-type hormone receptors with heterotrimeric G proteins is played by their third cytoplasmic loops. Studies in recent years have shown that synthetic peptides corresponding to the membrane-proximal parts of these loops may have selective influences on the transmission of hormone signals via their homologous receptors and trigger the signal cascade in the absence of hormone. We report the first synthesis of peptides derived from the C-terminal region of the third cytoplasmic loop of type 1B serotonin receptors, along with studies of their influences on the serotonin sensitivity of the adenylate cyclase system in the rat brain. Peptides 300–316 and 306–316 (numbered from amino acid positions in the rat serotonin subtype 1B receptor molecule) at micromolar concentrations, in the absence of hormone, stimulated GTP-binding Gi-proteins coupled with subtype 1B serotonin receptors and inhibited forskolin-stimulated adenylate cyclase activity. Studies using selective serotonin receptor agonists and antagonists showed that peptides 300–316 and 306–316 inhibit the transmission of the serotonin signal via the homologous receptor but have weak effects on other types of serotonin receptor. Peptide 300–316 were markedly more active than its shortened analog 306–316 in terms of the selectivity and efficacy of actions on the adenylate cyclase signaling system, which is regulated via subtype 1B serotonin receptors. These data provide evidence that region 300–316 of subtype 1B serotonin receptors is involved in the interaction with Gi proteins and includes the main molecular determinants responsible for transmission of the serotonin signal to adenylate cyclase.

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 β-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.

Receptors of Biogenic Amines in Muscle Membranes of Molluscs

In a number of molluscs belonging to different groups, serotonin and β-adrenergic agonists have a stimulatory effect on the sarcolemmal adenylyl cyclase (AC) activity, which is potentiated by guanine nucleotides. Catecholamines (isoprotenerol > adrenaline > noradrenaline) inhibit AC activity in Anodonta cygnea sarcolemma. In muscle membranes of Anodonta, Mizuhopecten and Spisula, a serotonin receptor (5-HTR) and a β-adrenoreceptor (β-AR) were identified. Guanine nucleotides lowered the affinities of 5-HTR and β-AR to agonists.