[BETA-ADRENERGIC REGULATION OF THE ADENYLYL CYCLASE SIGNALING SYSTEM IN MYOCARDIUM AND BRAIN OF RATS WITH OBESITY AND TYPES 2 DIABETES MELLITUS AND THE EFFECT OF LONG-TERM INTRANASAL INSULIN TREATMENT].

The stimulating effect of norepinephrine, isoproterenol and selective ß-adrenoceptor (ß3-AR) agonists BRL 37344 and CL 316.243 on the adenylyl cyclase signaling system (ACSS) in the brain and myocardium of young and mature rats (disease induction at 2 and 4 months, respectively) with experimental obesity and type 2 diabetes mellitus (DM2), and the influence of long-term treatment of animals with intranasal insulin (I-I) were studied. The AC stimulatory effects of ß-agonist isoproterenol in animals with obesity and DM2 was shown to be practically unchanged. The respective effects of norepinephrine on the AC activity were attenuated in the brain of young and mature rats and in the myocardium if mature rats, and the I-I treatment led to their partial recovery. In the brain and myocardium of mature rats with obesity and DM2, the enhancement of the AC stimulatory effects of ß3-AR agonists was observed, white in young rats the influence of the same pathological conditions was lacking. The I-I treatment decreased the AC stimulatory effects of ß3-agonists to their levels in the control. Since functional disruption of the adrenergic agonist-sensitive ACSS can lead to metabolic syndrome and DM2, the recovery of this system by the I-I treatment offers one of the ways to correct these diseases and their complications in the nervous and cardiovascular systems.

[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.

[A new conceptual approach for searching for molecular causes of diabetes mellitus, based on the study of functioning of hormonal signaling systems].

The review deals with analysis and generalization of the data obtained by authors on abnormalities in hormonal signal systems in diabetes mellitus (DM)--in rats with experimental models of DM of the types 1 and 2, in patients with DM, and in invertebrates (mollucs) with experimental diabetes-like state. Changes of functional state of hormonal signal systems regulated by different hormones, including biogenic amines and peptides of the insulin group, in a wide spectrum of tissues are discussed. The conclusion is made that disturbances in hormonal signal systems are the key molecular causes of physiological and metabolic abnormalities occurring in the types 1 and 2 DM. A concept on polyhormonal genesis of DM and the systemic nature of disturbances in the hormone-regulated signaling cascades under conditions of DM is formulated.

[Hypothesis of evolutionary origin of several human and animal diseases].

Studies of our Laboratory in the field of molecular and evolutionary endocrinology have allowed us to put forward a hypothesis about evolutionary origin of endocrine and other diseases of human and animals. This hypothesis is considered using a model of hormonal signal systems. It is based on the concept formulated by the authors about molecular defects in hormonal signal systems as the key causes of endocrine diseases; on evolutionary conservatism of hormonal signal systems, which stems logically from the authors' concept of the prokaryotic origin and endosymbiotic appearance in the course of evolution of chemosignal systems in the higher eukaryotes; from the fact that the process of formation of hormonal signal systems with participation of endosymbiosis including the horizontal transfer of genes is accompanied by transfer not only of normal, but also of the defected genetic material. There are considered examples of the principal possibility of transfer of defected genes between bacteria and eukaryotic organisms. Analysis of the current literature allows suggesting inheritance of pathogenic factors from evolutionary ancestors in the lineage prokaryotes--lower eukaryotes--higher eukaryotes.

[Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system of the infusorian Dileptus anser].

In some unicellular eukaryotes, cAMP performs functions not only of the second messenger, but also of hormone, the primary messenger. We have found that cAMP binds to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and enzyme adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K(D), 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N6,O2-dibutyryl-cAMP, a non-hydrolyzed cAMP analog, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated with the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.

The prokaryotic origin and evolution of eukaryotic chemosignaling systems.

Analysis of our own results and data published over the last two decades supports the authors' hypothesis of the prokaryotic origin and endosymbiotic mechanism of appearance of chemosignaling systems in higher eukaryotes. Comparison of the structural-functional organization of these information systems and their component blocks (receptors, GTP-binding proteins, enzymes with cyclase activity, protein kinases, etc.) in bacteria and eukaryotes revealed a whole series of similar characteristics pointing to evolutionary relatedness. This led to the conclusion that eukaryotic signal systems have prokaryotic roots. In terms of their architecture and functional properties, the signal transduction systems seen in unicellular eukaryotes represent a transitional stage in the evolution of chemosignaling systems between prokaryotes and higher eukaryotes. The propagation of chemosignaling systems in three kingdoms - Bacteria, Archaea, and Eukarya - occurred by horizontal transfer of bacterial genes and the coevolution of the components of these systems.

[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.

[Procaryotic genesis and evolution of chemosignaling systems of eukaryotes].

The analysis of own and literature data accumulated in the last two decades allowed to check and confirm the author's hypothesis about the prokaryotic origin and endosymbiotic genesis of chemosignalling systems of higher eukaryotes. The comparison of structural-functional organization of these information systems and their components (receptors, GTP-binding proteins, enzymes with cyclase activity, protein kinases etc.) in bacteria and eukaryotes revealed a number of similar features giving evidence for their evolutionary relationship. The conclusion was made that eukaryotic signaling systems have prokaryotic roots. The systems of signal transduction revealed in unicellular eukaryotes according to their architecture and functional properties represent a transient stage in the evolution of chemosignalling systems from prokaryotes to higher eukaryotes. The spreading of signalling systems among three super kingdoms--Bacteria, Archaea and Eukarya occurred as a result of horizontal transfer of bacterial genes and co-evolution of signalling components.

[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.

[Signal transduction systems of prokaryotes].

The main components of chemosignaling systems of prokaryotes are multifunctional receptor molecules that include both sensor domains specifically recognizing external signals and effector domains converting these signals into an adequate cell response. This review summarizes and analyzes data of structural-functional organization, molecular mechanisms of action, and regulation of receptor forms of histidine kinases, adenylyl cyclases, diguanylyl cyclases, and phosphodiesterases. These enzymes have been shown to be precursors of the receptor and effector components of the eukaryote hormonal signaling systems. This confirms the hypothesis developed by the authors about formation of the main archetypes of chemosignaling systems at the early evolution stages and about the evolutionary relationship of the signaling systems of prokaryotes and eukaryotes.

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.