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.     

Hypothesis about the Key Coordinating Role of Adenylyl Cyclase Signaling Mechanism and of cAMP in Regulatory Action of Peptides of the Insulin Superfamily on Fundamental Cellular Processes: Cell Growth, Apoptosis, Metabolism (Development of L.G. Leibson's Heritage)

A new signaling mechanism of action of insulin and other peptides of the insulin superfamily [1—5[, including the following signaling chain: receptor-tyrosine kinase → G_i-protein → phosphatidylinositol 3-kinase → protein kinase C (PKCζ ?) → G_s-protein → adenylyl cyclase → cAMP → protein kinase A, was detected and deciphered, which resulted in the hypothesis about the key coordinating role of this adenylyl cyclase signaling mechanism (ACSM) and cAMP that it produces in regulatory action of these peptides on fundamental processes in the cell. This hypothesis is based on the following assumptions: (1) ACSM and cAMP as the second messengers participate in realization of the mitogenetic, rather than metabolic, action of peptides of the insulin superfamily; (2) ACSM and cAMP are involved in realization of inhibitory effect of insulin and related peptides on processes of cell death (antiapoptotic effect); (3) ACSM and cAMP inhibit metabolic effects of peptides of the insulin nature by preventing their realization. On the basis of these postulates and available facts, our hypothesis can be formulated as follows. The ACSM- produced cAMP acts as the key coordinator and regulator of three simultaneous incompatible cellular processes: cell growth, apoptosis, and metabolism. It is cAMP that determines, at the corresponding stage of action of the insulin-related peptides, the choice of the pathway of transduction of the signal generated by them to coordinatedly initiate one (proliferative) program and switching-off two others that lead to the cell death and to predominance of anabolic processes in the cell. This coordinating role of cAMP can form the basis for the fine regulation of fundamental processes in the cell, which eventually provides cell survival.     

Adenylyl cyclase signaling mechanisms of relaxin and insulin action: similarities and differences

The adenylyl cyclase signaling mechanism (ACSM) of relaxin H2 action was discovered and deciphered in mammalian muscles. A study of signaling blocks involved in ACSM of relaxin in comparison with that of insulin previously detected showed a close similarity throughout the post-receptor signaling chain of both hormones. The inhibitory action of tyrosine kinase blockers on the hormone AC activating effect indicates that the relaxin receptor involved in ACSM is likely to be of the tyrosine kinase type. However, a recent discovery of a relaxin receptor with serpentine architecture leaves open the question concerning the existence of receptor of the tyrosine kinase type. The structural-functional organization of the ACSM due to the action of relaxin-shown here for the first time-can be presented as the following signaling sequence: relaxin receptor ==>G(i) protein (betagamma-dimer) ==>phosphatidylinositol 3-kinase ==>protein kinase Czeta ==>G(s) protein ==>adenylyl cyclase. According to our hypothesis, the regulatory action of the insulin superfamily peptides on cell processes (proliferation, apoptosis, and metabolism) is mediated via ACSM.     

On the mechanism of relaxin action: the involvement of adenylyl cyclase signalling system

The molecular mechanism of relaxin action was studied taking into account the evolutionary relationship of the peptides belonging to the insulin superfamily and using the authors' previous data on the involvement of the adenylyl cyclase (AC) signalling system in the action of insulin and related peptides. Human relaxin 2 (10(-12)-10(-8) M) has been shown to cause a dose-dependent activating effect on AC in the human myometrium (+370%), in rat skeletal muscles (+117%) and the smooth foot muscles of the bivalve mollusc Anodonta cygnea (+73%). In these tissues mammalian insulin and insulin-like growth factor-1 (IGF-1) also had the AC activating effect. The order of efficiency of the above peptides based upon their ability to induce the maximal AC activating effect was as follows: relaxin > IGF-1 > insulin (human myometrium); IGF-1 > relaxin > insulin (rat skeletal muscle); molluscan insulin-like peptide > IGF-I > insulin > relaxin (molluscan muscle). The relaxin AC activating effect was inhibited with a selective tyrosine kinase blocker tyrphostin 47 and potentiated with Gpp[NH]p providing evidence for the participation of the receptor-tyrosine kinase and G-protein of the stimulatory type (Gs) in the regulatory action of relaxin. The conclusion is that the signalling chain: receptor tyrosine kinase ==> Gs protein ==> AC is involved in the mechanism of relaxin action.     

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.     

Streptozotocin model of diabetes mellitus in the mollusc <Emphasis Type="Italic">Anodonta cygnea</Emphasis>: functional state of the adenylyl cyclase mechanisms of action of insulin superfamily peptides 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 streptozotocin (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 mollusc 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 G_s-protein and of its coupling with AC. Besides, administration of ST produced in the mollusc muscle 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.     

Involvement of adenylyn cyclase signaling mechanisms in relaxing effect of relaxin and insulin on the rat uterus, trachea and human myometrium

Participation of adenylyl cyclase signaling mechanisms of relaxin and insulin action in their regulating influence on the process of relaxation of the rat uterine and tracheal smooth muscles and human myometrium was shown. The study was based on the discovery of novel adenylyl cyclase signaling mechanisms of relaxin and insulin action in the muscle of vertebrates which involve: receptor --> Gi protein (betagamma dimer) --> phosphatidylinositol-3-kinase --> protein kinase Csigma (zeta) --> Gs protein --> adenylyl cyclase --> cAMP. In the rat uterus, trachea and human myometrium, relaxin, insulin and isoproterenol induced relaxation of KCl-contraction. The order of efficiency of the agents based upon their ability to induce the inhibiting effect on the KCl-contraction was as follows: relaxin = insulin > isoproterenol. The hormones induce activating effect on adenylyl cyclase leading to production of cAMP in the rat uterine and tracheal smooth muscles and human myometrium. It is shown that cAMP reproduces relaxing effect of the hormones under study. Thus, the involvement of novel adenylyl cyclase signaling mechanisms of relaxin and insulin action in realization of their relaxation effect on rat uterus, trachea and human myometrium was revealed for the first time.     

Adenylyl Cyclase Signaling Mechanism of Relaxin Action

In connection with our discovery of the adenylyl cyclase signaling mechanism (ACSM) of action of some peptides belonging to the insulin superfamily, a possibility of its involvement in action of another insulin superfamily peptide, relaxin, was studied. It was shown for the first time that human relaxin-2 (10^–12–10^–8 M) activated adenylyl cyclase (AC) in a dose-dependent manner. The maximal peptide effect was revealed at a concentration of 10^–8 M. Under condition of the hormonal action the basal enzyme activity increased by +310% in human myometrium, by +117%, in rat skeletal muscles, and by +49%, in foot smooth muscles of the bivalve mollusc Anodonta cygnea. Insulin and mammalian insulin-like growth factor-I (IGF-I) also produced the AC activating effect in these muscles. The order of efficiency of these peptides, based on their ability to induce the maximal AC stimulating effect, was as follows: relaxin > IGF-I > insulin (human myometrium); IGF-I > relaxin > insulin (rat skeletal muscle); insulin-like peptide of Anodonta (ILPA) > IGF-I > insulin > relaxin (molluscan muscle). The relaxin activating effect on AC was potentiated by a guanine nucleotide, the non-hydrolyzed analog of GTP, guanylylimidodiphosphate (Gpp[NH]p), which indicates participation of G_s-protein in realization of this effect. This effect was inhibited by a tyrosine kinase selective blocker, tyrphostin 47, and a phosphatidylinositol-3-kinase (PI-3-K) selective blocker, wortmannin. Thus, for the first time, participation of ACSM in the relaxin action has been established. This mechanism, as suggested at the present time state of its study, includes the following signal pathway: receptor-tyrosine kinase PI-3-K G_s-protein AC.     

Functional defects in adenylyl cyclase signaling mechanisms of insulin and relaxin in skeletal muscles of rat with streptozotocin type 1 diabetes

Functional disturbance in the novel adenylyl cyclase signaling mechanism (ACSM) of insulin and relaxin action in rat streptozotocin (STZ) type I diabetes was studied on the basis of the authors’ conception of molecular defects in hormonal signaling systems as the main causes of endocrine diseases. Studying the functional state of molecular components of the ACSM and the mechanism as a whole, the following changes were found in the skeletal muscles of diabetic rats compared with control animals: 1) increase of insulin receptor binding due to an increase in the number of insulin binding sites with high and low affinity; 2) increase of the basal adenylyl cyclase (AC) activity and the reduction of AC-activating effect of non-hormonal agents (guanine nucleotides, sodium fluoride, forskolin); 3) reduction of ACSM response to stimulatory action of insulin and relaxin; 4) decrease of the insulin-activating effect on the key enzymes of carbohydrate metabolism, glycogen synthase and glucose-6-phosphate dehydrogenase. Hence, the functional activity of GTP-binding protein of stimulatory type, AC and their functional coupling are decreased during experimental type 1 diabetes that leads to the impairment of the transduction of insulin and relaxin signals via ACSM.     

Comparative study of biological activity of insulins of lower vertebrates in the novel adenylyl cyclase test-system

The biological activity of insulins of lower vertebrates (teleosts-Oncorhynchus gorbuscha, Scorpaena porcus, chondrosteans-Acipenser guldenstaedti and cyclostomates-Lamperta fluviatilis) was studied and compared with that of standard pig insulin. The determination of biological activity was made using the novel adenylyl cyclase (AC) test-system based on the adenylyl cyclase signaling mechanism (ACSM) of insulin action discovered earlier by the authors. The biological activity of insulins was estimated as EC(50), i.e. concentration leading to half-maximal activating effect of the hormone (10(-11)-10(-7) M), in vitro, on adenylyl cyclase in two types of the target tissues: in membrane fractions of the muscles of rat and mollusc Anodonta cygnea. In rat, the efficiency of insulins was found to decrease in the following order: pig insulin>scorpaena insulin>gorbuscha insulin>sturgeon insulin>lamprey insulin. In the mollusc, the order was different: sturgeon insulin>scorpaena insulin>pig insulin>gorbuscha insulin. Lamprey insulin at the same concentrations did not apparently reach the maximal adenylyl cyclase activating effect. The suggestion was made that differences in the biological activity of insulins depend on the hormone structure and a number of indexes characteristic of the adenylyl cyclase test-system in the vertebrate and invertebrate tissues. The proposed adenylyl cyclase test-system is highly sensitive to insulin at physiological concentrations, has good reproduction and is easy to apply.     

A novel view on the mechanisms of action of insulin and other insulin superfamily peptides: involvement of adenylyl cyclase signaling system

A new signaling mechanism common to mammalian insulin, insulin-like growth factor I, relaxin and mollusc insulin-like peptide, and involving receptor-tyrosine kinase==>G(i) protein (betagamma)==>phosphatidylinositol-3-kinase==>protein kinase Czeta==>adenylyl cyclase==>protein kinase A was discovered in the muscles and some other tissues of vertebrates and invertebrates. The authors' data were used to reconsider the problem of participation of the adenylyl cyclase-cAMP system in the regulatory effects of insulin superfamily peptides. A hypothesis has been put forward according to which the adenylyl cyclase signaling mechanism producing cAMP has a triple co-ordinating role in the regulatory action of insulin superfamily peptides on the main cell processes, inducing the mitogenic and antiapoptotic effects and inhibitory influence on some metabolic effects of the peptides. It is suggested that cAMP is a key regulator responsible for choosing the transduction pathway by concerted launching of one (proliferative) program and switching off (suppression) of two others, which lead to cell death and to the predomination of anabolic processes in a cell. The original data obtained give grounds to conclude that the adenylyl cyclase signaling system is a mechanism of signal transduction not only of hormones with serpentine receptors, but also of those with receptors of the tyrosine kinase type (insulin superfamily peptides and some growth factors).     

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.     

Study of the Functional Organization of a Novel Adenylate Cyclase Signaling Mechanism of Insulin Action

In this study we continued decoding the adenylate cyclase signaling mechanism that underlies the effect of insulin and related peptides. We show for the first time that insulin signal transduction via an adenylate cyclase signaling mechanism, which is attended by adenylate cyclase activation, is blocked in the muscle tissues of the rat and the mollusk Anodonta cygnea in the presence of: 1) pertussis toxin, which impairs the action of the inhibitory GTP-binding protein (G_i); 2) wortmannin, a specific blocker of phosphatidylinositol 3-kinase; and 3) calphostin C, an inhibitor of different isoforms of protein kinase C. The treatment of sarcolemmal membrane fraction with cholera toxin increases basal adenylate cyclase activity and decreases the sensitivity of the enzyme to insulin. We suggest that the stimulating effect of insulin on adenylate cyclase involves the following stages of hormonal signal transduction cascade: receptor tyrosine kinase → G_iprotein (βγ) → phosphatidylinositol 3-kinase → protein kinase C (ζ?) → G_sprotein → adenylate cyclase → cAMP.     

Tyrosine kinase-mediated serine phosphorylation of adenylyl cyclase

Receptor tyrosine kinase (RTK) activation is associated with modulation of heptahelical receptor-stimulated adenylyl cyclase responses. The mechanisms underlying the RTK-mediated enhancement of adenylyl cyclase function remain unclear. In the present studies, we show that the tyrosine kinase-dependent enhancement of adenylyl cyclase isoform VI function parallels an enhancement in serine phosphorylation of the enzyme. This effect was mediated by both RTK activation, with IGF-1, and by tyrosine phosphatase inhibition, with sodium orthovanadate. This enhancement of adenylyl cyclase function was not attenuated by inhibitors of ERK, PKC, PKA, or PI3 kinase activity but was blunted by inhibition of endogenous p74(raf-1)() activity. To characterize the molecular site of this effect we identified multiple candidate serine residues in and adjacent to the adenylyl cyclase VI C1b catalytic region and performed serine-to-alanine site-directed mutagenesis using adenylyl cyclase VI as a template. Mutation of serine residues 603 and 608 or serine residues 744, 746, 750, and 754 attenuated both the tyrosine kinase-mediated enhancement of enzyme phosphorylation as well as the sensitization of function. Together, these data define a novel tyrosine kinase-mediated mechanism leading to serine phosphorylation of adenylyl cyclase isoform VI and the sensitization of adenylyl cyclase responsiveness.     

Insulin-Regulated Adenylyl Cyclase Signaling System in Rat Skeletal Muscles under Conditions of in vivo Insulin Administration and of Insulin Insufficiency Produced by Streptozotocin Diabetes

Possibility of the appearance of functional defects in the adenylyl cyclase (AC) signaling mechanism (ACSM) of insulin action, which was discovered by the authors earlier [1–3], is studied in skeletal muscles of rats with acute insulin insufficiency produced by streptozotocin diabetes (24 h). This ACSM includes the signaling chain: receptor-tyrosine kinase Gi-protein phosphatidylinositol 3-kinase protein kinase C-zeta Gs-protein adenylyl cyclase protein kinase A. At comparative evaluation of the functional state of individual molecular blocks of ACSM and the entire mechanism as a whole in skeletal muscles of diabetic rats in comparison with control animals, the following facts have been revealed: (1) an increase of the AC basal activity and a decrease of effects of non-hormonal activators of AC (guanine nucleotides, NaF, forskolin) ; (2) reduction of reactivity of the whole ACSM to insulin (10_–8 M, in vitro) and to combined action of the hormone and GIDP (10_–6 M) ; (3) a decrease of the activating action of insulin on key enzymes of carbohydrate metabolism—glycogen synthase and glucose-6-phosphate dehydrogenase (G6PDG). It is concluded that insulin insufficiency leads to several disturbances in the insulin ACSM: at the level of its catalytic component—AC, Gs protein and its coupling with AC, as well as to a decrease of regulatory metabolic effects of the hormone. These data indicate a decrease of sensitivity of skeletal muscles of diabetic rats to insulin and an involvement of this hormone in maintenance of functionally active status of the ACSM of insulin signal transduction.     

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

In the smooth muscles of mollusc Anodonta cygnea the regulatory action of hormones on adenylyl cyclase signaling system (ACSS) are realized through the receptors of serpentine type (biogenic amines, isoproterenol, glucagon) and receptor tyrosine kinase (insulin) type. Intracellular mechanisms of their interaction are interconnected. Application of hormones, their antagonists and pertussis toxin in combination with insulin and biogenic amines or glucagon on adenylyl cyclase (AC) activity allows revealing the possible sites of cross-linking in the mechanisms of their action. Combined influence of insulin and serotonin or glucagon leads to decreased stimulation of adenylyl cyclase (AC) by these hormones, whereas combined application of insulin and isoproterenol suppresses AC-stimulating effect of insulin, but AC-inhibiting effect of isoproterenol is maintained in the presence and absence of non-hydrolysable analog of GTP—guanylyl imido diphosphate (GIDP). The specific blockage of AC-stimulating effect of serotonin by cyproheptadine—antagonist of serotonin receptors, did not change AC stimulation by insulin. Beta-adrenoblockers (propranolol and alprenolol) prevent inhibition of AC activity by isoproterenol, but did not change AC stimulation by insulin. Pertussis toxin blocked AC-inhibiting effect of isoproterenol and weakened AC-stimulating action of insulin. Thus, in the muscles of Anodonta cygnea negative interaction between ACS have been revealed, which are realized under combined influence of insulin and serotonin or glucagon, most probably, at the level of receptor of serpentine type (serotonin, glucagon), whereas under action of insulin and isoproterenol at the level of G_i protein and AC interaction.     

Structural-functional characteristics of the adenylyl cyclase signaling system regulated by biogenic amines and peptide hormones in muscles of the earthworm 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 skin-muscle sac 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 mollusc 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 mollusc muscle, but much weaker that the action of these hormones on the rat myocardium AC activity. Use of Cterminal peptides of α-subunits of G-proteins of the stimulatory (385–394 Gαs) and inhibitory (346–355 Gαi2) types that disrupt selectively the hormonal signal transduction realized via Gsand Giproteins, 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 Gs-protein; the AC-inhibiting effect of somatostatin is realized via the receptor coupled with Gi-protein, whereas serotonin and tryptamine activate both types of G-proteins.     

The role of protein kinase C in insulin signal transduction via adenylyl cyclase signaling mechanism

Activation of proteinkinase C with diacylglycerol or phorbol-12-myristate-13-acetate in the rat muscle membrane or Anodonta cygnea mollusc blocks the insulin stimulating signal to adenylyl cyclase via tyrosinekinase type receptor. The same occurs with stimulating effect of biogenic amines to adenylyl cyclase via serpentine type receptor. Transduction of the inhibitory signal induced with isoproterenol to adenylyl cyclase remained unchanged in case of the proteinkinase C activation. The findings suggest that phorbol-sensitive proteinkinase C realizes a negative regulation of insulin-sensitive adenylyl cyclase signalling system. This negative regulation might prove a universal mechanism of the adenylyl cyclase system desensitisation.     

Insulin-Regulated Adenylyl Cyclase Signaling System in Cell Culture of Mouse Fibroblasts of L Line

In terms of the subsequent study of the earlier discovered adenylyl cyclase signaling mechanism of insulin action [1, 2], we studied participation in it of various isoforms of protein kinase C. As object of study, a culture of mouse fibroblast cells of the L line (LSM subline) was chosen. It has been shown that insulin and a non-hydrolyzed analog of GTP, Gpp[NH]p, stimulate the adenylyl cyclase (AC) activity in these cells both individually and in combination. Activators of phorbol-sensitive isoforms of protein kinase C, diacylglycerol and phorbol ester (phorbol-12-myristate-13-acetate) at their concentrations of 1–100 nM stimulate basal activity of AC. In their presence, a significant decrease of stimulating effects of insulin and Gpp[NH]p or their complete disappearance are observed. Calphostin C (1–100 nM), an inhibitor of both phorbol-sensitive and atypical, phorbol-insensitive isoforms of protein kinase C, somewhat increased the basal AC activity. However, the stimulating effects of insulin and Gpp[NH]p in the presence of calphostin C decreased markedly. On the whole, the obtained data allow us to suggest participation of various isoforms of protein kinase C (sensitive and insensitive to the phorbol esters) in regulation of the process of the insulin signal transduction in mouse fibroblasts through the adenylyl cyclase signaling mechanism. Thus, mechanisms of functioning of the insulin-regulated AC-system in fibroblasts, representatives of connective tissue cells, are similar to those that we described earlier in muscle tissues of vertebrate and invertebrate animals. Taken together, these data indicate the absence of tissue- and species-specificity in functioning of the insulin-regulated AC system and its wide spread in tissues of different animals.