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.     

A decrease in the sensitivity of adenylyl cyclase and heterotrimeric g proteins to chorionic gonadotrophin and peptide hormones action in the tissues of reproductive system of rats with experimental type 2 diabetes

Patients with different forms of the diabetes, particularly with insulin-independent type 2 diabetes, have a wide spectrum of the disturbances of the functions of reproductive system. It is suggested that the main reason of these disturbances is altered sensitivity of reproductive system tissues to the regulatory action of hormones. The aim of this study was the identification of the changes in functioning of adenylyl cyclase system (ACS) sensitive to human chorionic gonadotrophin (hCG) and the peptide hormones in the ovary, testes and uterus of rats with neonatal streptozotocin (STZ) diabetes that is similar to the type 2 diabetes in humans. The effects of hCG, PACAP-38 and relaxin, realizing their effects via stimulatory G proteins (G_s), and somatostatin, acting via the inhibitory G protein (G_i), on adenylyl cyclase (AC) activity and GTP binding to the G proteins were studied. In rats with STZ type 2 diabetes the regulatory effects of hCG and PACAP-38 decreased in the ovary and testes, while the effects of somatostatin decreased in all investigated tissues (especially in the uterus). This caused attenuation of the hormonal effects, stimulating (hCG and PACAP-38) or inhibiting (somatostatin) AC activity, and in the decrease of their stimulatory effect on the GTP binding. At the same time a significant decrease of ACS sensitivity to relaxin in the tissues of diabetic rats was not found. Data obtained suggest that one of the key reasons for impairments of reproductive functions in experimental type 2 diabetes is the decrease of ACS sensitivity to the hormones, hCG, PACAP-38 and somatostatin, which play an important role in the reproductive system functioning.     

Effect of intranasal insulin and serotonin on functional activity of the adenylyl cyclase system in myocardium, ovary, and uterus of rats with prolonged neonatal model of diabetes mellitus

The disturbances in hormonal signaling systems, adenylyl cyclase system (ACS) in particular, occur at the early stages of diabetes mellitus (DM) being one of the key causes of its complications. Since the correlation between the severity of DM and severity of disturbances in ACS is established, studying ACS activity can be used for monitoring DM and its complications and evaluating the effectiveness of their treatment. Recently, intranasal insulin (I-I) and the drugs increasing brain serotonin level, thus effectively restoring CNS functions, have begun to be used for the treatment of type 2 DM. However, the mechanisms of their action on peripheral tissues and organs at DM are not understood. The aim of this work was to study an influence of I-I and intranasal serotonin (I-S) on the functional activity of ACS in myocardium, ovary and uterus of rats with a neonatal model of type 2 DM. In the tissues of diabetic rats the changes in the regulation of adenylate cyclase (AC) by guanine nucleotides and hormones acting on enzyme in stimulatory and inhibitory manner were found, and these changes were characterized by receptor and tissue specificity. In diabetic rats I-I restored AC-stimulating effects of isoproterenol in the myocardium, that of guanine nucleotides and gonadotropin in the ovaries and relaxin in the uterus, as well as AC-inhibiting effects of somatostatin in all tissues and norepinephrine in the myocardium. Treatment with I-S led to a partial recovery of AC-inhibiting effect of norepinephrine in the diabetic myocardium, but did not affect the regulation of AC by other hormones. These data indicate that I-I normalizes the functional activity of ACS in the myocardium and in the tissues of reproductive system of female rats with neonatal DM, whereas the effect of I-S on ACS in the studied tissues is less pronounced. These results should be considered for the design and optimization of the strategy of I-I and I-S application for the treatment of DM and its complications.     

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.     

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.     

Opioid tolerance and the emergence of new opioid receptor-coupled signaling

Multiple cellular adaptations are elicited by chronic exposure to opioids. These include diminution of spare opioid receptors, decreased opioid receptor density, and G-protein content and coupling thereof. All imply that opioid tolerance is a manifestation of a loss of opioid function, i.e., desensitization. Recent observations challenge the exclusiveness of this formulation and indicate that opioid tolerance also results from qualitative changes in opioid signaling. In this article, Gintzler and Chakrabarti discuss the evidence that suggests that opioid tolerance results not only from impaired opioid receptor functionality, but also from altered consequences of coupling. Underlying the latter are fundamental changes in the nature of effectors that are coupled to the opioid receptor/G-protein signaling pathway. These molecular changes include the upregulation of adenylyl cyclase isoforms of the type II family as well as a substantial increase in their phosphorylation state. As a result, there is a shift in opioid receptor/G-protein signaling from predominantly G_iα inhibitory to G_βγ stimulatory following chronic in vivo morphine exposure. These adaptations to chronic morphine indicate the plasticity of opioid-signal transduction mechanisms and the ability of chronic morphine to augment new signaling strategies.     

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.     

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second‐messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein G_iα at a pseudo‐symmetric position to the stimulatory G_sα binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform‐specific modulatory effects on AC. Proteins 2016; 84:1844–1858. © 2016 Wiley Periodicals, Inc.     

Intranasal insulin affects adenyl cyclase system in rat tissues in neonatal diabetes

The changes in hormone-regulated adenylyl cyclase (AC) signaling system implicated in control of the nervous, cardiovascular and reproductive systems may contribute to complications of diabetes mellitus (DM). We investigated the functional state of AC system in the brain, myocardium, ovary and uterus of rats with neonatal DM and examined the influence of intranasally administered insulin on the sensitivity of this system to biogenic amines and polypeptide hormones. The regulatory effects of somatostatin and 5-HT_1BR-agonist 5-nonyloxytryptamine acting via G_i protein-coupled receptors were significantly decreased in DM and partially restored in insulin-treated rats. The effects of hormones, activators of AC, are changed in tissue- and receptorspecific manner, and intranasal insulin restored the effects rather close to the level in control. In insulin-treated non-diabetic rats, AC stimulating effects of isoproterenol and relaxin in the myocardium and of human chorionic gonadotropin in the ovaries were decreased, while the effects of hormones, inhibitors of AC, were increased. These data indicate that with intranasal insulin, Gi protein-mediated signaling pathways continue to gain strength. The obtained data on the influence of hormones on AC system in the brain, myocardium, ovary and uterus allow looking anew into the mechanisms of therapeutic effects of intranasal insulin.     

‘Cross-talk’ between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the α-subunits of the stimulatory and inhibitory G-proteins of AC (G_sα and G_i−2α) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_sα levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_sα levels by more than 50% at all time-points. G_i−2α levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein α-subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

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.     

The effect of long-term diabetes mellitus induced by treatment with streptozotocin in 6-week-old rats on functional activity of the adenylyl cyclase system

In type 1 diabetes mellitus (DM), changes occurring in the adenylyl cyclase signaling system (ACSS) are one of the key causes of complications of the disease. Since type 1 DM has been most often diagnosed in childhood and adolescence, the study of changes in ACSS in the early development of the disease is a genuine problem. For this, we developed a prolonged model of type 1 DM, which was induced by treatment of 6-week-old rats with moderate doses of streptozotocin (1.5M-DM), and studied the functional state of ACSS in the brain, myocardium, and testes of rats with this model of the disease 7 months after its start. The 1.5M-DM model was compared with the model that was induced by streptozotocin treatment of adult, 5-month-old animals (5M-DM). It was shown that, in 1.5M-DM, in the tissues of diabetic rats, the functional activity of ACSS sensitive to biogenic amines and polypeptide hormones was significantly changed. In rats with 1.5M-DM, the adenylate cyclase (AC) inhibitory effects of somatostatin (in all studied tissues), noradrenaline (in the myocardium and the brain), and agonists of type 1 serotonin receptor (in the brain) were weakened to the greatest degree. In the brain, the AC-stimulating effects of relaxin, isoproterenol, and agonists of G_s-protein-coupled serotonin receptors also decreased; in the myocardium, the corresponding effects of GppNHp, relaxin, and β-adrenergic agonists declined; and, in the testes, the AC effects of GppNHp and chorionic gonadotropin declined. When comparing the 1.5M-DM and 5M-DM models, the most pronounced differences between them were found in the effect of DM on hormonal regulation of ACSS in the brain, this being true both for AC-stimulating effects of dopamine and PACAP-38 and for AC-inhibiting effects of bromocriptine and somatostatin. These results indicate significant changes in hormonal regulation of the nervous, cardiovascular, and reproductive systems in rats with early induction of type 1 DM, in some cases more severe changes as compared with late model of 5M-DM. These changes may be the basis for development of diabetic cardiomyopathy, cognitive deficiency, and hypogonadotropic states, which are often detected in children and adolescents with type 1 DM.     

Alterations of Tubulin Function Caused by Chronic Antidepressant Treatment in Rat Brain

1. Antidepressants have been used clinically for many years; however, the neurochemical mechanism for their therapeutic effect has not been clarified yet. Recent reports indicate that chronic antidepressant treatment directly affects the postsynaptic membrane to increase the coupling between the stimulatory GTP-binding (G) protein, G_s, and adenylyl cyclase. Tubulin, a cytoskeletal element, is involved in the stimulatory and inhibitory regulation of adenylyl cyclase in rat cerebral cortex via direct transfer of GTP to G proteins. In this study, we investigated whether the functional change of the adenylyl cyclase system caused by chronic antidepressant treatment involves an alteration of tubulin function in the regulation of adenylyl cyclase activity.2. Male Sprague–Dawley rats were treated once daily with amitriptyline or saline by intraperitoneal injection (10 mg/kg) for 21 days, and their cerebral cortex membranes and GppNHp-liganded tubulin (tubulin-GppNHp) were prepared for what.3. GppNHp-stimulated adenylyl cyclase activity in cortex membranes from amitriptyline-treated rats was significantly higher than that in control membranes. Furthermore, tubulin–GppNHp prepared from amitriptyline-treated rats was more potent than that from control rats in the stimulation of adenylyl cyclase activity in the cortex membranes of the controls. However, there was no significant difference in manganese-stimulated adenylyl cyclase activity between control and amitriptyline-treated rats.4. The present results suggest that chronic antidepressant treatment enhances not only the coupling between G_s and the catalytic subunit of adenylyl cyclase but also tubulin interaction with G_s in the cerebral cortex of the rat.     

Molecular causes of changes in sensitivity of adenylyl cyclase signaling system to biogenic amines in the heart muscle during experimental streptozotocin diabetes

Changes in hormonal sensitivity of the adenylyl cyclase signaling system (ACS) and their possible molecular causes in the heart muscle of rats with experimental streptozotocin diabetes (type I diabetes) are investigated. An increase in stimulating effects of noradrenaline and isoproterenol on adenylyl cyclase (AC) activity have been shown. In the case of noradrenaline, this increase is due to suppression of Gi-protein function and Gi-coupled inhibitory AC signaling pathway. Meanwhile, in diabetic rats the influence of C-terminal peptide 346-355 of alphai2-subunit on hormonal activation of AC and GTP-binding is diminished. In the case of isoproterenol, along with its stimulating effect, at micromolar concentrations this hormone exerts inhibitory action, realized, presu- mably, through beta3-adrenergic receptors. Effect of isoproterenol on AC and GTP-binding in the heart of diabetic animals is modified by peptide 385-394 alphas, blocking Gs-coupled signaling pathways, and by peptide 346-355 alphai2, blocking transduction of inhibitory signals. In addition, a decrease in serotonin stimulating effect on components of ACS in diabetic animals was shown. The data obtained provide evidence for changes in ACS function in diabetes, which can be detected mainly at the G-protein level. The proposed peptide strategy is a new and perspective approach for studying molecular causes of functional violations in hormonal signaling systems arising at endocrine pathology.     

The effect of prolonged intranasal administration of serotonin on the activity of hypothalamic signaling systems in male rats with neonatal diabetes

The functioning of the serotonergic system of the brain is impaired in type II diabetes (T2D), and this leads to metabolic and hormonal dysfunction. The elevation of serotonin level in the CNS is one of the approaches for correcting of the serotonergic system of the brain. The aim of the present work was to investigate the effect of intranasal serotonin (InS) administration for 5 weeks at a daily dose of 20 μg on the metabolic parameters and functional activity of adenylate cyclase signaling system (ACSS) sensitive to peptide hormones and biogenic amines in the hypothalamus of male rats with neonatal T2D. Neonatal model of T2D was induced by injecting streptozotocin (70 mg/kg) into 5-day-old rat pups. Four-month-old animals with apparent T2D manifestations were divided into two groups: an untreated group (D0, n = 6) and a group that received InS treatment (DIS, n = 6). InS administration to diabetic rats restored ACSS regulation by the agonists of type 2 dopamine receptors (DA₂R) and type 4 melanocortin receptors (MC₄R) and enhanced the inhibitory effect of serotonin on adenylate cyclase activity. Elevated expression of genes encoding DA₂R, MC₄R, and serotonin receptor of the 1B subtype (5-HT_1BR) was among the main causes of this change. The relative activity of signaling cascades involving various types of serotonin (G_s-coupled 5-HT_4,6,7R/G_i-coupled 5-HT₁R), dopamine (DA₁R/ DA₂R), and melanocortin (MC₃R/MC₄R) receptors involved in ACSS regulation was also altered in the animals of the DIS group. InS administration restored hormonal regulation in the hypothalamus, improved glucose tolerance, and increased the sensitivity of tissues to insulin. The data obtained show that the elevation of serotonin level in the CNS is a promising approach for the recovery of hypothalamic signaling pathways in T2D and correction of the metabolic disturbances dependent on these pathways.     

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian <Emphasis Type="Italic">Dileptis anser</Emphasis>

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 μM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca²⁺ concentrations to 100 μM 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 μM Ca²⁺ with IC₅₀ values of 35, 137, and 174 M, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 μM Ca²⁺, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 μM calcium ions, but were markedly inhibited by 100 μM 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 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 depends on calcium cations and possibly is regulated by Ca²⁺/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of α-subunits for G_i-1 which were almost twice those in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the α-subunits of G_i-2 and G_i-3, and 42 and 45 kDa forms of G_s and for G-protein β-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forsckolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE₁ and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of G₁-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.     

Development of non-hormonal regulators of the adenylyl cyclase signaling system based on the peptides,derivatives of the third intracellular loop of somatostatin receptors

In most serpentine type receptors the third intracellular loop (ICL-3) is responsible for the interaction with heterotrimeric G proteins and for transduction of a hormonal signal to the enzymes, generators of the second messengers. It was found that the peptides corresponding to ICL-3 influence functional activity of hormonal signaling systems in the absence of the hormone and, consequently, may be considered as prototypes for the development of selective regulators of these systems. We have originally synthesized peptides corresponding to the C-terminal regions 255–269 and 240–254 of ICL-3 of type 1 and 2 rat somatostatin receptors (Som₁R and Som₂R). Micromolar concentrations of these peptides activated G _i proteins and inhibited forskolin-stimulated activity of adenylyl cyclase (AC) in rat brain tissues. The peptide 255–269 of Som₁R is a selective antagonist of Som₁R, and the peptide 240–254 of Som₂R is an agonist of Som₁R. The peptide 255–269 of Som₁R decreased the regulatory effects of somatostatin and the selective Som₁R agonist, CH-275, realized via the homologous receptor, while the peptide 240–254 of Som₂R, on the contrary, increased the AC inhibitory effect of CH-275. Both peptides insignificantly influenced regulatory effects of the Som₂R agonist octreotide. Thus, the peptides studied by us are selective regulators of the somatostatin-sensitive AC system. Using the peptides we have demonstrated that ICL-3 of both Som₁R and Som₂R includes the main molecular determinants that are responsible for activation of G _i proteins and regulation of the AC system by somatostatin and its analogues.     

Receptor of the serpentine-type and heterotrimeric G protein as targets of action of polylysine dendrimers

Molecular processes of the action of polycationic peptides that represent polylysine homo- and heterodendrimers on the functional activity of the biogenic amine- and peptide hormone-sensitive adenylyl cyclase signaling system (AC system) in rat myocardium and brains were studied. An intended use of these peptides is that of highly effective polymer carriers for biologically active substances. The polylysine homodendrimers of the third [(NH₂)₁₆(Lys)₈(Lys)₄(Lys)₂Lys-Ala-NH₂] (I), fourth [(NH₂)₃₂(Lys)₁₆(Lys)₈(Lys)₄(Lys)₂Lys-Ala-NH₂] (II), and fifth [(NH₂)₆₄(Lys)₃₂(Lys)₁₆(Lys)₈(Lys)₄(Lys)₂Lys-Ala-NH₂] (III) generations, as well as polylysine heterodendrimers of the fifth generation, [(NH₂)₆₄(Lys-Glu)₃₂(Lys-Glu)₁₆(Lys-Glu)₈(Lys-Glu)₄(Lys-Glu)₂Lys-Ala-Ala-Lys(ClAc)-Ala-NH₂] (IV), [(NH₂)₆₄(Lys-Ala)₃₂(Lys-Ala)₁₆(Lys-Ala)₈(Lys-Ala)₄(Lys-Ala)₂Lys-Ala-Lys(ClAc)-Ala-Ala-NH₂] (V) and [(NH₂)₆₄(Lys-Gly-Gly)₃₂(Lys-Gly-Gly)₁₆(Lys-Gly-Gly)₈(Lys-Gly-Gly)₄(Lys-Gly-Gly)₂Lys-Gly-Gly-Lys(ClAc)-Ala-Ala-NH₂] (VI), interact with the C-terminal regions of α subunits of the heterotrimeric G proteins, preferably of the inhibitor type, and stimulate its activity in respector-independent manner. The most effective G-protein activators were homodendrimers II and III and heterodendrimer V. The polylysine dendrimers disturbed the functional coupling of receptors of biogenic amines and peptide hormones with G_i proteins and, to a lesser extent, G_s proteins. This was manifested as a decrease in the regulatory effects of the hormones on AC activity and the GTP binding of the G protein, as well as by a decrease in the affinity of receptors to agonists in the presence of polylysine dendrimers, which is a consequence of the dissociation of the receptor-G protein complex. It has also been shown that, based on their molecular mechanisms and selectivity of action on G proteins, polylysine dendrimers are similar to mastoparan and melittin, which are natural toxins of insect venom.     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.