Effect of synthetic cationic peptides on activation of the adenylyl cyclase signaling system by biogenic amines in muscle tissue of molluscs and rats

The hormone-sensitive adenylyl cyclase signaling system (ACS), made of serpentine receptor, heterotrimeric G-protein and enzyme adenylyl cyclase (AC), regulates a wide spectrum of growth and metabolic processes in the cell. Molecular mechanisms of functional coupling of ACS components still remain obscure. We examined the influence of synthetic cationic peptides Ac-Ala-His(Ala)2-His-Ala-NH2 (I), Ac-Ala-His-(Ala)3-His-(Ala)2-His-Ala-NH2 (II), and Ac-(Pro)2-His-(Ala)2-His-(Ala)3-His-(Ala)2-His-Ala-NH2 (III) on the basal AC activity and that stimulated by nonhormonal (NaF) and hormonal reagents (serotonin--molluscs, beta-isoproterenol--rats) in smooth muscles of the freshwater bivalve molluscs Anodonta cygnea and in skeletal muscles of rats. Peptides II and III (the latter more effective) were shown to decrease hormone-stimulated AC activity in both tissues, in a dose-dependent manner. Peptide III strongly reduced NaF stimulating effect to AC, which suggests the involvement of this peptide in the functional coupling of both receptors with G-proteins, and of G-proteins with AC. A correlation was found between the efficacy of peptide action on the functional activity of ACS components and peptide length. As shown by IR-spectroscopy, in water all peptides can form helical structures. However, alpha-helicity of peptides I and II was higher than that of peptide III, which does not conform to a power series in efficacy of these peptides. Thus, it is the length of cationic peptides that plays a key role in hormonal regulation of the functional activity of ACS, especially on the step of receptor-G-protein coupling.     

Molecular mechanisms of action of dendrons, containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of the adenylyl cyclase signaling systems

For the aims of studying molecular mechanisms of functioning of adenylyl cyclase signaling systems (ACS), we investigated the influence of synthetic polycationic peptides of the star-like structure (dendrons), containing 48-60 sequence of HIV-1 TAT-protein, on the functional activity of ACS components in smooth muscles of the mollusc Anodonta cygnea and in rat skeletal muscles. It has been shown that the following peptides (Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx(= epsilon-aminohexanoic acid)-Cys(Acm), referred to as peptide I, (Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys(Acm) (peptide II), [(Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide III), and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln)2-Lys-epsilonAhx-Cys]2 (peptide IV) inhibit in a dose-dependent manner the adenylyl cyclase (AC) activity stimulated by both nonhormanal agents (GppNHp and forskolin) and hormones, such as serotonin (mollusc) and isoproterenol (rat). Peptides III and IV (tetrameric dendrons) were most effective in comparison with peptides I and II (dimeric dendrons). The AC activity stimulated by hormones and forskolin was most sensitive to the action of dendrons. All dendrons stimulated GTP-binding activity of G-proteins: dimeric dendrons were most effective at 10(-5) M concentration, whereas tetrameric dendrons at 10(-6) M. In the presence of dendrons, the affinity of beta-antagonist [3H]-dihydroalprenolol to P-adrenergic receptor in rat muscle mem- branes was unchanged. At the same time, the affinity of beta-agonist isoproterenol to the receptor decreased, and no shift to the right was observed on the curve of isoproterenol-induced [3H]-dihydroalprenolol displacement in the presence of GTP. The obtained data show the disturbance of the coupling between the receptor and G-protein, which is the main reason of dendron inhibitory action on AC stimulation by hormones. Besides, these data demonstrated that hormones could disturb the functional activity of AC, i.e. a catalytic component of ACS.     

Involvement of SH-groups in the regulation of adenylate cyclase signal system by isoproterenol and serotonin in cultured murine fibroblasts L (subline LSM)

The adenylyl cyclase system (ACS) plays a key role in transduction of a hormonal signal into eukaryotic cells. The functional activity of the system depends on SH-groups of proteins involved in the ACS: receptor, G-protein, and enzyme adenylyl cyclase (AC). We studied the influence of thiols and SH-blockers on the regulation of AC activity by nonhormonal (NaF and Gpp[NH]p) and hormonal (biogenic amines isoproterenol and serotonin) agents in homogenates of cultured murine fibroblasts of line L (subline LSM). In the presence of thiols 2-mercaptoethanol (5 mM) and dithiothreitol (1 mM) the basal AC activity somewhat increased, whereas the stimulating effects of NaF, Gpp[NH]p, and hormones decreased. No potentiating action of Gpp[NH]p on hormonal effect in this case was found. The SH-blockers 25 mkM p-chloromercuribenzoic acid (CMBA) and 0.2 mM N-ethylmaleimide significantly inhibited both the basal AC activity and that stimulated by different agents. Thiols partially restored CMBA inhibited AC activity (in the case of N-ethylmaleimide restoring effects of thiols were insignificant). This, the ACS of murine fibroblasts of subline LSM is SH-sensitive. The forms of SH-groups in proteins involved in the ACS determine their functional activities and a possibility of transduction of the hormonal signal on the effector systems.     

Effect of C-terminal peptide of G-protein alfa(s)-subunit on regulation of adenylate cyclase and protein kinase A activities by biogenic amines and glucagon in mollusc and rat muscles

The peptide synthesised by us: 387-394-amide (10(-7)-10(-4) M), in a dose dependent manner decreases activities of adenyl cyclase and proteinkinase A evoked by serotonine and glucagon in smooth muscles of the freshwater bivalve mollusc Anodonta cygnea and that evoked by beta-agonist isoproterenol--in the rat skeletal muscles. Even in concentration 10(-7) M, the peptide completely eliminates potentiation of the hormones' stimulating effect on adenylyl cyclase activity with the non-hydrolizable analogue of guanine nucleotides (Gpp[NH]p). At the same time, the peptide does not affect stimulation of the adenylyl cyclase activity with non-hormonal agents (NaF, Gpp[NH]p and forkolin). In the presence of the peptide, inhibiting effects of the hormones on activities of adenylyl cyclase and protein kinase A will be preserved. The findings reveal the importance of the G-protein alpha s-subunit's C-terminal regional of a stimulating type for its functional coupling with receptors of a serpentine type, and elucidate the molecular mechanisms of interaction between the G-protein and the receptor.     

Inhibitory action of polycationic peptides on hormonal regulation of adenylyl cyclase of the ciliate Dileptus anser

To analyse molecular mechanisms of regulatory action of different hormones on the activity of the adenylyl cyclase signaling system (ACS) of the ciliate Dileptus anser, we studied the influence on this process of six synthetic polycationic peptides and peptides, corresponding to C-terminal regions of mammalian G-protein 385-394 alphas- and 346-355 alphai2-subunits. As we reported earlier, these peptides block hormonal signal transduction in tissues of the higher eukaryotes. Now it has been found that both polycationic peptides, containing hydrophobic C to-radicals, and branched peptides decrease regulatory effects of peptide hormones (insulin, relaxin) and biogenic amines (serotonin, adrenaline) on adenylyl cyclase (AC) activity and GTP-binding. In regard to the following peptides Cys-epsilonAhx-Trp-Lys-Lys(C10)-Lys2-Lys(C10)-Lys3-Lys(C10)-Tyr-Lys-Lys(C10)-Lys-Lys-amide and [(Gly-Arg-Gly-Asp-Ser-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro- Pro-Gly)2-Lys-EAhx-Cys]2 (epsilonAhx - E-aminocaproyl, C10 - caprinoyl group) their dose-dependent inhibitory action is shown. In cell culture of D. anser with a lower basal AC activity, both hydrophobic and branched peptides stimulated AC and GTP-binding without hormones. The data give evidence that these peptides can activate ACS of ciliates in a receptor-independent manner. No influence of peptides 385-394 alphas and 346-355 alphai2 on hormonal signal transduction in D. anser was observed, due, presumably, to some structural differences of G-proteins of the lower and higher eukaryotes. A conclusion was made about an important role of polycationic regions for functional coupling of hormone-activated receptor and G-proteins in the ciliate D. anser.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

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

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

The hormone-sensitive adenylyl cyclase system of the ciliate Dileptus anser

The hormone-sensitive adenylyl cyclase system (AC system) was found and characterized for unicellular eukaryotes--the ciliatae Dileptus anser. It has been first shown that hormones of higher eukaryotes--biogenic amines (adrenalin, isoproterenol and serotonin) and peptide glucagon--stimulate in dose-dependent manner the activity of adenylyl cyclase (AC) of D. anser. The enzymatic activity was stimulated also by guanine nucleotides--GTP and their non-hydrolysable analogue Gpp[NH]p. Stimulating effects of hormones and guanine nucleotides strongly depend on the level of AC basal activity, which is relatively easy to reach (1430 to 3900 pmol cAMP/min per 1 mg of protein). The sensitivity of D. anser AC system to hormones and guanine nucleotides shows the presence of receptor or receptor-related molecules, capable of interacting with the hormone and activating AC through heterotrimeric G-proteins, in ciliatae. On the base of obtained data, a conclusion is made about the similarity of the structural-functional organization of AC systems of D. anser and higher eukaryotes.     

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.     

Role of cysteine sulfhydryl groups in activation of adenylyl cyclase signaling system by biogenic amines in molluscan and rat tissues

It has been shown that in smooth muscles of the freshwater bivalve molluscAnodonta cygnea as well as in skeletal muscles and brain striatum of rats a blocker of SH-groups,para-chlormercury benzoate (ChMB), and an alkylating agent,N-ethylmaleimide, inhibit both the basal adenylyl cyclase (AC) activity and the activity of the enzyme stimulated by non-hormonal agents (NaF, Gpp[NH]p) and by hormonal agents such as serotonin (mollusc muscles, rat brain) or isoproterenol (rat muscles and rat brain). The inhibitory effects of ChMB andN-ethylmaleimide on AC are partly eliminated by an SH-group containing reagent, β-mercaptoethanol (ME, 5 mM). Restoration of the basal and of the stimulated enzyme activity inhibited by ME is better in the case of the ChMB than of theN-ethylmaleimide action. It has also been found that ME stimulates both the basal and the stimulated by non-hormonal agents AC activity. In the presence of ME the hormonal stimulating effects on the enzyme are also preserved, except for the effect of isoproterenol on AC in rat skeletal muscles; this effect is inhibited by ME. Potentiation of the stimulating effect of the hormones on AC by Gpp[NH]p is only preserved in the molluscan smooth muscles (the effect of serotonin—90%). The data obtained indicate that cysteine sulfhydryl groups play a key role in hormonal regulation of the functional activity of the components of the hormone-sensitive adenylyl cyclase signaling system.     

Regulatory Action of Synthetic Cationic Peptides Containing Residues of Glutamic Acid on Functional Activity of Components of the Adenylyl Cyclase Signal System

One of the most important stages of hormonal signal transduction in cells through the hormone-sensitive adenylyl cyclase signal system (ACS) is functional coupling of receptor of the serpentine type to heterotrimeric GTP-binding protein (G-protein). The main role in realization of such coupling is played by spiralized regions of the receptor cytoplasmic loops proximal in relation to membrane, most of them carrying positive charge. To study molecular mechanisms of interaction of the receptor with G-protein, we compared effects of synthetic cationic peptides containing residues of glutamic acid on the process of regulation of ACS by hormones (biogenic amines) and non-hormonal agents in smooth muscles of the freshwater bivalve mollusc Anodonta cygnea and skeletal muscles of rat. All peptides had the clearly expressed ability to form -helices. Peptides H-(Leu-His-Glu-Lys)₄-Leu-NH₂ (I), H-(Leu-His-Glu-Lys)₃-Lys-His-Glu-Lys-Leu-NH₂ (II), H-(Leu-Lys-Glu-Lys)₄-Leu-NH₂ (III), and H-(Ile-His-Glu-Lys)₄-Ala-NH₂ (IV) at concentrations of 10^–6–10^–3 M reduced dose-dependently the value of stimulating effects of serotonin (in mollusc muscles) and isoproterenol (in rat muscles) on the adenylyl cyclase (AC) and protein kinase A (PKA) activities. Values of concentration of these peptide causing a 50% decrease of the hormone-stimulating effect (IC₅₀) vary from 150 to 750 µM. According to the degree of this inhibitory action on stimulating effects of hormones, they may be arranged in the following series: III II > IV I. The peptides I–IV were more effective than the peptide H-(Glu-Lys)₈-Ala-NH₂ (V) with the charge close to zero, but much less effective than the studied earlier cationic peptides containing only positively charged amino acid residues. The inhibitory effect of the peptides I-IV on stimulation of AC by non-hormonal agents, NaF, Gpp[NH]p, and forskolin, was essentially less pronounced and was marked only at 10^–4–10^–3 M concentrations. Thus, the inclusion of negatively charged amino acid residues in the primary structure of polycationic peptides leads to a decrease in their ability to inhibit hormonal stimulation of AC and PKA, which indicates importance both of the total positive charge of peptides and of distribution of the charged amino acids in the formed helices for realization of the uncoupling action on the ACS components—the receptor and G-protein.     

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).     

The role of sulfhydryl groups in functioning of components of adenylate cyclase signal system in smooth muscles of the mollusk Anodonta cygnea (effect of N-ethylmaleimide and p-chloromercuribenzoic acid)]

The alkylating agent N-ethylameimide and the sulfhydryl group blocker p-chloromercuribenzoic acid (CPMA) inhibited in dose-dependent manner both basal activity of adenylyl cyclase (AC) and its activity stimulated by non-hormonal substances (forskolin, sodium fluoride, guanylilimidodiphosphate) in smooth muscles of the freshwater bivalve mollusk Anodonta cygnea. The double increase (from 30 to 60 min) in the time of preincubation of a sarcolemmal membrane fraction with ethylmaleimide and CPMA led to an essential increase in enzyme inhibition (especially for CPMA). 50 mM SH-containing reagent beta-mercaptoethanol (ME) partially restored the AC activity, inhibited by N-ethylmaleimide and CPMA, except when these two latter reagents were in high concentrations (1-10 and 0.5 mM, respectively). The data obtained point to the key role of cysteine SH-groups in regulation of the functional activity of proteins, components of the adenylyl cyclase system--AC and heterotrimeric G-proteins.     

Adenylyl cyclase regulates signal onset via the inhibitory GTP-binding protein, Gi

Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, G(s) and G(i), respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Galpha(s) and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric G(s) (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328-1331). This latter action of ACV would facilitate the rapid onset of signaling via G(s). Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Galpha(i), we investigated whether the receptor-mediated activation of heterotrimeric G(i) was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric G(i) such that the amount of peptide required to stimulate G(i) in steady-state GTPase activity assays was 3-4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) to G(i) was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPgammaS binding to G(i) in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc(-) cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous G(i) and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of G(i).     

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.     

Adenylyl cyclase stimulation by VIP in rat seminal vesicle membranes.

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in membranes from rat seminal vesicle. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 microM. The stimulation of adenylyl cyclase by VIP was time and temperature dependent. The response was linear with time up to 15 min at 30 degrees C. Half-maximal adenylyl cyclase activation (in the presence of 10 microM GTP) was achieved at 3.0 nM VIP. The enzyme activity increased about 150% with respect to basal values at the maximal VIP concentration tested (1 microM). The relative potency of peptides upon stimulation of adenylyl cyclase activity was: VIP greater than helodermin greater than peptide histidine isoleucinamide greater than rat growth hormone-releasing factor. Other agents like GTP (0.1 mM), GppNHp (0.1 mM), forskolin (0.1 mM) and sodium fluoride (10 mM) increased the adenylyl cyclase activity 1.8-, 4.4-, 6.7- and 2.4-fold, respectively. Taken together, the presence of VIP in nerve terminals innervating the seminal vesicle of rats and the existence of VIP receptors coupled to adenylyl cyclase strongly suggest a physiological role for this neuropeptide in the modulation of seminal vesicle cell function.     

Inhibition of functional activity of the adenylyl cyclase signaling system of the ciliate Dileptus anser by colchicine and vinblastine

Cytoskeleton plays a key role in the functioning of hormonal signaling systems in vertebrate animals. However, data on the effect of cytoskeletal components, in particular microtubules, on the functional activity of chemosignaling systems of unicellular organisms are currently lacking. The goal of this work consisted of studying the effects of microtubule-disrupting agents, colchicine and vinblastine, on the adenylyl cyclase system of free living infusoria Dileptus anser. The incubation of D. anser with colchicine and vinblastine (10^?5–10^?6 M) weakly affected the basal activity of adenylyl cyclase (AC), but led to a significant decrease in or complete block of AC stimulation with nonhormonal (GppNHp, sodium fluoride) and hormonal agents (adrenaline, serotonin, glucagon). The basal level of GTP binding in heterotrimeric G proteins decreased and there was observed inhibition of stimulation of G proteins by hormones. Colchicine and vinblastine have been shown to interrupt adrenalin-produced AC stimulation achieved through G_s-protein, but weakly affect its inhibiting AC effect caused by the G_i-protein. Thus, it has been established for the first time that, in unicellular organisms, i.e., infusoria D. anser, microtubules are involved in the regulation of the functional activity of the AC system and their action is realized at the level of G proteins, which is similar to Gs-proteins in vertebrate animals.     

Effect of SH-Specific Reagent 5,5′-Dithobis(2-Nitrobenzoic Acid) on Functional Activity of Components of Adenylyl Cyclase Signal System

Sensitivity of adenylyl cyclase signal system to 5,5′-dithobis(2-nitrobenzoic acid) (DTNB) oxidizing SH-groups of cystein residues to disulfide bonds was studied. It was shown that treatment of plasma membranes fractions of smooth muscles of the mollusc Anodonta cygnea and of rat skeletal muscles as well as of homogenate of mouse fibroblasts culture of L strain with micromole concentrations of DTNB led to a decrease of activity of adenylyl cyclase (AC) stimulated by GIDP, sodium fluoride, and, to a lesser degree, forskolin. Dithiothreitol (DTT) partly restored the stimulating effects of GIDP, NaF, and forskolin, the effect of this dithiol being dose-dependent. AC stimulated by biogenic amines—serotonin in mollusc muscles, isoproterenol in rat muscles, and both hormones in mouse fibroblasts—is more sensitive to DTNB than the enzyme stimulated by non-hormonal agents. Thus, the stimulatory effects of hormones decreased dose-dependently in the presence of 10–100 μM DTNB and were almost completely blocked by 250 μM reagent. These effects were partly restored in the presence of 5 mM DTT. The obtained data indicate a high sensitivity of the hormone-stimulated AC to action of the reagents interacting specifically with SH-groups of the proteins components of the AC system. In the rat muscle membranes treated with 25 μM DTNB, no significant rightward shift was observed of the curve of competitive replacement of the β-adrenergic receptor antagonist [³H]-dihydroalprenolol by the β-agonist isoproterenol in the presence of GTP and the affinity of the agonist to the receptor somewhat decreased, which indicates a disturbance of functional coupling of the β-adrenergic receptor with G-protein after treatment with DTNB.     

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.     

Receptor and tissue specificity of the effects of peptides corresponding to intracellular regions of the serpentine type receptors

Proximal regions of the third intracellular loop (ICL-3) are responsible for the interaction with heterotrimeric G proteins in most of the serpentine type receptors. The peptides corresponding to these regions are able to activate G proteins in the absence of hormone and to alter the transduction of hormonal signal via the respective homologous receptor. However, the molecular mechanisms of action of the peptides, their specificity to receptors and target tissues are currently not well understood. The goal of this work was to study the receptor and tissue specificity of peptides-derivatives of C-terminal regions of the ICL-3 of luteinizing hormone receptor (LHR), type 1 relaxin receptor (RXFP1), somatostatin receptors of types 1 and 2 (Som₁R and Som₂R), and 5-hydroxytryptamine receptors of subtype 1B and type 6 (5-HT_1BR and 5-HT₆R) on the functional activity of adenylyl cyclase (AC) and GppNHp-binding of G proteins in the brain, myocardium, and testis of rats. It was shown that the influence of peptides on AC and G proteins is well detected in tissues enriched in homologous receptors. The effects stimulating AC and GppNHp-binding were most pronounced in the testes for LHR peptide, in the brain for peptide 5-HT₆R, and in all of the tested tissues (but mainly in the myocardium) for the RXFP1 peptide. The AC-inhibiting effects of peptides Som₁R, Som₂R and 5-HT_1BR, as well as the stimulation of GppNHp binding induced by these peptides, were most pronounced in the brain. In the presence of the peptides, the AC effects of hormones acting via homologous receptors were significantly attenuated, while the AC effects of other hormones changed insignificantly. The findings suggest that biological activity of the peptides depends on their interaction with complementary regions of homologous receptors, which should be taken into account when developing highly selective regulators of hormonal signaling systems on the basis of these peptides.