The study of molecular mechanisms of action of natural amino acids and serotonin on adenylyl and guanylyl cyclases of the ciliates

It has been previously shown that some amino acids and their derivatives are capable of regulating the activity of adenylyl cyclase (AC) and guanylate cyclase (GC) in free-living ciliates Dileptus anser and Tetrahymena. The aim of this work was to study the molecular mechanisms of action of methionine, tyrosine, alanine and neurohormone serotonin on the activity of enzymes-cyclases and the identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both ciliates, and the AC effect of serotonin in T. pyriformis was carried out with the participation of Ca2+-dependent form of AC and heterotrimetic G proteins. AC stimulating effect of tyrosine and alanine was expressed weakly and only detected in D. anser. Serotonin is both ciliates and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine had no effect on GC. Methionine and serotonin bind to surface receptors of the ciliates with high affinity. K(D) for [methyl-3H] methionine binding to D. anser and T. pyriformis were 7.5 and 35.6 nM, and for [3H] serotonin binding were 2.7 and 4.7 nM, respectively. Alanine and tyrosine bind to the ciliates with low affinity. Thus, ciliates D. anser and T. pyriformis have chemosignaling systems regulated by amino acids and their derivatives and including the enzymes with cyclase activity. There is an assumption that these systems are similar to hormonal signaling systems of higher eukaryotes and are their predecessors.     

The adenylyl and guanylyl cyclase superfamily

New structures solved in 1997 revealed that the adenylyl cyclase core consists of a pair of catalytic domains arranged in a wreath. Homologous catalytic domains are arranged in diverse adenylyl and guanylyl cyclases as symmetric homodimers or pseudosymmetric heterodimers. The kinship of the adenylyl and guanylyl cyclases has been confirmed by the structure-based interconversion of their nucleotide specificities. Catalysis is activated when two metal-binding aspartate residues on one domain are juxtaposed with a key aspargine—arginine pair on the other. Allosteric activators of mammalian adenylyl cyclase, forskolin and the stimulatory G protein α subunit, promote the catalytically optimal juxtaposition of the two domains.     

Conversion of a guanylyl cyclase to an adenylyl cyclase

Guanylyl cyclases catalyze the formation of cGMP from GTP, but display extensive identity at the catalytic domain primary amino acid level with the adenylyl cyclases. The recent solving of the crystal structures of soluble forms of adenylyl cyclase has resulted in predictions of those amino acids important for substrate specificity. Modeling of a membrane-bound homodimeric guanylyl cyclase predicted the comparable amino acids that would interact with the guanine ring. Based on these structural data, the replacement of three key residues in the heterodimeric form of soluble guanylyl cyclase has led to a complete conversion in substrate specificity. Furthermore, the mutant enzyme remained fully sensitive to sodium nitroprusside, a nitric oxide donor.     

Regulatory properties of adenylyl and guanylyl cyclase in human spermatozoa

Completion of maturation of spermatozoa (capacitation) occurs in the female genital tract. As a result, spermatozoa acquire the high motility and the capability for acrosomal reaction, which determines their fertility. There are evidences that adenylyl cyclase and guanylyl cyclase signaling systems detected in human and mammalian spermatozoa are involved in these processes. The goal of the present study was characterization of these systems in human ejaculate spermatozoa (ES) and in human fertile spermatozoa (FS) isolated by a density gradient centrifugation. In FS homogenate the basal activity of the adenylyl cyclase (AC) was significantly higher as compared with ES (47 ± 5 vs. 28 ± 3 pmol cAMP/min per mg of protein). At the same time, the AC stimulatory effects of non-hormonal activators of soluble and membrane-bound forms of AC (NaHCO₃, Mn²⁺, forskolin, and non-hydrolyzable GTP analogue—GppNHp) in FS were lower as compared with ES. Isoproterenol, serotonin, PACAP-38, and, to the lesser extent, noradrenalin and adenosine stimulated the AC activity in ES. Among hormones inhibiting AC, only adenosine decreased the enzyme activity. At the same time, in FS the inhibitory AC effects of adenosine, noradrenalin, and serotonin were markedly expressed, and the stimulatory effects of these hormones were decreased or absent. The basal activity of guanylyl cyclase (GC) in ES and FS homogenates was 27 ± 3 and 21 ± 2 pmol cGMP for 1 min per 1 mg protein, respectively, and was significantly increased in the presence of 10 mM Mn²⁺. The stimulatory GC effects of natriuretic peptides—ANP and CNP, activators of receptor forms of GC, was significantly higher in ES than in FS, and the effect of ANP was more pronounced as compared with CNP. The data indicate the multiplicity of cAMP- and cGMP-dependent signaling cascades regulating fertility of human spermatozoa. We found that the sensitivity of AC and GC to hormones in the common pool of ES and in the fraction of highly motile FS isolated by centrifugation was essentially different, which is to be considered when using FS for accessory reproductive technology.     

The Dictyostelium homologue of mammalian soluble adenylyl cyclase encodes a guanylyl cyclase

A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.     

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptus anser

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca2+ was studied and it was found that calcium cations at concentrations of 0.5-10 microM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 microM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca2+. Calmodulin antagonists--chlorpromazine, W-7, and W-5--caused a dose-dependent decrease of the enzyme activity stimulated by 5 microM Ca2+ with IC50 values of 35, 137, and 174 microM, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 microM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 microM calcium ions, but were markedly inhibited by 100 microM Ca2+. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca2+, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or were restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity does not D. depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

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.     

Inhibition of adenylyl and guanylyl cyclase isoforms by the antiviral drug foscarnet

The pyrophosphate (PP(i)) analog foscarnet inhibits viral DNA-polymerases and is used to treat cytomegalovirus and human immunodeficiency vius infections. Nucleotide cyclases and DNA-polymerases catalyze analogous reactions, i.e. a phosphodiester bond formation, and have similar topologies in their active sites. Inhibition by foscarnet of adenylyl cyclase isoforms was therefore tested with (i) purified catalytic domains C1 and C2 of types I and VII (IC1 and VIIC1) and of type II (IIC2) and (ii) membrane-bound holoenzymes (from mammalian tissues and types I, II, and V heterologously expressed in Sf9 cell membranes). Foscarnet was more potent than PP(i) in suppressing forskolin-stimulated catalysis by both, IC1/IIC2 and VIIC1/IIC2. Stimulation of VIIC1/IIC2 by Galpha(s) relieved the inhibition by foscarnet but not that by PP(i). The IC(50) of foscarnet on membrane-bound adenylyl cyclases also depended on their mode of regulation. These findings predict that receptor-dependent cAMP formation is sensitive to inhibition by foscarnet in some, but not all, cells. This was verified with two cell lines; foscarnet blocked cAMP accumulation after A(2A)-adenosine receptor stimulation in PC12 but not in HEK-A(2A) cells. Foscarnet also inhibited soluble and, to a lesser extent, particulate guanylyl cylase. Thus, foscarnet interferes with the generation of cyclic nucleotides, an effect which may give rise to clinical side effects. The extent of inhibition varies with the enzyme isoform and with the regulatory input.     

GTPgammaS regulation of a 12-transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) triphosphate (GTPgammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPgammaS stimulated the adenylyl cyclase activity of DdGCA(kqd) with properties similar to those of the wild-type enzyme (decrease of K(m) and increase of V(max)), demonstrating that GTPgammaS stimulation is independent of substrate specificity. Furthermore, GTPgammaS activation of DdGCA(kqd) is retained in several null mutants of Galpha and Gbeta proteins, indicating that GTPgammaS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.     

Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.     

Molecular mechanisms of regulatory action of adrenergic receptor agonists on functional activity of adenylyl cyclase signaling system of the ciliates Dileptus anser and Tetrahymena pyriformis

Adenylyl cyclase signaling system (ACS) of the higher eukaryotes involves the following main components: receptor, heterotrimeric G protein, adenylyl cyclase (AC), and protein kinase A. At present, these components have been found in cells of different species of the lower eukaryotes. Hence, the signal transduction through ACS of unicellular eukaryotes may have some features in common with those of the higher eukaryotes. We showed earlier that agonists of adrenergic receptors (ARs) regulate AC activity of ciliates Dileptus anser and Tetrahymena pyriformis. The aim of this work was to study molecular mechanisms of AR ligand action on the functional activity of different components of ACS of the ciliates. It has been shown that beta-AR antagonist [3H]-dihydroalprenolol binds membranes of the ciliates with a comparatively lower affinity than those of the higher eukaryotes (Kd for D. anser was 13.4 nM, for T. pyriformis--27 nM). Beta-AR ligands--agonist (-)-isoproterenol and antagonists propranolol and atenolol in competition manner displace [3H]-dihydroalprenolol with IC50 that are 10-100 times higher than corresponding IC50 of beta-AR of the higher eukaryotes. In the presence of GTP, the right shift of competition curves of [3H]-dihydroalprenolol displacement by isoproterenol was obtained, being most considerable in the case of D. anser. Adrenaline and isoproterenol in a dose-dependent manner stimulated GTP-binding in cell cultures of D. anser and T. pyriformis. Suramin (10(-5) M), the inhibitor of heterotrimeric G proteins, completely blocked effects of these hormones. In D. anser culture, adrenaline and isoproterenol in a dose-dependent manner, stimulated AC activity, and its stimulating effects in the presence of beta-AR blockers vanished (propranolol) or decreased to a great extent (atenolol). At the same time the effects were unchanged in the presence of alpha2-AR antagonists yohimbine and idazoxan. These data show the involvement of G protein-coupled beta-AR in signal transduction induced by AR agonists in D. anser cells. In cell culture of T. pyriformis isoproterenol weakly stimulated AC activity, and its effect was completely blocked by beta-AR blockers. Adrenaline in T. pyriformis cells in a dose-dependent manner inhibited AC activity. Inhibiting effect of hormone was decreased in the presence of alpha2-AR blockers. On the basis of the obtained data we concluded that adrenaline in T. pyriformis cells inhibited AC activity through G protein-coupled receptor, being close to alpha2-AR of vertebrate animals.     

CyaG, a novel cyanobacterial adenylyl cyclase and a possible ancestor of mammalian guanylyl cyclases

A novel gene encoding an adenylyl cyclase, designated cyaG, was identified in the filamentous cyanobacterium Spirulina platensis. The predicted amino acid sequence of the C-terminal region of cyaG was similar to the catalytic domains of Class III adenylyl and guanylyl cyclases. The N-terminal region next to the catalytic domain of CyaG was similar to the dimerization domain, which is highly conserved among guanylyl cyclases. As a whole, CyaG is more closely related to guanylyl cyclases than to adenylyl cyclases in its primary structure. The catalytic domain of CyaG was expressed in Escherichia coli and partially purified. CyaG showed adenylyl cyclase (but not guanylyl cyclase) activity. By site-directed mutagenesis of three amino acid residues (Lys(533), Ile(603), and Asp(605)) within the purine ring recognition site of CyaG to Glu, Arg, and Cys, respectively, CyaG was transformed to a guanylyl cyclase that produced cGMP instead of cAMP. Thus having properties of both cyclases, CyaG may therefore represent a critical position in the evolution of Class III adenylyl and guanylyl cyclases.     

Effects of guanosine on insulin secretion and adenylyl and guanylyl cyclase activities of isolated rat islets of Langerhans

The effect of guanosine on insulin secretion adenylyl and guanylyl cyclase activities of isolated rat islets of Langerhans was investigated. Guanosine (1–100 μM) inhibited glucose, tolbutamide, theophylline and prostaglandin E₂-stimulated insulin secretion although it failed to affect glucagon stimulated secretion. Prostaglandin E₂-stimulated adenylyl cyclase of islets was inhibited by guanosine although guanosine had no effect on basal, fluoride, glucagon or GTP-stimulated activity. Guanosine markedly decreased basal guanylyl cyclase activity of islets.These results suggest that guanosine may affect insulin release by inhibiting adenylyl and guanylyl cyclase activities in the ß-cell thereby decreasing the intracellular concentrations of cyclic nucleotides.This effect may be important in modulating the secretory response of the islets to a variety of hormonal agents.     

Effect of natural amino acids and sugars on cyclase activities in infusoria Tetrahymena pyriformis and Dileptus anser

Natural amino acids and sugars in intracellular eukaryotes are known to regulate adenylyl cyclase (AC) and guanylyl cyclase (GC) systems that control the most important cell processes. The goal of the present work consisted in study of effects of natural amino acids and sugars and some of their derivatives on AC and GC activities of infusoria Tetrahymena pyriformis and Dileptus anser. Methionine, arginine, lysine, and tryptamine stimulated basic AC activity of T. pyriformis, whereas alanine, thyrosine, and cysteine decreased it. Methionine, glycine, alanine, thyrosine, arginine, and to the lesser degree tryptamine and histidine stimulated AC of D. anser. The GC activity of T. pyriformis are increased in the presence of tryptamine, tryptophane, histidine, arginine, and lysine, whereas glycine and aspartic acid, on the contrary, decreased it. Tryptamine, tryptophan, leucine, glutamic acid, serine, histidine, and alanine stimulated the GC activity of D. anser. Glucose, fructose, and sucrose stimulated the basal AC activity of both infusorians and GC of T. pyriformis, with glucose and sucrose increasing AC of T. pyriformis twice, while that of D. anser 4.5 times. Lactose stimulated AC and GC of T. pyriformis and was inefficient with respect to the D. anser cyclases, whereas mannose and galactose did not affect the enzyme activities in both infusorians. The study of the chemotactic response of infusorians to amino acids and sugars indicates that involved in realization of this response can be signaling pathways both dependent on and independent of cyclic nucleotides. Thus, it has been established for the first time that several amino acids and sugars affect functional activity of enzymes with cyclase activity of the infusorians T. pyriformis and D. anser. This confirms the hypothesis that at early stages of evolution the large spectrum of comparatively simple natural molecules has a hormone-like action.     

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.     

Guinea-pig lung adenylyl and guanylyl cyclase and PDE activities associated with airway hyper- and hypo-reactivity following LPS inhalation

The relationships between changes in in vivo airway reactivity and levels cyclicAMP and cyclicGMP were determined in guinea-pig lungs after exposure to inhaled lipopolysaccharide (LPS). After LPS (30 microg.ml(-1), 1 h), guinea-pigs displayed in vivo airway hyperreactivity (AHR) at 1 h and hyporeactivity (AHOR) at 48 h, to inhaled (20 s) histamine (1 or 3 mM, respectively). Isoprenaline-stimulated cAMP or SNAP-stimulated cGMP were determined in the lungs isolated from guinea-pigs exposed to LPS inhalation to determine whether there was a relationship between AHR or AHOR and adenylyl/guanylyl cyclase and phosphodiesterase (PDE) activities. Assays were performed in the absence and presence of the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Levels of cAMP and cGMP in its presence indicated adenylyl and guanylyl cyclase activities, respectively. The difference between cAMP and cGMP levels, in the absence and presence of IBMX, reflected relevant PDE activity. In vivo AHR was associated with increased PDE activity towards cAMP and cGMP (67 and 278%, respectively) and also increased adenylyl (47%) and guanylyl (210%) cyclase activities. In vivo AHOR at 48 h after LPS inhalation was also associated with raised cyclase activity (p < 0.05), whereas relevant PDE activity declined by 79 and 68%, compared with 48 h after vehicle. Although net stimulated cGMP levels increased during AHR and AHOR and net stimulated cAMP increased during AHOR, our index of PDE activity increased during AHR and decreased during AHOR. These results therefore support the rationale for the use of PDE-inhibitors in the treatment of respiratory diseases associated with AHR.