cyclic-gmp and mastoparan

Simple photoreceptors, namely photoresponsive neurons, designated as A-P-1, Es-1, Ip-2 and Ip-1, exist in the sea slug Onchidium ganglion. Previous works has shown that, of these, Ip-2 and Ip-1 respond to light with a hyperpolarizing receptor potential, caused by the opening of light-dependent, cGMP-gated K+ channels, whereas A-P-1 and Es-1 are depolarized by light, owing to the closing of the same K+ channels. The present study of Ip-2 or Ip-1 cells was undertaken to identify the G-proteins that couple light to the activation of guanylate cyclase (GC), thereby leading to the opening of K+ channels and the consequent hyperpolarizing photocurrents. The specific channel blocker, 4-aminopyridine (4-AP), and a GC inhibitor, LY-83583, both suppressed this hyperpolarizing photocurrent. N-ethylmaleimide and GDP-beta-S also inhibited this photocurrent, consistent with the involvement of G-proteins. Mastoparan an activator of both Go- and Gi-type G-proteins, induced an outward current. Furthermore, benzalkonium chloride (C(16)BAC), a selective activator of Go, dose-dependently generated an outward current similar to that induced by mastoparan. Both of these outward currents were susceptible to 4-AP, LY-83583 and N-ethylmaleimide. Taken together, these results suggest that phototransduction in Ip-2 or Ip-1 cells is triggered by a Go-type G-protein coupled to GC. Thus, this new cGMP cascade contrasts with the conventional phototransduction cGMP cascade mediated by the Gt-type G-protein coupled to phosphodiesterase, seen in the vertebrate photoreceptors and the above A-P-1 or Es-1 cells.

Topics: Animals; Cyclic GMP; Drug Interactions; Enzyme Inhibitors; Ethylmaleimide; Ganglia, Invertebrate; GTP-Binding Protein alpha Subunits, Gi-Go; Guanylate Cyclase; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Membrane Potentials; Mollusca; Patch-Clamp Techniques; Peptides; Photic Stimulation; Photoreceptor Cells; Vision, Ocular; Wasp Venoms

The rat pineal gland with its circadian noradrenaline-regulated melatonin rhythm is an excellent model for studying adrenergic signal transduction with respect to cAMP and cGMP formation. The stimulatory G(s) proteins play a well-established role in this process. In contrast, the potential roles of the inhibitory G(i) proteins, the functionally unclear other G(o) proteins, and a number of G protein subtypes are not known. The present study examines the effects on beta(1)- and beta(1)-plus-alpha(1)-stimulated cAMP and cGMP formation of a number of G protein modulators in rat pinealocyte suspension cultures. The effects of the nitric oxide donor sodium nitroprusside on cGMP were also examined. The results showed that drugs that activate G proteins of the G(i)/G(o) family, i.e., pertussis toxin, mastoparan, and compound 48/80, had no effect on unstimulated, isoproterenol (beta(1))-stimulated, or combined isoproterenol/phenylephrine (beta(1)-plus()-alpha(1))-stimulated cAMP and cGMP accumulation. However, in this experimental paradigm, the inhibitors of sulfhydryl G proteins (N-ethylmaleimide) and those of phospholipase A2-related G proteins (isotetrandrine) exerted a clear inhibitory effect. Sodium-nitroprusside-stimulated cGMP accumulation was also inhibited. These results confirm a previous report that members of the G(i)/G(o) family, which are present in the rat pineal gland, do not play a major role in adrenergic signal transduction. The new finding that sulfhydryl G proteins and phospholipase A2-associated G proteins exert a clear stimulatory effect on adrenergic signal transduction suggests that they are subtypes of G(s) proteins.

Topics: Adrenergic alpha-Agonists; Adrenergic beta-Agonists; Alkaloids; Animals; Arylamine N-Acetyltransferase; Benzylisoquinolines; Cyclic AMP; Cyclic GMP; Ethylmaleimide; GTP-Binding Proteins; Intercellular Signaling Peptides and Proteins; Isoproterenol; Male; Nitric Oxide; Nitroprusside; p-Methoxy-N-methylphenethylamine; Peptides; Pertussis Toxin; Phenylephrine; Phospholipases A; Phospholipases A2; Pineal Gland; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic; Signal Transduction; Sulfhydryl Reagents; Virulence Factors, Bordetella; Wasp Venoms

Arachidonic acid and nitric oxide (NO) act as retrograde and intercellular messengers in the nervous system. Regulation of cyclooxygenase is well established, but regulation of phospholipase A(2), the enzyme responsible for the liberation of arachidonic acid, by NO has not been thoroughly investigated. Using the PC12 cell line as a neuronal model, we studied the effects of exogenous NO compounds on arachidonic acid release. Incubation with Ca(2+) ionophores or mastoparan (wasp venom peptide) stimulated [3H]arachidonic acid release from prelabeled PC12 cells. [3H]Arachidonic acid release was inhibited by cytosolic phospholipase A(2) inhibitors, but not by dithiothreitol. A cytosolic phospholipase A(2) protein band with a molecular mass of approximately 100 kDa was detected by immunoblotting. S-Nitroso-cysteine inhibited basal and stimulated [3H]arachidonic acid release in concentration-dependent manners. Other NO compounds such as sodium nitroprusside and S-nitroso-N-acetylpenicillamine did not affect [3H]arachidonic acid release. N-Ethylmaleimide also inhibited [3H]arachidonic acid release. The inhibitory effects of S-nitroso-cysteine and N-ethylmaleimide were irreversible, because [3H]arachidonic acid release from PC12 cells preincubated with S-nitroso-cysteine or N-ethylmaleimide was much lower than that from nontreated cells. These findings suggest (a) cytosolic phospholipase A(2) is activated by Ca(2+) or mastoparan, and inhibited by S-nitroso-cysteine in a cyclic GMP-independent manner, (b) N-ethylmaleimide also inhibits cytosolic phospholipase A(2) and arachidonic acid release in PC12 cells. S-Nitroso-cysteine can regulate the production of other retrograde messenger arachidonic acid.

Topics: Animals; Arachidonic Acid; Cyclic GMP; Cysteine; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Ethylmaleimide; Intercellular Signaling Peptides and Proteins; Nitroso Compounds; PC12 Cells; Peptides; Phospholipases A; Rats; S-Nitrosothiols; Tritium; Wasp Venoms

Activation of membrane-bound guanylate cyclase GC-A by atrial natriuretic factor (ANF) may require the involvement of accessory proteins. To identify these postulated proteins, we isolated a 1. 0-kb cDNA clone from a rat brain expression library using a polyclonal antiserum against mastoparan. The 1.0-kb cDNA encodes a protein of 111 amino acids. Expression of this cDNA in COS-7 cells potentiated ANF-stimulated GC-A activity. Therefore, the 1.0-kb gene encodes a guanylate cyclase regulatory protein (GCRP). Fluorescence microscopy studies using the fusion protein of GCRP with green fluorescence protein (GFP) indicated that GCRP was present in the cytosol in PC12 cells, but translocated toward the plasma membrane in the presence of ANF. Coimmunoprecipitation experiments indicate that GCRP associates with GC-A in the presence of ANF. These results suggest that ANF induces the association of GCRP with GC-A and this association contributes to the activation of GC-A.

Topics: Amino Acid Sequence; Amino Acids; Animals; Base Sequence; Blotting, Western; Brain; Cell Membrane; CHO Cells; Cloning, Molecular; COS Cells; Cricetinae; Cyclic GMP; DNA, Complementary; Dose-Response Relationship, Drug; Enzyme Activation; Gene Library; Green Fluorescent Proteins; Guanylate Cyclase; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Luminescent Proteins; Microscopy, Fluorescence; Molecular Sequence Data; PC12 Cells; Peptides; Plasmids; Precipitin Tests; Protein Binding; Proteins; Rats; Receptors, Enterotoxin; Receptors, Guanylate Cyclase-Coupled; Receptors, Peptide; Recombinant Fusion Proteins; Tissue Distribution; Translocation, Genetic; Wasp Venoms

The cGMP-gated cation channel of rod photoreceptor cells plays a central role in the phototransduction process by controlling the influx of cations into the rod outer segment in response to changes in cGMP levels. Previous studies have shown that the cGMP-gated channel in native rod outer segment membrane vesicles is modulated by calmodulin in a calcium-dependent manner. In this study we report that the immunoaffinity-purified channel consisting of the 63-kDa alpha-subunit and a 240-kDa protein is also modulated by calmodulin when reconstituted into lipid vesicles. In the absence of calmodulin, the purified channel had an apparent Km of 33 microM and a Hill coefficient of 3.3 for cGMP-dependent efflux of Ca2+ from reconstituted lipid vesicles. In the presence of calmodulin, the Km increased to 44 microM without affecting the Hill coefficient or maximum velocity of ion efflux. Calmodulin modulation of the channel is inhibited by the calmodulin antagonist, mastoparan. In the absence of mastoparan, the half-maximum inhibition of channel activity (IC50) occurred at 1.85 +/- 0.25 nM calmodulin at a cGMP concentration of 12.5 microM; in the presence of mastoparan, the IC50 value increased to 20.3 +/- 3.8 nM calmodulin. Based on the strong, selective interaction of calmodulin with the channel, an efficient, general method has been developed to isolate functionally active cGMP-gated channels from mammalian and amphibian photoreceptor membranes. Calmodulin extraction studies, Western blotting, and channel activity measurements indicate that endogenous rod outer segment calmodulin modulates the activity of the channel through its binding to the 240-kDa protein. From these studies we conclude that the 240-kDa protein of the cGMP-gated channel is a major calmodulin target protein of rod outer segment membranes.

Topics: Animals; Calmodulin; Calmodulin-Binding Proteins; Cattle; Chromatography, Affinity; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Intercellular Signaling Peptides and Proteins; Ion Channel Gating; Ion Channels; Peptides; Rod Cell Outer Segment; Wasp Venoms

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dystrophin with a monoclonal antiphosphotyrosine antibody. Sequence analysis indicates that dystrophin contains seven potential phosphorylation sites for cyclic AMP- and cyclic GMP-dependent protein kinases (all localized in the central rod domain of the molecule) as well as several sites for protein kinase C and casein kinase II. Interestingly, potential sites of phosphorylation by protein kinase C and casein kinase II are located in the proximity of the actin-binding site. These results suggest, by analogy with what has been demonstrated in the case of other cytoskeletal proteins, that the phosphorylation of dystrophin by endogenous protein kinases may modulate both self assembly and interaction of dystrophin with other cytoskeletal proteins in vivo.

Topics: Animals; Antibodies, Monoclonal; Calcium; Calmodulin; Cyclic AMP; Cyclic GMP; Dystrophin; Heparin; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Peptides; Phosphorylation; Protein Kinases; Rabbits; Wasp Venoms

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenosine Triphosphate; Adenylyl Cyclases; Animals; Cyclic AMP; Cyclic GMP; Cytosol; Glucagon; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Triphosphate; Guanylate Cyclase; Heart Ventricles; Intercellular Signaling Peptides and Proteins; Kinetics; Myocardium; Peptides; Pertussis Toxin; Rana esculenta; Virulence Factors, Bordetella; Wasp Venoms