guanosine-triphosphate and calmidazolium

1. We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the beta-adrenergic stimulated L-type calcium current (ICa-L) in rabbit pacemaker (sino-atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO-mediated cholinergic response. 2. Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3. The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane-permeable chelator BAPTA-AM (10 microM) significantly reduced (and in some cases abolished) the attenuation of ICa-L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh-induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 microM) also abolished the attenuation of ICa-L by CCh, with no change in the activation of IK,ACh. 4. Neither thapsigargin nor ryanodine (5-10 microM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa-L by CCh. 5. Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa-L and the activation of IK,ACh. This establishes that a PTX-sensitive GTP-binding protein links the muscarinic receptor to NO synthase activation in SAN cells. 6. Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)-activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa-L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), blocks the effect of CCh on ICa-L, but not on IK,ACh. Using reverse transcriptase-polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Base Sequence; Biological Clocks; Calcium; Calmodulin; Chelating Agents; Cholecystokinin; Cholinergic Fibers; Cloning, Molecular; Egtazic Acid; Electrophysiology; Enzyme Inhibitors; Guanosine Triphosphate; Imidazoles; Isoenzymes; Molecular Sequence Data; Nitric Oxide; Nitric Oxide Synthase; Pertussis Toxin; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Polymerase Chain Reaction; Protein Kinase C; Rabbits; Receptors, Muscarinic; Sinoatrial Node; Sulfonamides; Thapsigargin; Vasodilator Agents; Virulence Factors, Bordetella

Plasma membranes isolated from dispersed gastric muscle cells exhibited calmodulin-dependent NOS activity that was stimulated by Ca2+ in the range 0.1-1 mM (maximum 10 microM). Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) (in the presence of GTP), and GTP gamma S (guanosine 5'-O-(gamma-thio)triphosphate) stimulated NOS activity in a concentration-dependent fashion above that maximally stimulated by Ca2+. The increase in NOS activity induced by VIP, PACAP, and GTP gamma S was abolished by GDP beta S (guanosine 5'-O-(beta-thio)diphosphate), which had no effect on NOS activity stimulated by Ca2+. The NOS inhibitor NG-nitro-L-arginine and the calmodulin antagonist calmidazolium abolished NOS activity stimulated by all agents including Ca2+. NOS activity stimulated by GTP gamma S, VIP, and PACAP was inhibited by Gi alpha 1-2 antibody but not by Gq alpha, Gs alpha, and Gi alpha 3 antibodies. NOS activity stimulated by VIP and PACAP was inhibited by 80-83% in membranes derived from pertussis toxin-treated cells. We conclude that a Ca2+/calmodulin-dependent NOS present in plasma membranes of gastric muscle cells is activated by two homologous peptide transmitters, VIP and PACAP, via a common receptor coupled to pertussis toxin (PTx)-sensitive Gi1-2. The study provides the first evidence of receptor-mediated G protein activation of NOS in smooth muscle cells.

Topics: Adenylate Cyclase Toxin; Amino Acid Oxidoreductases; Animals; Calcium; Calmodulin; Cell Membrane; Cell Separation; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Imidazoles; Membrane Proteins; Muscle, Smooth; NADP; Neuropeptides; Nitric Oxide Synthase; Pertussis Toxin; Pituitary Adenylate Cyclase-Activating Polypeptide; Rabbits; Stomach; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

The Ca2+/calmodulin (CaM) dependence of adenylate cyclase activity in Manduca sexta prothoracic glands was investigated. Membrane fractions from two developmental stages were used, day 3 of the last larval instar and day 0 of the pupal stage, both of which respond to the neuropeptide prothoracicotropic hormone (PTTH) with increased cAMP production dependent on extracellular Ca2+. The data revealed that both larval and pupal prothoracic gland membrane fractions have a Ca2+/CaM-dependent adenylate cyclase which is inhibited by CaM antagonists and EGTA. The larval adenylate cyclase shows a multiphasic response to Ca2+/CaM, with a 2-fold stimulation between 0.02 and 0.01 microM, a further increase in adenylate cyclase activity at concentrations greater than 2 microM and a potentiation of NaF-stimulated activity at doses greater than 0.1 microM Ca2+/CaM. Pupal prothoracic gland membrane fractions exhibit only the second phase of stimulation. Stimulation by the GTP analogs GTP-gamma-S and Gpp(NH)p is dependent on CaM in larval, but not in pupal membrane fractions, suggesting a role for CaM in Gs protein-mediated regulation of adenylate cyclase. However, adenylate cyclase activity in glands from both stages is dependent on CaM, supporting our initial premise that Ca2+ is required for cAMP synthesis in the prothoracic glands.

Topics: Adenylyl Cyclases; Animals; Calcium; Calcium Channels; Calmodulin; Cyclic AMP; Guanosine Triphosphate; Imidazoles; Insect Hormones; Insecta; Thorax; Trifluoperazine