Neuronal nitric oxide synthase is refractory to mechanism-based inactivation in GH3 pituitary cells.
Nitric oxide formation by GH3 pituitary cells is stimulated by depolarizing concentrations of K+ and by the L-channel Ca2+ agonist Bay kappa 8644 in an additive manner that depends on extracellular Ca2+. Ca(2+)-dependent NO formation at 100 microM arginine was inhibited 50% over a 30-min period by 5 microM NG-amino-L-arginine, 30 microM N6-iminoethyl-L-ornithine (NIO) and 520 microM N5-iminoethyl-L-lysine (NIL) but required concentrations of aminoguanidine (AG) greater than 3 mM. As measured at 100 microM extracellular arginine, intracellular neuronal nitric oxide synthase (nNOS) was inactivated 50% over a 30-min period by 150 microM NG-amino-L-arginine and 1500 microM NIO, but required concentrations of NIL or AG greater than 5 mM. The inactivation of nNOS by these agents occurred only under conditions that mobilized extracellular Ca2+ and was inhibited by increased extracellular arginine. Thus these agents inhibit cellular Ca(2+)-dependent NO formation at concentrations far lower than those required to inactivate the cellular nNOS. Inhibition of NO formation by these agents was not attributable to effects on cellular arginine uptake. In contrast diphenyliodonium chloride produced a rapid and complete inactivation of cellular NO formation and nNOS activity. These inactivations produced by diphenyliodonium chloride occurred with identical kinetics but displayed no requirement for Ca2+. These data support the assertion that neuronal NO synthase is refractory to mechanism-based inactivation in GH3 pituitary cells.
Cooper
G R
GR
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854, USA.
Barr
A
A
Wolff
D J
DJ
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D001713:Biphenyl Compounds / Q000494:pharmacology; D002460:Cell Line; D016207:Cytokines / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D009861:Onium Compounds / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D051381:Rats
D001713:Biphenyl Compounds; D016207:Cytokines; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D009861:Onium Compounds; C031291:diphenyliodonium; D009569:Nitric Oxide; C063904:N(G)-aminoarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; C496322:Nos1 protein, rat