Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways.
We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a messenger in the molluscan CNS, and that cGMP acts as one of its effectors.
Bodnárová
Michaela
M
Whitney Laboratory for Marine Bioscience, University of Florida, 9505 Ocean Shore Blvd., St. Augustine, FL 32080, USA.
Martásek
Pavel
P
Moroz
Leonid L
LL
D000818:Animals; D001048:Aplysia / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002490:Central Nervous System / Q000201:enzymology*; D002956:Citrulline / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D020033:Protein Isoforms; D051381:Rats
D002147:Calmodulin; D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D010726:Phosphodiesterase Inhibitors; D020033:Protein Isoforms; D002956:Citrulline; D009249:NADP; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium