Cellular and enzymatic studies of N(omega)-propyl-l-arginine and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea as reversible, slowly dissociating inhibitors selective for the neuronal nitric oxide synthase isoform.
N(omega)propyl-l-arginine (NPA) and S-ethyl-N-[4-(trifluoromethyl)phenyl]isothiourea (TFMPITU) inhibit selectively the neuronal nitric oxide (NO) synthase (nNOS) isoform. In the presence of Ca(2+) and calmodulin (CaM), NPA and TFMPITU produce a time- and concentration-dependent suppression of nNOS catalyzed NO formation. This suppression of activity occurs by a first order kinetic process as revealed from linear Kitz-Wilson plots but does not depend on catalytic turnover since it occurs in the absence of NADPH. Following full suppression of NO synthetic activity by either NPA or TFMPITU, NO synthesis can be restored slowly by excess arginine or by dilution, indicating that the effects of these agents are reversible. This behavior is consistent with a dissociation of NPA and TFMPITU from nNOS slowed by a conformational transition produced by Ca(2+) CaM-binding. NPA and TFMPITU bind to nNOS rapidly producing a heme-substrate interaction as revealed by difference spectrophotometry. At physiological conditions (100 microM extracellular arginine), NPA and TFMPITU inhibit Ca(2+)-dependent NO formation by GH(3) pituitary cells with IC(50) values of 19 and 47 microM, respectively, but require millimolar concentrations to inhibit NO formation by cytokine-induced RAW 264.7 murine macrophages. The inhibition of NO formation by these agents in GH(3) cells is rapidly reversible and not due to suppression of cellular arginine uptake.
Cooper
G R
GR
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey, 08854, USA.
Mialkowski
K
K
Wolff
D J
DJ
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002956:Citrulline / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology; D016207:Cytokines; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052248:Nitric Oxide Synthase Type I; D019335:Nitroarginine / Q000494:pharmacology; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects; D051381:Rats; D013379:Substrate Specificity / Q000187:drug effects; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
D002147:Calmodulin; D016207:Cytokines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C109599:N(omega)-propylarginine; C106598:S-ethyl N-(4-(trifluoromethyl)phenyl)isothiourea; D019335:Nitroarginine; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; C496322:Nos1 protein, rat; D013890:Thiourea; C086717:S-methylthiocitrulline; D002118:Calcium