title: Kinetics, pharmacology, and autoradiographic distribution of L-[3H]nitroarginine binding sites in rat cerebellum.
abstract: The kinetics and pharmacology of NG-nitro-L-[2,3,4,5-3H]arginine (L-[3H]NOARG) binding to rat cerebellum were investigated using in vitro radioligand binding. Specific L-[3H]NOARG binding in cerebellum was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. Specific binding was Ca2+ dependent and sensitive to pH (with an optimum of 5.5-7.0). Added calmodulin (1.5-40 micrograms/ml) had no influence on specific L-[3H]NOARG binding. However, the calmodulin antagonists W-5, W-13, and calmidazolium inhibited L-[3H]-NOARG binding with IC50 values in the micromolar range, and calmodulin (10 micrograms/ml) competitively reversed this inhibition. Nitric oxide synthase (NOS) inhibitors (NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine acetate) and L-arginine displaced L-[3H]NOARG binding with IC50 values in the nanomolar range, whereas D-arginine and basic amino acids (L-lysine and L-histidine) displaced L-[3H]NOARG binding with IC50 values in the millimolar range. A comparison of the NOS functional assay with L-[3H]NOARG binding in rat cerebellum showed similar profiles of Ca2+ dependency and inhibitory kinetics. Quantitative autoradiographic distribution of L-[3H]NOARG binding sites was significantly higher in the molecular layer than in the granular layer of cerebellum. These studies confirm the potential use of L-[3H]NOARG binding to study the regional distribution and functional properties of NOS.
authors.affiliation: Neuroscience Research Unit, Hŏpital Saint-Luc (University of Montreal), Quebec, Canada.
authors.forename: V L
authors.initials: VL
authors.lastname: Rao
authors.forename: R F
authors.initials: RF
authors.lastname: Butterworth
mesh_terms: D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001345:Autoradiography; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002531:Cerebellum / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D019335:Nitroarginine; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution; D014316:Tritium
chemical_list: D002147:Calmodulin; D009419:Nerve Tissue Proteins; D014316:Tritium; D019335:Nitroarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D002118:Calcium