Inactivation of nitric oxide by rat cerebellar slices.
Nitric oxide (NO) functions as an intercellular messenger throughout the brain. For this role to be performed efficiently, there must be a mechanism for neutralizing NO, but whether an active biological process exists, or whether NO is lost mainly through diffusion is unclear. To investigate this issue, rat cerebellar slices were exposed to constant levels of NO and the cGMP generated within the slice used as an indicator of NO concentrations therein. NO was about 1000-fold less potent in slices (EC50, 1 microM) than in separated cells from the same tissue (EC50, 1.6 nM), consistent with access of NO to the slice interior being greatly hindered by inactivation. Supporting this interpretation, immunohistochemical analysis indicated a marked concentration gradient of cGMP across the thickness of slices exposed to subsaturating NO concentrations, signifying a marked NO gradient. Several known NO-degrading processes, including reaction with lipid peroxyl radicals, erythrocytes and superoxide ions, were eliminated as contributing factors, indicating a novel mechanism. A diffusion-inactivation model was used to estimate the kinetics of NO consumption by the slices. The best fits to experimental data indicated a Michaelis-Menten-type reaction having a Vmax of 1-2 microM s-1 and a Km of around 10 nM. The rates predict that inactivation would impose a very short half-life (<10 ms) on NO in physiological concentrations (up to 10 nM) and that it would play an important role in shaping the NO concentration profiles when it is synthesized by multiple nearby sites.
Hall
C N
CN
Wolfson Institute for Biomedical Research, University College London, Cruciform Building, Gower Street, London WC1E 6BT, UK. catherine.hall@ucl.ac.uk
Garthwaite
J
J
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004048:Diethylamines / Q000494:pharmacology; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D006207:Half-Life; D066298:In Vitro Techniques; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013096:Spermine / Q000031:analogs & derivatives / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors
D000975:Antioxidants; D016861:Cyclooxygenase Inhibitors; D004048:Diethylamines; D004791:Enzyme Inhibitors; D016859:Lipoxygenase Inhibitors; D020030:Nitric Oxide Donors; D013481:Superoxides; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; C087229:diethylamine dinitric oxide adduct; D019001:Nitric Oxide Synthase; D006152:Cyclic GMP