A rapid and transient synthesis of nitric oxide (NO) by a constitutively expressed type II NO synthase in the guinea-pig suprachiasmatic nucleus.
1. We have measured extracellular NO/NO(2)(-) concentrations in guinea-pig suprachiasmatic nucleus (SCN) brain slices using fast cyclic voltammetry. A rapid and transient signal equivalent to 2.2+/-0.2 microM NO/NO(2)(-) (mean+/-s.e.mean, n=13) was detected at 1.26 V, the peak oxidation potential for NO, following local electrical stimulation (five pulses of 0.1 ms duration at 100 Hz, delivered every 5 min). 2. The NO/NO(2)(-) signal was inhibited by the non-selective nitric oxide synthase (NOS) inhibitors L-NAME, L-NMMA and the highly selective type II NOS (iNOS) inhibitor 1400 W (Garvey et al., 1997) in a concentration-dependent manner. IC(50) values were 229 microM (65 - 801, n=3, geomean and 95% confidence intervals (C.I.)), 452 nM (88 - 2310, n=5), and 14.2 microM (3.6 - 54.4, n=5), with maximum inhibitions of 82.8+/-6.7, 46.0+/-8.1, and 90.6+/-3.6%, respectively. 3. Exposure of the slices to the protein synthesis inhibitor cyclohexamide or the inhibitor of type II NOS induction dexamethasone immediately following slice cutting, and for a subsequent 4 - 5 h, did not inhibit the NO/NO(2)(-) signal. 4. The evoked NO/NO(2)(-) signal was not reduced following 6 h perfusion in Ca(2+)-free media, consistent with a Ca(2+)-independent type II NOS activity. 5. PCR for type II NOS revealed the presence of this isotype in the SCN, even immediately following removal of the brain. 6. These studies provide the first evidence to suggest a functional, constitutively-active type II NOS within the brain of normal, healthy adult animals, and add type II NOS to the multiple isotypes of NO synthase playing a role within the mammalian SCN.
Starkey
S J
SJ
Neuropharmacology II, Centre of Excellence for drug discovery in Psychiatry, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY. SJS14150@Glaxo.Wellcome.CO.UK
Grant
A L
AL
Hagan
R M
RM
D000578:Amidines / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006168:Guinea Pigs; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013493:Suprachiasmatic Nucleus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D014018:Tissue Distribution; D019323:omega-N-Methylarginine / Q000494:pharmacology
D000578:Amidines; D001596:Benzylamines; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D009573:Nitrites; D012333:RNA, Messenger; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester