A rapid transient synthesis of nitric oxide (NO) by a constitutively expressed type IINO synthase in the guinea-pig suprachiasmatic nucleus

1 We have measured extracellular NO/NO2- concentrations in guinea-pig suprachiasmatic nucleus (SCN) brain slices using fast cyclic voltammetry. A rapid and transient signal equivalent to 2.2 +/-0.2 muM NO/NO2- (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/NO2- 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. IC50 values were 229 muM (65-801, n=3, geomean and 95% confidence intervals (C.I.)), 452 nM (88-2310, n=5), and 14.2 muM (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/NO2- signal. 4 The evoked NO/NO2- signal was not reduced following 6 h perfusion in Ca2+-free media, consistent with a Ca2+-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.