INHIBITION OF RAT CEREBELLAR NITRIC-OXIDE SYNTHASE BY 7-NITRO INDAZOLE AND RELATED SUBSTITUTED INDAZOLES

1 7-Nitro indazole (7-NI) produces potent. inhibition of rat cerebellar nitric oxide synthase (NOS) with an IC50 of 0.9 +/- 0.1 mum (n = 6). NOS activity is dependent on the presence of both exogenous CaCl2 and NADPH. The inhibitory potency of 7-NI remained unaltered in the presence of different concentrations of either CaCl2 (0.75-7.5 mM) or NADPH (0.05-5.0 mm). 2 Kinetic (Lineweaver-Burke) analysis of the effect of 7-NI on rat cerebellar NOS revealed that inhibition was of a competitive nature with a K(i) value of 5.6 muM. The K(m) of cerebellar NOS with respect to L-arginine was 2.5 muM. 3 The following indazole derivatives (IC50 values shown in parentheses, all n = 6) caused concentration-related inhibition of rat cerebellar NOS in vitro: 6-nitro indazole (31.6 +/- 3.4 muM), 5-nitro indazole (47.3 +/- 2.3 muM), 3-chloro indazole (100.0 +/- 5.5 muM), 3-chloro 5-nitro indazole (158.4 +/- 2.1 muM) and indazole (177.8 +/- 2.1 muM). The IC50 values for 5-amino indazole, 6-amino indazole and 6-sulphanilimido indazole were in excess of 1 mm; 3-indazolinone was inactive. 4 7-NI (10 mg kg-1) administered i.p. to rats produced 60 min thereafter a significant inhibition of NOS activity in cerebellum (31.1 +/- 3.2%, n = 6), cerebral cortex (38.2 +/- 5.6%, n = 6), hippocampus (37.0 +/- 2.8%, n = 6) and adrenal gland (23.7 +/- 3.0%, n = 6). NOS activity in olfactory bulb and stomach fundus were unchanged. 5 These results indicate that 7-NI is a potent and competitive inhibitor of rat brain NOS in vitro and also inhibits NOS in different brain regions and in the adrenal gland in vivo. Inhibition of NOS is a characteristic property of the indazole nucleus. Nitration of the indazole ring at positions 5, 6 and 7 results in a graded increase in inhibitory potency. Indazole-based inhibitors of NOS may prove useful tools with which to evaluate the biological roles of nitric oxide in the central nervous system.