Philip Bland‐Ward

1. 7-Nitro indazole (7-NI) produces potent inhibition of rat cerebellar nitric oxide synthase (NOS) with an IC50 of 0.9 +/- 0.1 microM (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 Ki value of 5.6 microM. The Km of of cerebellar NOS with respect to L-arginine was 2.5 microM. 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 microM), 5-nitro indazole (47.3 +/- 2.3 microM), 3-chloro indazole (100.0 +/- 5.5 microM), 3-chloro 5-nitro indazole (158.4 +/- 2.1 microM) and indazole (177.8 +/- 2.1 microM). 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.

1. The functional consequences of P2X receptor activation on peripheral sensory neurones have been investigated in vivo. Behavioural indices of acute nociception were monitored in the conscious rat following subplantar injection of adenosine 5'-triphosphate (ATP), alpha,beta-methylene ATP, adenosine 5'-diphosphate (ADP) and adenosine. 2. Signs of overt nociception, i.e. hindpaw lifting and licking, were apparent in animals injected subplantar with the P2X receptor agonist, alpha,beta-methylene ATP. Nociceptive behaviours continued for 15 min following administration of alpha,beta-methylene ATP (200 nmol) and were dose-related (0-5 min hindpaw lifting times after injection of alpha,beta-methylene ATP 100 nmol and 1000 nmol were 89 +/- 26 s and 232 +/- 11 s, respectively). Subplantar ATP evoked a modest response only at the highest dose tested (1000 nmol; 0-5 min hindpaw lifting time 66 +/- 19 s) whilst ADP or adenosine (both 600 nmol) elicited negligible spontaneous nociceptive activity. 3. Morphine (3 mg kg-1, i.v.) abolished hindpaw licking behaviour induced by subplantar injection of either alpha,beta-methylene ATP (600 nmol) or bradykinin (1 nmol) and substantially reduced (88 +/- 5%) paw licking in formalin (0.5%, 0.1 ml) injected animals. In contrast, hindpaw lifting was only modestly inhibited (34 +/- 11%) in morphine-pretreated animals that had received subplantar bradykinin and was unaffected in rats in which the noxious stimulus was either subplantar alpha,beta-methylene ATP or formalin. Pretreatment of hindpaws with subplantar bupivacaine (1% w/v, 0.1 ml) abolished alpha,beta-methylene ATP-evoked nociceptive behaviours. 4. Hindpaw lifting and licking mediated by alpha,beta-methylene ATP (600 nmol, subplantar) were inhibited (72 +/- 15% and 95 +/- 5%, respectively) by 30 min local pretreatment with 600 nmol alpha,beta-methylene ATP. Subplantar alpha,beta-methylene ATP pretreatment did not inhibit behaviour stimulated by subsequent bradykinin (1 nmol) or formalin (0.5%, 0.1 ml) injection into the hindpaw. 5. Desensitization of small diameter sensory neurones with a single subplantar injection of capsaicin (100 micrograms) abolished all behaviours indicative of spontaneous nociceptive sensation in animals subsequently injected with alpha,beta-methylene ATP (600 nmol), bradykinin (1 nmol) or formalin (0.5%, 0.1 ml). 6. We conclude that activation of P2X receptors present on small diameter (capsaicin-sensitive) primary afferent neurones in the rat hindpaw mediates behaviour indicative of acute nociception.

7-Nitro indazole (7-NI) inhibits rat striatal, cerebellar, hippocampal, cerebral cortex and olfactory bulb nitric oxide synthase (NOS) in vitro with IC50 values of 0.68 +/- 0.01 microM, 0.64 +/- 0.03 microM, 1.53 +/- 0.05 microM, 0.93 +/- 0.04 microM and 1.05 +/- 0.02 microM respectively (n = 6). Intraperitoneal (i.p.) or oral administration of 7-NI (30 mg kg-1) to rats inhibited NOS enzyme activity measured ex vivo in all five brain regions (n = 5-6). NOS inhibition (maximal effect, 0.5 h post-injection) was transient with complete recovery at either 4 h (oral administration) or 24 h (i.p. administration). Repeated i.p. injection of 7-NI (30 mg kg-1, every 4 h for 20 h) inhibited NOS enzyme activity at 24 h by 51-61% in all brain regions. The relatively transient NOS inhibitory effect of 7-NI following parenteral administration should be taken into account when using this drug to evaluate the central effects of nitric oxide.

1. After a period of myocardial ischaemia, reperfusion of the myocardium can elicit cardiac arrhythmias. Susceptibility to these arrhythmias declines with time, such that a preceding period of more than approximately 40 min ischaemia is associated with few reperfusion-induced arrhythmias. We have tested the hypothesis that this decline in susceptibility occurs, in part, because of protection by endogenous guanosine 3':5'-cyclic monophosphate (cyclic GMP). 2. Rat isolated hearts were subjected to 60 min left regional ischaemia followed by reperfusion (n = 10 per group). Methylene blue (20 microM), a soluble guanylate cyclase inhibitor, raised the incidence of reperfusion-induced ventricular fibrillation (VF) from 10% in control hearts to 80% (P < 0.05). This effect of methylene blue was abolished by co-perfusion with zaprinast (100 microM), a phosphodiesterase inhibitor which, in the rat heart, is cyclic GMP-specific (specific for the type-V phosphodiesterase isozyme). 3. Methylene blue reduced cyclic GMP levels in the ischaemic, non-ischaemic and reperfused myocardium (P < 0.05) to 50 +/- 10, 52 +/- 12 and 70 +/- 7 fmol mg-1 tissue wet weight, respectively from control values of 143 +/- 38, 147 +/- 43 and 156 +/- 15 fmol mg-1. Co-perfusion with zaprinast prevented this effect, and cyclic GMP levels were actually elevated (P < 0.05) to 366 +/- 102, 396 +/- 130 and 293 +/- 22 fmol mg-1 in ischaemic, non-ischaemic and reperfused myocardium, respectively. Zaprinast by itself also elevated cyclic GMP content. Cyclic AMP levels were not affected by zaprinast or methylene blue. 4. In conclusion, when endogenous cardiac cyclic GMP synthesis is reduced, susceptibility to reperfusion-induced VF after sustained ischaemia is substantially increased. The effect is prevented by inhibiting cyclic GMP degradation. Therefore cyclic GMP appears to be an endogenous intracellular cardioprotectant, and its actions may account for the low susceptibility to VF normally encountered in hearts reperfused after sustained ischaemia.