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RESEARCH PRODUCT

Tonic inhibitory action by nitric oxide on spontaneous mechanical activity in rat proximal colon: involvement of cyclic GMP and apamin-sensitive K+ channels

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The cellular mechanisms by which endogenous nitric oxide (NO) modulates spontaneous motility were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. Apamin (1–100 nM) produced a concentration-dependent increase in the amplitude of the spontaneous pressure waves. The maximal contractile effect was of the same degree as that produced by Nω-nitro-L-arginine methyl ester (L-NAME) (100 μM) and the joint application of apamin plus L-NAME had no additive effects. Apamin (0.1 μM) reduced the inhibitory effects (i.e. reduction in the amplitude of the pressure waves) induced by sodium nitroprusside (SNP) (1 nM–10 μM) or 8-Br-cyclic GMP (1–100 μM). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (0.1–5 μM), inhibitor of NO-stimulated guanylate cyclase, produced a concentration-dependent increase of the spontaneous contractions. ODQ (1 μM) in the presence of apamin (0.1 μM) did not produce any further increase in the contraction amplitude, whereas after L-NAME (100 μM) it decreased the spontaneous contractions. ODQ (1 μM) reduced the SNP inhibitory effects. Zaprinast (1–50 μM), inhibitor of cyclic GMP phosphodiesterase, produced a concentration-dependent decrease of the spontaneous contractions. The effects of zaprinast were significantly reduced in the presence of apamin (0.1 μM) or L-NAME (100 μM). These results suggest that small conductance Ca2+-dependent K+ channels and cyclic GMP are involved in the modulation of the spontaneous contractile activity in rat proximal colon. Cyclic GMP production system and opening of apamin-sensitive K+ channels appear to work sequentially in transducing an endogenous NO signal. Keywords: Neural tonic inhibition, nitric oxide, apamin-sensitive K+ channels, cyclic GMP, ODQ, colon, gastrointestinal motility Introduction A growing body of evidence suggests a role for endogenous nitric oxide (NO) not only as a non adrenergic, non cholinergic (NANC) inhibitory neurotransmitter released by electrical field stimulation, but also as a tonic neural modulator of the spontaneous motility in a wide range of gastrointestinal tissues (Sanders & Ward, 1992), including rat preparations (Hata et al., 1990; Li & Rand 1990; Irie et al., 1991; Kanada et al., 1992; Suthamnatpong et al., 1993a; Postorino et al., 1995; Serio et al., 1995; Martinez-Cuesta et al., 1996; Mule et al., 1998a). In particular, a previous study has indicated that basal neural release of NO exerts a tonic inhibitory influence on circular muscle of rat proximal colon (Mule et al., 1998a). Different transduction pathways have been proposed for inhibitory actions of NO (see Shuttleworth & Sanders, 1996 for review). One is a pathway involving a guanosine 3′,5′-cyclic monophosphate (cyclic GMP) generating system. According to this hypothesis, production of cyclic GMP, and perhaps phosphorylation of cellular proteins by cyclic GMP-dependent protein kinase, transduces the NO signal and produces relaxation of smooth muscle. Another possible mechanism concerns the enhancement by NO of the open probability of K+ channels which mediate the hyperpolarization response to inhibitory neurotransmission. In fact, at least a portion of the relaxation induced by NO appears to be due to hyperpolarization of membrane potential or inhibition of electrical activity and subsequent reduction of Ca2+ influx through voltage-dependent Ca2+ channels. However, the activation of K+ channels can be due to direct stimulation by NO or mediated indirectly by a cyclic GMP generating system (Bolotina et al.,1994; Koh et al., 1995). Therefore, biochemical and electrical mechanisms may coexist and together mediate the inhibitory effects of NO. In particular, we have provided evidence that, in the circular muscle of rat proximal colon, the inhibitory junction potentials (IJPs) are largely dependent on the synthesis of NO and due to the activation of apamin-sensitive Ca2+-dependent K+ channels (Serio et al., 1992, 1995). In contrast, in the same preparation, NO has been reported to mediate NANC relaxation by a mechanism independent of changes in membrane potentials (Suthamnatpong et al., 1994) and of changes in cyclic GMP content (Takeuchi et al., 1996), although the same investigators had previously reported an association between the cyclic GMP level and the NO-induced relaxation (Suthamnatpong et al., 1993b; Maehara et al.,1994). In any case, the mechanism by which NO produces a tonic inhibition of circular muscle has not been studied in rat colon. In this report we have investigated the mechanisms involved in the tonic inhibitory action of NO in circular muscle of rat proximal colon. So, our specific objectives were: (1) to verify a possible involvement of the small conductance Ca2+- dependent K+ channels in the modulation of the spontaneous contractile activity and in the hypermotility induced by Nω-nitro-L-arginine methyl ester (L-NAME); (2) to determine the role of cyclic GMP in NO tonic inhibition; (3) to analyse the possible interaction among NO basal synthesis, cyclic GMP production system and opening of K+ channels. Preliminary accounts of part of this work have been given (Mule et al., 1998b).