A possible effect of sulfhydryl reagents on the contractile activity of the rat detrusor muscle

Abstract

We aimed to investigate the effect of sulfhydryl (SH) inactivating agents, ethacrynic acid and N-ethylmaleimide, on the contractile activity of rat detrusor muscle. Wistar Kyoto rats weighing 150–250 g were anaesthetized with ketamine and bled to death. The urinary bladders were surgically removed and detrusor strips were mounted under 0.5 g tension in organ baths. The responses were recorded with isotonic transducers on polygraph paper. After an equilibrium period, the tissues were contracted by electrical field stimulation, acetylcholine, ethacrynic acid or N-ethylmaleimide and the effects of l-cysteine, glutathione, verapamil, Ca²⁺-free solution, sodium nitroprusside or atropine were then examined on these contractions. Verapamil, Ca²⁺-free solution or atropine significantly reduced the contractions elicited by electrical field stimulation and acetylcholine whereas l-cysteine, glutathione or sodium nitroprusside had no effect on the contractions in response to these stimuli. l-Cysteine, glutathione, verapamil or Ca²⁺-free solution significantly inhibited the contractions induced by ethacrynic acid or N-ethylmaleimide. Sodium nitroprusside slightly inhibited only the contraction induced by ethacrynic acid but not that with N-ethylmaleimide. Atropine has no action on the contractions in response to these SH reagents. These findings suggest that SH reagents may play a role in the contractile activity of rat detrusor muscle and this action seems to be related to the gating of Ca²⁺ channels. Further experiments are needed to determine the cellular mechanism(s) of action by which these SH reagents act on the detrusor smooth muscle.

Introduction

A role of N-ethylmaleimide and ethacrynic acid, sulfhydryl (SH) alkylating agents Rapoport and Murad, 1988, Lacampagne et al., 1995 in contraction of some tissues, such as guinea pig ileum (Salimi et al., 1979), rabbit ear arteries (Neering and Glover, 1979) and rat diaphragm muscle (Roed, 1989) has been demonstrated. Recent studies suggested a role of free SH groups in gating of the Ca²⁺ channels in some tissues, such as cardiac myocytes Lacampagne et al., 1995, Suzuki et al., 1998, vascular endothelial cells Elliott and Koliwad, 1997, Az-ma et al., 1999, rabbit heart sarcolemma and skeletal muscle (Murphy et al., 1990). SH reagents have been used to establish the role of SH groups in the function of Ca²⁺ channels and it has been shown that SH alkylating agents induced a Ca²⁺ current leading to an increase of tension in skeletal muscle fibers of a crustacean Zuazaga and Del Castillo, 1985, Lizardi et al., 1992. It was also shown that N-ethylmaleimide increased the Ca²⁺ current in heart cells (Nakajima et al., 1990). It has been reported from a recent study that SH reagents exerted a stimulatory effect on the L-type Ca²⁺ current on frog ventricular myocytes and it was concluded that there are sites on the Ca²⁺ channels that are subject to direct modification by SH reagents (Yamaoka et al., 2000).

In the urinary bladder, the Ca²⁺ channels seem to be mainly of the L type (Anderson, 1993). Several experimental studies of animal detrusor tissue demonstrated the importance of extracellular Ca²⁺ entry through L-type Ca²⁺ channels and mobilisation of intracellular Ca²⁺ for tonic activity Mostwin, 1986, Huddart and Butler, 1986, Batra et al., 1987, Kishii et al., 1992. Extracellular Ca²⁺ removal and L-type Ca²⁺ channel blockers reduce contractile activity (Andersson and Forman, 1986).

These results prompted us to investigate whether there is an actual contribution of SH reagents to the contractile activity of rat detrusor muscle or not. We thus compared the effect of N-ethylmaleimide and ethacrynic acid on the basal tonus of the isolated rat detrusor muscle with the neurogenic contractions induced by electrical field stimulation and myogenic contractions induced by acetylcholine and then examined the actions of l-cysteine, glutathione, Ca²⁺-free medium, verapamil, sodium nitroprusside or atropine on the responses of the tissue to these stimuli.