The role of constitutive and inducible nitric oxide synthase in senna- and cascara-induced diarrhoea in the rat

doi:10.1016/S0014-2999(97)00023-X

European Journal of Pharmacology

Volume 323, Issue 1, 26 March 1997, Pages 93-97

https://doi.org/10.1016/S0014-2999(97)00023-X Get rights and content

Abstract

The role of constitutive and inducible nitric oxide (NO) synthase in rats treated with senna and cascara was studied. Senna (60 mg/kg p.o.) and cascara (800 mg/kg p.o.) ex vivo significantly increased Ca²⁺-dependent constitutive NO synthase activity in the rat colon. Induction of NO synthase (12% of the total NO synthase) was associated with cascara, but not senna, administration. Dexamethasone (0.03–0.3 mg/kg i.p.), which inhibits the expression of inducible NO synthase, significantly and dose-dependently reduced cascara- (but not senna-) induced diarrhoea and colonic fluid secretion. These findings suggest that senna probably exerts its laxative effect through stimulation of the constitutive isoform of NO synthase, while the inducible isoform of NO synthase also seems to be involved in the laxative effect of cascara. © 1997 Elsevier Science B.V. All rights reserved.

Introduction

Senna and cascara are laxative anthraquinone drugs containing glycosides as active ingredients (Gaginella, 1994). Being large polar molecules, the glycosides are poorly absorbed in the small intestine. As they appear in the large intestine, colonic bacteria liberate the aglycone which is then reduced to the active anthrol form (Van Os, 1976; Lemli and Lemmens, 1980).

Several mechanisms of action have been proposed to explain the laxative effect of anthraquinones. Mechanisms include inhibition of Na⁺,K⁺-ATPase (see Gaginella, 1994), stimulation of prostaglandin (Beubler and Kollar, 1988), histamine (Capasso et al., 1986) or serotonin (Beubler and Schirgi-Degen, 1993) biosynthesis and other non-specific metabolic effects on the epithelial cells (Verhaeren, 1980). However, despite the fact that numerous mechanisms of action have been proposed to explain the laxative effect of senna and cascara, their precise mechanism of action has not been clearly elucidated.

Recently it has been shown that the nitric oxide (NO) synthase inhibitor, N^G-nitro-l-arginine methyl ester (l-NAME) reduces senna- and cascara-induced diarrhoea and fluid secretion (Izzo et al., 1996). NO, an endogenous mediator synthesized by the enzyme NO synthase from the amino acid l-arginine, is now recognized as an important regulator of gut functions (Miller and Gaginella, 1995). NO synthase can exist as a constitutive (Ca²⁺/calmodulin-dependent) and an inducible (Ca²⁺/calmodulin-independent) isoform in many tissues, including the gastrointestinal tract (Moncada and Higgs, 1993; Boughton Smith et al., 1993; Nichols et al., 1993). The expression, but not the activity, of the inducible NO synthase is inhibited by glucocorticoids such as dexamethasone. This action is distinct from that of arginine analogues, such as l-NAME, which are inhibitors of both the constitutive and inducible NO synthases (Moncada and Higgs, 1993).

The aim of the present study was to evaluate the role of constitutive and inducible NO in senna- and cascara-induced intestinal fluid secretion and diarrhoea. For this purpose we examined the activity of the constitutive and the inducible NO synthase following senna or cascara administration. The effect of dexamethasone, which inhibits the expression of the inducible NO synthase, was also evaluated.

Section snippets

Animals

Male Wistar (Nossan) rats, weighing 160–180 g, were used after one week for adaptation to the housing conditions. Standard food (Morini) was withheld 20 h before the experiments but there was free access to drinking water.

Nitric oxide synthase activity

The animals were killed by CO₂ 8 h after oral administration of senna 60 mg/kg or cascara 800 mg/kg. Full thickness segments of the colon were homogenized at 4°C in 4 vols. of 20 mM HEPES buffer (pH 7.2) containing 320 mM sucrose, 1 mM dl-dithiothreitol, 10 μg/ml soybean

Nitric oxide synthase activity

NO synthase activity, that was abolished by incubation in vitro with l-NMMA (1 mM), was detected in the supernatants of colonic homogenates of control rats, and was 0.506±0.07 pmol/min per g tissue. This activity was abolished by incubation with EGTA (1 mM, Fig. 1).

Eight hours after senna (60 mg/kg) or cascara (800 mg/kg) administration, a significant increase of total NO synthase activity (abolished in vitro by l-NMMA 1 mM) was detected (Fig. 1). The NO synthase activity in the supernatant

Discussion

We have shown that possible mediators for the laxative effect of senna and cascara include NO, which stimulates intestinal secretion in vitro (MacNaughton, 1993; Tamai and Gaginella, 1993; Wilson et al., 1993) and in vivo (Gaginella et al., 1994; Izzo et al., 1994; Miller et al., 1993a). We have recently shown that l-NAME, a reversible inhibitor of NO synthase, reduces cascara- and senna-induced colonic fluid secretion and diarrhoea (Izzo et al., 1996). In addition, the present results indicate

Acknowledgements

This research was supported by CNR (Rome), Murst 40% and 60% and Regione Campania. We are grateful to Dr. Angela Tosco for the revision of the manuscript.

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