Nitric oxide counteracts 5-hydroxytryptamine- and cholera toxin-induced fluid secretion and enhances the effect of oral rehydration solution.

The effects of pharmacological modulation of the nitric oxide (NO) pathway on intestinal fluid transport were studied in a model of ligated jejunal loops of anaesthetized rats in vivo. Close intraarterial infusion of 5-hydroxytryptamine (5-HT) (0.16 microg/min) induced net fluid secretion. Intravenous infusion of the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (0.55 mg/kg per min) reversed net fluid absorption in controls to net secretion and significantly enhanced 5-HT-induced fluid secretion. 5-HT-induced net fluid secretion was inhibited by intravenous infusion of L-arginine (8.88 mg/kg per min), sodium nitroprusside (22.2 microg/kg per min), or 3-morpholino sydnonimine (SIN-1) (22.2 microg/kg per min). Intraluminal instillation of cholera toxin (0.5 microg/ml) induced net secretion, which was significantly enhanced by L-NAME and reduced by L-arginine. Another series of experiments was performed using a model of luminally perfused jejunal loops. Cholera toxin (10 microg/ml) induced profuse net fluid secretion also in this model. L-Arginine and sodium nitroprusside significantly enhanced net fluid absorption compared to controls and abolished the secretory effect of cholera toxin. Luminal perfusion with oral rehydration solution enhanced net absorption of fluid in controls and reversed cholera toxin-induced secretion to absorption. Intravenous infusion, but not intraluminal administration, of L-arginine significantly enhanced the antisecretory effect of oral rehydration solution. These results give further support to the existence of an intestinal NO-mediated proabsorptive tone, which also downregulates fluid secretion elicited by different enterotoxins or mediators of secretion. Intravenous administration of exogenous sources of NO counteracts intestinal fluid accumulation and augments the antisecretory effect of oral rehydration solution, findings which may lead to therapeutic consequences.

Involvement of K+ channel modulation in the proabsorptive effect of nitric oxide in the rat jejunum in vivo.

The role of K+ channels in the mediation of the nitric oxide(NO)-induced proabsorptive effect in intestinal fluid transport was investigated in a functional study, using a model of ligated jejunal loops of anaesthetized rats in vivo. The K+ channel opener cromakalim and the K+ channel blocker glibenclamide were administered under basal conditions as well as under conditions, when fluid secretion was influenced by N omega-nitro-L-arginine methyl ester (L-NAME), prostaglandin E2, Escherichia coli heat stable enterotoxin a (E. coli STa) or L-arginine. Intravenous infusion of cromakalim (63.5 micrograms/kg per min) significantly enhanced net fluid absorption compared to controls, totally abolished net fluid secretion induced by L-NAME (0.55 mg/kg per min), reversed net fluid secretion induced by intraluminal instillation of E. coli STa (10 units/ml) to absorption, but did not influence fluid secretion elicited by close i.a. infusion of prostaglandin E2 (79 ng/min). Close i.a. infusion of glibenclamide (0.16 mg/kg per min) reversed net fluid absorption to net secretion, blocked the absorptive effect of L-arginine (8.88 mg/kg per min) and reduced the proabsorptive effect of cromakalim. The secretory effect of L-NAME was not further enhanced by glibenclamide. These results suggest that modulation of basolateral K+ channels by NO is involved in the mediation of its proabsorptive effect, since opening and closure of K+ channels mimicked, respectively counteracted, the action of NO-donors and inhibitors of NO-synthesis on intestinal fluid transport. The role of prostaglandins in the proabsorptive effect of NO remains to be elucidated. These results furthermore support the role of K+ channel openers as potential new antidiarrheal drugs.

Significance of nitric oxide in the stimulation of intestinal fluid absorption in the rat jejunum in vivo.

1. The effects of inhibiting nitric oxide (NO)-synthase on fluid transport, mucosal cyclic GMP and cyclic AMP levels and intraluminal prostaglandin E2 (PGE2)-release were studied in a model of ligated jejunal loops of anaesthetized rats in vivo. Experiments were performed under basal conditions as well as under conditions, when net fluid secretion was induced by Escherichia coli heat stable enterotoxin a (E. coli STa) or PGE2. 2. Intravenous infusion of the NO-synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 0.25-50 mg kg-1, 45 min) dose-dependently reversed net fluid absorption to net secretion, whereas infusion of D-NAME, the inactive enantiomer of L-NAME, in corresponding doses did not influence net fluid transport. N omega-nitro-L-arginine (L-NOARG, 25 mg kg-1), another NO-synthase inhibitor, also elicited net secretion of fluid. 3. L-NAME (25 mg kg-1)-induced net fluid secretion was reversed to net absorption by infusion of L-arginine (400 mg kg-1) or sodium nitroprusside (1 mg kg-1) and s.c. administration of indomethacin (10 mg kg-1). Hexamethonium (1 mg kg-1, s.c.), a ganglionic blocker and granisetron (100 micrograms kg-1, s.c.), a 5-HT3-receptor antagonist, did not influence L-NAME-induced net secretion. 4. Net fluid secretion induced by intraluminal instillation of E. coli STa (10 units ml-1) was enhanced by infusion of L-NAME (25 mg kg-1) and was inhibited by infusion of L-arginine (400 mg kg-1) and sodium nitroprusside (1 mg kg-1). D-Arginine (400 mg kg-1) did not influence E. coli STa-induced fluid secretion. Likewise, net fluid secretion induced by i.a. infusion of PGE2 (79 ng ml-1, 30 min) was enhanced by infusion of L-NAME and was inhibited by L-arginine and sodium nitroprusside. D-Arginine(400 mg kg-1) did not influence PGE2-induced fluid secretion.5. PGE2 levels in intraluminal fluid were not elevated after infusion of L-NAME (25mgkg-1) compared to controls.6. Mucosal cyclic GMP and cyclic AMP levels after L-NAME-treatment were not different from control values.7. These results indicate that nitric oxide plays an important role in the regulation of intestinal fluid transport. The data suggest a nitric oxide-dependent proabsorptive tone in the intestine, which possibly involves the enteric nervous system and suppression of prostaglandin formation. This proabsorptive tone also may downregulate fluid secretion induced by E. coli STa or PGE2.

Serotonin antagonists inhibit sennoside-induced fluid secretion and diarrhea.

The aim of this study was to investigate whether 5-hydroxytryptamine (serotonin, 5-HT) is involved in the mediation of sennoside-induced colonic fluid secretion and diarrhea. Oral administration of purified sennosides (25, 40 and 64 mg/kg) dose-dependently reversed net fluid absorption to net fluid secretion, enhanced the incidence of diarrhea and stimulated the release of 5-HT into the colonic lumen from 7.1 to 17.3 ng/g wet weight. The 5-HT2 antagonist ketanserin and the 5-HT3 antagonist tropisetron dose-dependently but only partially reduced sennoside (40 mg/kg)-induced fluid secretion whereas the 5-HT3 antagonist granisetron dose-dependently reduced and at 300 micrograms/kg totally abolished sennoside-induced secretion. Granisetron, but not ketanserin and tropisetron, reduced the incidence of diarrhea in sennoside-treated rats, indicating the involvement of 5-HT also in acceleration of large intestinal transit. It is concluded that 5-HT is an important mediator both of sennoside-induced fluid secretion in the rat colon and of diarrhea.

Antisecretory activities of orally administered loperamide and loperamide oxide on intestinal secretion in rats.

In-vivo experiments in the rat jejunum have been performed to compare the antisecretory effect of orally administered loperamide with the effect of its pro-drug, loperamide oxide. Both loperamide and loperamide oxide, administered orally, reduced the secretory effect of prostaglandin E2 (32 ng min-1, intra-arterially) in the jejunum and the colon. Differences between the two drugs as to time course and dose response can be seen. Loperamide oxide shows its antisecretory effect in the jejunum, and at a dose of 2 mg kg-1 also shows its effect in the colon 1 h after administration. The effect was maximal after 2 h and decreased after 4 h. A dose-response relationship was demonstrated at 2 h in the jejunum and the colon. In comparison, the effect of loperamide started later, and a good dose-response relationship was not observed in the jejunum or in the colon, higher doses always appearing less effective than lower doses.

Inhibition of 5-hydroxytryptamine- and enterotoxin-induced fluid secretion by 5-HT receptor antagonists in the rat jejunum.

The effects of cholera toxin and heat stable Escherichia coli (E. coli) enterotoxin on intestinal fluid secretion are commonly considered to be mediated by cyclic nucleotides. It was demonstrated recently, by using the 5-hydroxytryptamine (5-HT)2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist tropisetron, that 5-HT acts as an important mediator in cholera toxin- and heat stable E. coli enterotoxin-induced fluid secretion. In the present investigation ketanserin and tropisetron were compared with the newer 5-HT3 receptor antagonists ondansetron and granisetron versus 5-HT-, cholera toxin- and heat stable E. coli enterotoxin-induced fluid secretion in the rat jejunum in vivo. Both ondansetron and granisetron dose-dependently inhibited 5-HT- and enterotoxin-induced fluid secretion. Ketanserin blocked 5-HT-induced fluid secretion, but only diminished enterotoxin-induced effects even at higher doses. Tropisetron inhibited 5-HT- and cholera toxin-induced effects at high dose but only diminished heat stable E. coli enterotoxin-induced effects. We conclude that 5-HT3 receptors, located on enterochromaffin cells and nervous structures, are more important in mediating fluid secretion than 5-HT2 receptors, located on the epithelial cells.

Stimulation of enterocyte protein kinase C by laxatives in-vitro.

To elucidate the role of protein kinase C in the mechanism of action of stimulatory laxatives, experiments were performed with preparations of rat lysed enterocytes. The phorbol ester 4-beta-phorbol 12-myristate 13-acetate (PMA) concentration-dependently (2-200 micrograms mL-1) stimulated the activity of protein kinase C in this preparation. Ricinoleic acid, the active principle of castor oil, deacetylbisacodyl, the active principle of bisacodyl, and deoxycholic acid exerted the same effect, although less efficiently. This reflects their potency for inducing intestinal fluid secretion and prostaglandin release, effects that are also induced more potently by PMA. Accordingly, the potency of the three C18 fatty acids, ricinoleic acid, stearic acid and oleic acid on protein kinase C activity in-vitro, on prostaglandin E2 release and on net fluid secretion in-vivo runs in parallel. It is therefore concluded that stimulatory laxatives activate protein kinase C, leading to prostaglandin E2 release, thus resulting in net fluid secretion.

Effects of purified Clostridium difficile toxin A in the small intestine of the rat in vivo.

The action of highly purified Clostridium difficile toxin A was studied in the jejunum of rats in vivo. C. difficile toxin A reversed dose-dependently net fluid absorption into net fluid secretion, accompanied by an increase in prostaglandin E2 but not 5-hydroxytryptamine output into the gut lumen. Accordingly, indomethacin but not the 5-hydroxytryptamine receptor antagonists ketanserin plus tropisetron were able to inhibit toxin A-induced fluid secretion. Atropine and hexamethonium were without effect on the action of toxin A, such excluding a nervous mechanism. The cyclic nucleotides cyclic AMP and cyclic GMP appear not to be involved in the mediation of the secretory response. The reduced cyclic GMP levels are most likely the result of a complete destruction of the villus membranes, where the guanylate cyclase is located. Histological studies revealed massive damage to intestinal villi, whereas the majority of the crypts seem to be unaffected. In conclusion, toxin A-induced intestinal fluid secretion appears to be caused mainly by severe mucosal damage. PGE2-release may be the consequence of the inflammation accompanying this damage. The mechanism seems to be completely different to those of cholera toxin or Escherichia coli heat stable enterotoxin.

5-HT receptor antagonists and heat-stable Escherichia coli enterotoxin-induced effects in the rat.

The effect of heat-stable E. coli enterotoxin on intestinal fluid secretion is commonly considered to be mediated by stimulation of mucosal cyclic guanosine monophosphate (cGMP). It was demonstrated recently that 5-hydroxytryptamine (5-HT) acts as an important mediator in cholera toxin-induced fluid secretion. To elucidate the possible involvement of 5-HT in the secretory response to heat-stable E. coli enterotoxin, in vivo experiments were performed in the rat jejunum. The inhibitory effects of the 5-HT2 receptor antagonist ketanserin, the 5-HT3 receptor antagonist tropisetron and indomethacin were studied in heat-stable E. coli enterotoxin-induced fluid secretion. Tropisetron and ketanserin (100 micrograms/kg each) alone only partially reduced the secretory effect of the toxin. However, in combination, the two blockers (100 plus 100 micrograms/kg) significantly reduced and at 200 plus 200 micrograms/kg totally abolished heat-stable E. coli enterotoxin-induced secretion without influencing the enterotoxin-induced increase in cGMP. Pretreatment with indomethacin (10 mg/kg) reduced the secretory response to the enterotoxin by about 50%. These results support the concept that 5-HT is an important mediator in intestinal fluid secretion induced by heat-stable E. coli enterotoxin. The enterotoxin may use 5-HT to stimulate prostaglandin formation via 5-HT2 receptors and to activate neuronal structures via 5-HT3 receptors.