Eckhard Beubler

Involvement of 5-Hydroxytryptamine, Prostaglandin E2, and Cyclic Adenosine Monophosphate in Cholera Toxin-Induced Fluid Secretion in the Small Intestine of the Rat In Vivo

The diarrhea of cholera is considered to rely solely on a cyclic adenosine monophosphate-mediated secretory mechanism. However, both 5-hydroxytryptamine and prostaglandin E2 have been proposed to be involved in the pathogenesis of cholera. In vivo experiments were performed, therefore, in the rat jejunum to investigate the influence of purified cholera toxin on fluid secretion, luminal release of 5-hydroxytryptamine and prostaglandin E2, and formation of mucosal cyclic adenosine monophosphate. Also the effects of ketanserin, indomethacin, verapamil, and nifedipine on the named parameters were studied. Cholera toxin dose-dependently (0.1-0.5 microgram/ml) and time-dependently (1-5 h) increased mean net fluid secretion with a maximum response at 4 h. It also caused a significant (p less than 0.01) rise in release of 5-hydroxytryptamine and prostaglandin E2, in addition to formation of cyclic adenosine monophosphate. The dose-response curve for cholera toxin-induced fluid secretion was shifted to the right by indomethacin (10 mg/kg s.c.) and ketanserin (200 micrograms/kg s.c.), none of which caused a change in cholera toxin-induced release of 5-hydroxytryptamine. However, both agents significantly decreased the release of prostaglandin E2. Verapamil (0.2-9.5 micrograms/min i.a.) and nifedipine (0.05-0.5 microgram/min i.a.) dose-dependently reduced cholera toxin-induced fluid secretion. The estimated local concentrations at half-maximal inhibition were 5 x 10(-7) M verapamil and 5 x 10(-8) M nifedipine, respectively. The cholera toxin-induced increase in release of 5-hydroxytryptamine and prostaglandin E2 and formation of cyclic adenosine monophosphate was unaffected by verapamil. These results support the concept that cholera toxin-induced fluid secretion in vivo is caused, in part, by release of 5-hydroxytryptamine, which in turn stimulates formation of prostaglandin E2.

Effect of ricinoleic acid and other laxatives on net water flux and prostaglandin E release by the rat colon

Ricinoleic acid, oleic acid, dioctyl sodium sulphosuccinate, deoxycholic acid, sennoside A + B and mannitol reduced or reversed water flux from lumen to blood in rat colon in situ. Stearinic acid was without any effect. Ricinoleic acid, oleic acid, dioctyl sodium sulphosuccinate, deoxycholic acid and sennoside A + B stimulated release of PGE‐like material into the colonic lumen whereas the osmotic laxative mannitol and stearinic acid did not. Inhibition of PGE biosynthesis by pretreatment of the rats with indomethacin significantly reduced (but did not abolish) the effect of ricinoleic, oleic and deoxycholic acids on net water flux and PGE release. Indomethacin reduced the effect of dioctyl sodium sulphosuccinate and of sennoside A + B on PGE release but not their effect on the net water flux. The effect of mannitol was not influenced by indomethacin. The amount of PGE release in experiments with ricinoleic acid, oleic acid, stearinic acid and dioctyl sodium sulphosuccinate (with and without indomethacin) showed a good correlation (r = 0·99) with the change in net water flux. Deoxycholic acid, sennoside A + B and mannitol did not show this correlation. It is assumed that the action of non‐osmotic laxatives is partially mediated by PGE, although other mechanisms also seem to be involved in their mode of action.

5-HT2 and 5-HT3 receptor subtypes mediate cholera toxin-induced intestinal fluid secretion in the rat

The mechanisms of diarrhea in Asiatic cholera have been studied extensively. Cyclic adenosine monophosphate, 5-hydroxytryptamine (5-HT), prostaglandins, and the function of neuronal structures have been implicated in the pathogenesis of cholera. To elucidate the action of 5-HT in mediating cholera secretion, in vivo experiments were performed in the rat jejunum. The inhibitory effects of the 5-HT2 receptor antagonist ketanserin and the 5-HT3 receptor antagonist ICS 205-930 were studied in cholera toxin- and 5-HT-induced fluid secretion. Both ketanserin and ICS 205-930 dose-dependently but only partially reduced the secretory effect of cholera toxin. The combination of the two blockers totally abolished cholera toxin-induced secretion without any influence on cholera toxin-induced increase in cyclic adenosine monophosphate. Prostaglandin E2- and bisacodyl-induced secretion was not affected by the combined administration of 5-HT2 and 5-HT3 antagonists. The present results provide evidence for an important role of 5-HT in cholera toxin-induced secretion. The data suggest a model in which cholera toxin may initiate the release of 5-HT from enterochromaffin cells. 5-Hydroxytryptamine may then cause prostaglandin E2 formation via 5-HT2 receptors and activation of neuronal structures via 5-HT3 receptors. These two effects may finally lead to the profuse fluid secretion which can be totally blocked by the combination of a 5-HT2 blocker and a 5-HT3 blocker.

Inhibition of stimulated fluid secretion in the rat small and large intestine by opiate agonists

Summary1.The influence of morphine, levorphanol, dextrorphan, loperamide and naloxone on the increase in intestinal fluid volume (IFV) caused by PGE1, vasoactive intestinal peptide (VIP), carbachol and bethanechol was investigated in the small intestine of rats.2.Morphine reduced the basal IFV and the increase in IFV caused by PGE1, VIP and carbachol. The effect of bethanechol was not altered by morphine.3.The effects of levorphanol were comparable to those of morphine, whereas dextrorphan was inactive.4.Loperamide reduced the increase in IFV caused by PGE1, but not that caused by carbachol.5.Naloxone enhanced the effect of PGE1 and VIP on IFV but neither altered basal IFV nor the effect of carbachol. This result may suggest an inhibitory influence of intestinal enkephalins on PGE1-or VIP-induced intestinal secretion.6.The influence of opiates was further tested on the effect of the diphenolic laxative, bisacodyl and of the osmotic laxative, mannitol on water net flux in the tied off colon of the rat in situ. Morphine, levorphanol and loperamide reduced the effect of bisacodyl, whereas the effect of mannitol and the net water absorption in controls remained unchanged.7.The experiments show an antisecretory effect of opiates on different types of stimulation of fluid secretion in the small and large intestine. It is concluded that the antidiarrhoeal action of opiates can be better explained by antisecretory than by smooth muscle action on the gut.

Neuropeptide Y (NPY) and peptide YY (PYY) inhibit prostaglandin E2-induced intestinal fluid and electrolyte secretion in the rat Jejunum in vivo

NPY, a recently discovered peptide consisting of 36 amino acids, is present in intrinsic intestinal nerves and in extrinsic noradrenergic nerves innervating intestinal blood vessels. We have investigated the influence of NPY and of the structurally related peptide YY (PYY) on the effect of PGE2-induced fluid and electrolyte secretion in the tied-off rat jejunum in vivo. Close intraarterial infusion of PGE2 (4.5-450 pmol X min-1) dose dependently reversed the net absorption of fluid, sodium and chloride into net secretion (P less than 0.01 for all three parameters). Additional i.a. infusion of NPY significantly inhibited the effect of PGE2 (45 pmol X min-1) on fluid transport at infusion rates of 0.4 and 4.0 pmol X min-1 (P less than 0.01). Infusion of 0.04 pmol X min-1 NPY was without effect. PGE2-induced sodium and chloride secretion were also significantly reduced by NPY at an infusion rate of 0.4 but not of 0.04 pmol X min-1. NPY alone was without any effect on fluid or electrolyte absorption in the controls. PYY, which is present in endocrine cells but not in nerves in the gut, was without effect at 0.4 pmol X min-1 and slightly but significantly reduced PGE2-induced fluid secretion at 4.0 pmol X min-1. It is concluded that NPY is a potent inhibitory factor in the neuronal control of intestinal fluid and electrolyte transport.

Significance of calcium for the prostaglandin E2-mediated secretory response to 5-hydroxytryptamine in the small intestine of the rat in vivo

5-Hydroxytryptamine (5-HT) has been claimed to mediate intestinal secretion in morphine withdrawal diarrhea through stimulation of local prostaglandin formation without involving cyclic adenosine monophosphate. Therefore, experiments were performed to study (a) the effects of exogenous 5-HT and the cyclic adenosine monophosphate-dependent secretagogue, vasoactive intestinal polypeptide, on intestinal prostaglandin E2 (PGE2) formation and (b) the involvement of calcium in the secretory response to close intraarterial infusion of 5-HT, PGE2, or vasoactive intestinal polypeptide in tied-off loops of rat jejunum in vivo. 5-Hydroxytryptamine and vasoactive intestinal polypeptide reversed fluid absorption to net secretion (p less than 0.01), but only 5-HT caused an increase in luminal PGE2 output (p less than 0.01). Indomethacin and d,l-verapamil prevented only the secretory effect of 5-HT. Exogenous PGE2 (1.6-160 ng/min) reversed absorption to secretion (p less than 0.01) in a dose-dependent manner, irrespective of whether the rats were pretreated with indomethacin or not. Racemic and l-verapamil, but not d-verapamil, markedly reduced (p less than 0.01) the secretory effect of physiologically low doses of PGE2 (1.6 and 16 ng/min), whereas high doses of PGE2 (160 ng/min), which caused a significant increase in mucosal cyclic adenosine monophosphate (p less than 0.005), were not inhibited by verapamil. These data suggest that PGE2 may be an important intermediate in the transduction mechanism leading to 5-HT-induced intestinal secretion, and that physiologic doses of PGE2 may act by facilitating calcium entry, rather than by increasing intracellular calcium through activation of the adenylate cyclase.

Protein Carbamylation Renders High-Density Lipoprotein Dysfunctional

Carbamylation of proteins through reactive cyanate has been demonstrated to predict an increased cardiovascular risk. Cyanate is formed in vivo by breakdown of urea and at sites of inflammation by the phagocyte protein myeloperoxidase. Because myeloperoxidase (MPO) associates with high-density lipoprotein (HDL) in human atherosclerotic intima, we examined in the present study whether cyanate specifically targets HDL. Mass spectrometry analysis revealed that protein carbamylation is a major posttranslational modification of HDL. The carbamyllysine content of lesion-derived HDL was more than 20-fold higher in comparison with 3-chlorotyrosine levels, a specific oxidation product of MPO. Notably, the carbamyllysine content of lesion-derived HDL was five- to eightfold higher when compared with lesion-derived low-density lipoprotein (LDL) or total lesion protein and increased with lesion severity. The carbamyllysine content of HDL, but not of LDL, correlated with levels of 3-chlorotyrosine, suggesting that MPO mediated carbamylation in the vessel wall. Remarkably, one carbamyllysine residue per HDL-associated apolipoprotein A-I was sufficient to induce cholesterol accumulation and lipid-droplet formation in macrophages through a pathway requiring the HDL-receptor scavenger receptor class B, type I. The present results raise the possibility that HDL carbamylation contributes to foam cell formation in atherosclerotic lesions.

Involvement of μ- and κ-, but not δ-, opioid receptors in the peristaltic motor depression caused by endogenous and exogenous opioids in the guinea-pig intestine

Opiates inhibit gastrointestinal propulsion, but it is not clear which opioid receptor types are involved in this action. For this reason, the effect of opioid receptor – selective agonists and antagonists on intestinal peristalsis was studied. Peristalsis in isolated segments of the guinea‐pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the intraluminal pressure changes associated with the peristaltic waves. μ‐Opioid receptor agonists (DAMGO, morphine), κ‐opioid receptor agonists (ICI‐204,448 and BRL‐52,537) and a δ‐opioid receptor agonist (SNC‐80) inhibited peristalsis in a concentration‐related manner as deduced from a rise of the peristaltic pressure threshold (PPT) and a diminution of peristaltic effectiveness. Experiments with the δ‐opioid receptor antagonists naltrindole (30 nM) and HS‐378 (1 μM), the κ‐opioid receptor antagonist nor‐binaltorphimine (30 nM) and the μ‐opioid receptor antagonist cyprodime (10 μM) revealed that the antiperistaltic effect of ICI‐204,448 and BRL‐52,537 was mediated by κ‐opioid receptors and that of morphine and DAMGO by μ‐opioid receptors. In contrast, the peristaltic motor inhibition caused by SNC‐80 was unrelated to δ‐opioid receptor activation. Cyprodime and nor‐binaltorphimine, but not naltrindole and HS‐378, were per se able to stimulate intestinal peristalsis as deduced from a decrease in PPT. The results show that the neural circuits controlling peristalsis in the guinea‐pig small intestine are inhibited by endogenous and exogenous opioids acting viaμ‐ and κ‐, but not δ‐, opioid receptors.

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 Nω‐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. Nω‐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 μg 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 (25 mg kg−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.

INHIBITION BY MORPHINE OF PROSTAGLANDIN E1‐STIMULATED SECRETION AND CYCLIC ADENOSINE 3′,5′‐MONOPHOSPHATE FORMATION IN THE RAT JEJUNUM in vivo

1 The effects were studied of prostaglandin E1 (PGE1), theophylline and morphine on net water flux and mucosal cyclic adenosine 3′,5′‐monophosphate (cyclic AMP) levels in the jejunum of anaesthetized rats in vivo. 2 Infusion of PGE1 (3.2 μg/min, i.a.) caused a reversal from net water absorption to net secretion and enhanced the mucosal cyclic AMP content by 54%. 3 Theophylline (5 mg/ml, intraluminal) similarly produced a reversal from net water absorption to net secretion and increased mucosal cyclic AMP content by 54%. Additional intra‐arterial infusion of PGE1 resulted in a massive increase in net water secretion and an increase in mucosal cyclic AMP content by about 200%. 4 Pretreatment with morphine (10 mg/kg, s.c.) reduced the effect of PGE1 on net water flux and completely inhibited its effect on the mucosal cyclic AMP content. Naloxone (10 mg/kg, s.c.) abolished both effects of morphine. 5 A good correlation (r = 0.99) was demonstrated between mucosal cyclic AMP levels and net water flux. 6 The present results demonstrate that PGE1 stimulates intestinal fluid secretion by increasing mucosal cyclic AMP levels. The antidiarrhoeal effect of morphine can be explained by its inhibition of the PGE‐mediated increase in cyclic AMP levels, which, in turn, leads to a reduction in intestinal secretion.