The inhibition of cholera toxin-induced 5-HT release by the 5-HT(3) receptor antagonist, granisetron, in the rat

1. The secretagogue 5-hydroxytryptamine (5-HT) is implicated in the pathophysiology of cholera. 5-HT released from enterochromaffin cells after cholera toxin exposure is thought to activate non-neuronally (5-HT(2) dependent) and neuronally (5-HT(3) dependent) mediated water and electrolyte secretion. CT-secretion can be reduced by preventing the release of 5-HT. Enterochromaffin cells possess numerous receptors that, under basal conditions, modulate 5-HT release. 2. These include basolateral 5-HT(3) receptors, the activation of which is known to enhance 5-HT release. 3. Until now, 5-HT(3) receptor antagonists (e.g. granisetron) have been thought to inhibit cholera toxin-induced fluid secretion by blockading 5-HT(3) receptors on secretory enteric neurones. Instead we postulated that they act by inhibiting cholera toxin-induced enterochromaffin cell degranulation. 4. Isolated intestinal segments in anaesthetized male Wistar rats, pre-treated with granisetron 75 microg kg(-1), lidoocaine 6 mg kg(-1) or saline, were instilled with a supramaximal dose of cholera toxin or saline. Net fluid movement was determined by small intestinal perfusion or gravimetry and small intestinal and luminal fluid 5-HT levels were determined by HPLC with fluorimetric detection. 5. Intraluminal 5-HT release was proportional to the reduction in tissue 5-HT levels and to the onset of water and electrolyte secretion, suggesting that luminal 5-HT levels reflect enterochromaffin cell activity. 6. Both lidocaine and granisetron inhibited fluid secretion. However, granisetron alone, and proportionately, reduced 5-HT release. 7. The simultaneous inhibition of 5-HT release and fluid secretion by granisetron suggests that 5-HT release from enterochromaffin cells is potentiated by endogenous 5-HT(3) receptors. The accentuated 5-HT release promotes cholera toxin-induced fluid secretion.     

GANGLION BLOCKING ACTIVITY OF CIMETIDINE IN THE ANAESTHETIZED CAT

Cimetidine produced a dose-dependent and reversible inhibition of contractions of the nictitating membrane elicited through stimulation of the preganglionic nerve supplying the superior cervical ganglion in the anaesthetized cat. Postganglionic nerve stimulation resulted in only a slight and variable inhibition of the contractions. Both hexamethonium and cimetidine also produced a dose-dependent and reversible fall in arterial blood pressure. The ganglion blocking activity of cimetidine was much weaker than that of hexamethonium; the ED50 ratio (cimetidine:hexamethonium) calculated from the cumulative log dose-response curves for the two drugs was 64. The possible mechanism(s) of ganglion blockade produced by cimetidine is discussed including a possible action at nicotinic receptors, either directly or indirectly (via its anticholinesterase activity), and ion channel blockade. The clinical implications of cimetidine-induced ganglionic blockade are also discussed, especially with respect to sexual impotence associated with the use of cimetidine.     

Effects of chronic chlorisondamine administration on the sympatho-adrenal axis

The chronic administration of chlorisondamine, a ganglionic blocking agent, for periods up to 1 week produced dose-dependent increases in rat adrenal catecholamines (CA), tyrosine hydroxylase TH) activity and dopamine β-hydroxylase (DBH) activity, and also increased plasma levels of circulating DBH. Uptake of [³H]epinephrine into isolated adrenal medullary storage vesicles from rats given chlorisondamine was reduced compared to controls. Prior denervation of the adrenal eliminated the chlorisondamine-induced increases in CA and TH, and reduced but did not eliminate the increases in DBH. Atropine pretreatment did not reduce the stimulatory effects of chlorisondamine. Ganglionic blockade was effective throughout the period of chronic chlorisondamine administration. as confirmed by the failure of insulin to evoke CA secretion even when given at the longest time period between chlorisondamine injections. These data suggest that chlorisondamine produces a mixture of direct and trans-synaptic sympatho-adrenal stimulation as well as ganglionic blockade, and that the mixed effect persists upon chronic administration.     

Involvement of serotonin and calcium channels in the intestinal fluid secretion evoked by bile salt and cholera toxin.

1. The enteric nervous system (ENS) is activated when exposing the intestinal mucosa to cholera toxin or certain bile salts. Cholera toxin stimulates ENS, at least in part, by the release of 5-hydroxytryptamine (5-HT) from the enterochromaffin cells. Calcium channel blockers of the L-type markedly attenuate the fluid secretion and the luminal release of 5-HT caused by cholera toxin. 2. The objective of the present study was to elucidate if sodium deoxycholate activated ENS in a similar manner as cholera toxin. Furthermore, the effect of several calcium channel blockers was tested on the fluid secretion caused by cholera toxin or bile salt. 3. Sodium deoxycholate (4 mM) caused a release of 5-HT into the intestinal lumen, which was inhibited by calcium channel blockade. Granisetron, a 5-HT3 receptor blocker, partly inhibited the fluid secretion caused by bile salt. 4. The effects of nifedipine, felodipine, R-felodipine, H186/86 (t-butyl analogue of felodipine) on the fluid secretion caused by cholera toxin or sodium deoxycholate were studied. Both secretory states were markedly attenuated in a dose dependent manner by all calcium channel blockers tested regardless of their effects on arterial pressure. 5. It is concluded that both cholera toxin and bile salt activate ENS, at least in part, via a release of 5-HT from the enterochromaffin cells. The antisecretory effect calcium channel blockers is partly explained by an inhibition of this release of 5-HT.     

Role of mast cells and pro-inflammatory mediators on the intestinal secretion induced by cholera toxin.

Recent data suggest that diarrhea caused by Vibrio cholerae involves a pro-inflammatory mediators release, such as cytokines, prostaglandin and nitric oxide. The aim of this study was to investigate the role of mast cells and their mediators in the intestinal secretion induced by cholera toxin. We examined the dose responses, time course and role of mast cells and pro-inflammatory mediators in cholera toxin intestinal secretory response, in vivo. Cholera toxin caused a dose-dependent secretion, in ligated small intestine loops, at 18 h. Rats treated with 48/80 compound or ketotifen had a significant decrease in the intestinal secretory response. Cholera toxin secretion was significantly reduced by an unspecific histamine/serotonin receptor antagonist, histamine receptor antagonist, phospholipase A2 and cyclooxygenase inhibitors, platelet-activating factor (PAF) receptor antagonists and TNF-alpha synthesis blockers. On the other hand, pretreatment with a specific serotonin receptor antagonist and lipoxygenase inhibitors failed to block this effect. Analysis of the intestinal fluid from rats injected with cholera toxin, revealed that cholera toxin induces the release of IL-1beta and TNF-alpha into fluid. The data suggest that, at least in part, mast cells are involved in cholera toxin-induced secretion, as well as point to the importance of histamine, prostaglandins, PAF, IL-1beta and TNF-alpha in this process.     

A STUDY OF GASTRIC SECRETION IN DOGS USING PILOCARPINE

• 1 Pilocarpine (40 μg/min i.v.) stimulated acid and pepsin secretion from Heidenhain pouches but did not stimulate secretion from the innervated stomach.
• 2 Pilocarpine significantly increased acid secretion from the innervated main stomach after the vagal nerves were bilaterally blocked with lidocaine.
• 3 The effect of pilocarpine on secretion from the Heidenhain pouch was depressed by ganglionic blockade (hexamethonium), by dopamine and by isoprenaline.
• 4 The stimulatory action of histamine on gastric secretion from either pouch or gastric fistulae was uninfluenced by dopamine.
• 5 The action of pilocarpine was augmented by the muscarinic ganglionic stimulant AHR 602. Considering this effect, as well as the secretory depression of isoprenaline, pilocarpine resembles pentagastrin rather than methacholine in its action.

     

Activation of the micturition reflex by NK2 receptor stimulation in the anaesthetized guinea-pig.

1. The mechanisms underlying stimulation of bladder contractions and bronchoconstriction by the selective NK2 receptor agonist, [beta-Ala8]NKA(4-10), were examined in the anaesthetized guinea-pig. 2. Atropine, alpha,beta-methylene-ATP and ganglion blocking agents were used to examine the contribution of reflex arc activation and/or potentiation of efferent mechanisms to the NK2 receptor-mediated responses seen in these two tissues. 3. [beta-Ala8]NKA(4-10)-induced bronchoconstriction was immediate, dose-dependent and was unaffected by pretreatment with ganglion blockers (hexamethonium or chlorisondamine), blockade of muscarinic receptors by atropine, or desensitization of P2 purinoceptors by alpha,beta-methylene-ATP. 4. At does of 5 micrograms kg-1 and above, [beta-Ala8]NKA(4-10) induced bladder contractions that appeared to be of an 'all-or-nothing' nature. These contractions occurred after a delay of 10 to 30 s and were often biphasic, comprised of an initial rapid component followed by a slower tonic component. 5. Pretreatment of the animals with either atropine or the desensitizing purinoceptor agonist alpha,beta-methylene-ATP, resulted in partial inhibition of bladder contractile responses to [beta-Ala8]NKA(4-10). The combination of atropine and alpha,beta-methylene-ATP pretreatment resulted in additive inhibition leading to complete blockade of the response. 6. The bladder responses to [beta-Ala8]NKA(4-10) (5 micrograms kg-1) were inhibited by pretreatment with the ganglion blockers, hexamethonium and chlorisondamine, indicating a preganglionic mechanism of action. 7. These findings demonstrate the indirect nature of the bladder contractions induced by activation of NK2 receptors in the anaesthetized guinea-pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular effects of brevetoxins in dogs

In anesthetized spontaneously breathing dogs, brevetoxin caused dose-dependent periods of apnea and, at high doses, respiratory arrest. In artificially ventilated dogs, i.v. brevetoxin caused complex dose-related cardiovascular changes consisting of: (1) bradycardia; (2) triphasic blood pressure changes, sequentially characterized by depressor/pressor/depressor phases; (3) cardiac arrhythmias including ventricular fibrillation; (4) muscle fasciculations. Prevention of bradycardia by atropine, vagotomy or ganglionic blockage unmasked a tachycardic action of the toxin. Ganglionic blockade, but not atropine or vagotomy, reduced the initial depressor effect of toxin. Phentolamine prevented the toxin-induced initial hypotension and secondary hypertension. Propranolol prevented the tachycardic and late depressor effects of toxin. In reserpinized dogs, low doses of toxin caused muscle fasciculations but none of the above cardiovascular effects; large doses caused bradycardia preventable by atropine, but not by vagotomy or chlorisondamine. These results suggest that brevetoxin: elicits the Bezold-Jarisch effect, i.e. initial hypotension, bradycardia and apnea; releases catecholamines, probably adrenal epinephrine, causing tachycardic, secondary pressor and late depressor effects; in large doses, releases vagal acetylcholine; -induced catecholamine and acetylcholine release is not nicotinic; produces effects, like those reported for veratridine, attributable to a common action on excitable membranes.     

Inhibition of cholera toxin-induced intestinal secretion by the 5-HT3 receptor antagonist ICS 205–930

Summary The secretory effect of cholera toxin on the gut has been ascribed to the activation of submucosal 5-hydroxytryptamine receptors by 5-hydroxytryptamine released from the enterochromaffin cells. The hypothesis that neuronally located 5-HT₃ receptors are involved in cholera toxin-induced diarrhoea has now been tested. Intestinal secretion was stimulated in mice by oral administration of pure cholera toxin and the effects of ICS 205-930, a potent and selective antagonist at 5-HT₃ receptors, and of ketanserin, a compound that blocks 5-HT₂ receptors, were investigated.Oral administration of cholera toxin resulted in a significant accumulation of fluid in the intestine. Pretreatment of the animals with ICS 205–930 partly prevented this effect and a maximal reduction of about 50% was achieved with doses of 0.3 mg/kg i. p. and higher. Ketanserin had only minimal effects up to a high dose of 1 mg/kg i. p. when a 20% reduction of fluid accumulation was seen. The data support the view that activation of 5-HT3 receptors plays a major role in the secretory effect of cholera toxin in the gut.     

Regional differences in neurogenic signal transduction pathway of cholera toxin-induced fluid, electrolyte and serotonin accumulation in the porcine jejunum

Serotonin, acetylcholine and substance P are mediators involved in the secretory response to cholera toxin in the small intestine. The aim of this study was to investigate the regional difference in the effect of a serotonin receptor type 3 antagonist (ondansetron), a nicotinic receptor antagonist (hexamethonium), and a substance P antagonist (the neurokinin receptor type 1 antagonist, CP 99,994) on the cholera toxin-induced fluid accumulation in the porcine jejunum. A dose-range of cholera toxin (0.32-56.00 microg/loop) was instilled for 4 hr in ligated loops in two regions of the proximal jejunum in 6-8-week-old pigs. Ondansetron (200 microg/kg), hexamethonium (10 mg/kg), CP 99,994 (1 mg/kg), or saline alone (control) were given intravenously 10 min. before cholera toxin instillation. Cardiovascular parameters, blood gas data, net fluid accumulation, serotonin and electrolyte concentration in the accumulated fluid were measured. Cardiovascular and blood gas parameters were within the normal range in all treatments. The apparent maximal response in fluid accumulation was reduced 20% in case of ondansetron, and by 33% using CP 99,994 in the aboral region compared to control, whereas no effect was observed in the oral region. Hexamethonium reduced the apparent maximal secretory response in both the oral and aboral regions by 45%. None of the treatments with antagonists changed the luminal content of serotonin or the electrolyte concentrations in the accumulated fluid. The results demonstrate that the involvement of serotonin receptor type 3 and neurokinin type 1 receptors in the transductional pathway of cholera toxin-induced fluid accumulation vary significantly within the jejunum, while the cholinergic (nicotinic) transmission plays an even role.     

Some pharmacological effects of the nematocide, morantel

1. In isolated nerve-muscle preparations as well as in nerve-muscle preparations in intact anaesthetized animals, morantel exhibited neuromuscular blocking properties similar to those of depolarizing blockers. The drug also caused spastic paralysis of 3 day-old chicks and contracture of the isolated toad rectus abdominis muscle.

2. Morantel caused contraction of the guinea-pig and rabbit isolated small intestine. This effect was antagonized by atropine and hexamethonium.

3. Morantel caused an increase in the blood pressure of the anaesthetized rat and cat and contraction of the nictitating membrane of the anaesthetized cat. These effects were antagonized by hexamethonium.

4. It was concluded that morantel (like the related compound pyrantel) has acetylcholine-like action and that its structure is consistent with such action.

     

Blockade by prostaglandins E2 and F1α of the response of the rabbit ileum to stimulation of sympathetic nerve and its reversal by some antihistamines,dexamphetamine and methylphenidate

Prostaglandins E₂ and F_1α, 0.05–1.0 μg/ml, blocked the inhibitory response of the isolated rabbit ileum to stimulation of periarterial (sympathetic) nerve without affecting responses to noradrenaline. The sympathetic blockade by PGE₂ and PGF_1α had a quick onset, did not increase much by time, and was more effective in blocking responses to stimulation of periarterial nerves at low frequencies (3, 5 and 7 shocks/sec) than at high frequencies (10, 20 and 60 shocks/sec). The blockade of sympathetic neurotransmission by PGE₂ and PGF_1α was easily reversed by brief washing, by raising the calcium ion concentration, and by the addition of dexamphetamine, methylphenidate, chlorpheniramine and diphenhydramine but not by antazoline. The sympathetic blockade by PGE₂ and PGF_1α is most probably through reduction in the amount of the transmitter released.     

Tetrodotoxin inhibits directly acting stimulants of intestinal fluid secretion

Intravenous injection of tetrodotoxin (20 μg kg^?1) to anaesthetized rats blocks PGE₁- and inhibits VIP-induced fluid secretion from the mucosa of the jejunum without affecting the physiological rates of net water and glucose absorption. It is suggested that the toxin might release an endogenous inhibitor of secretion or that it might possess direct antisecretory activity on the epithelial cells of the jejunum. It is further suggested that results where tetrodotoxin has been used to investigate secretagogue action should be treated with caution.     

The effects of PGE1 on responses to cardiac vagus nerve stimulation and acetylcholine release

PGE₁, 2.5 × 10^?9 to 5 × 10^?9 g/ml, reduced the negative chronotropic responses to vagal stimulation and to acetylcholine in isolated guinea-pig atria. Neither ganglionic transmission nor axonal impulse conduction were depressed by the compound in anaesthetized cats. PGE₁ did not affect acetylcholine release from the parasympathetic nerve terminals of the guinea-pig ileum. These findings are in disagreement with the view that PGE₁ modulates parasympathetic transmission by reducing the release of the cholinergic transmitter. It reduces vagal bradycardia by antagonizing the effect of acetylcholine on the atrial cells. This ‘anticholinergic’ action may be related to the cyclic AMP system.     

DOCA-SALT INDUCED MYOCARDIAL SENSITIZATION TO VENTRICULAR FIBRILLATION BY ISOPRENALINE IN RATS. ROLE OF THE AUTONOMIC NERVOUS SYSTME

• 1 The possible role of the autonomic nervous system in the development of myocardial sensitization to isoprenaline produced by DOCA-salt pretreatment was evaluated in bilaterally unanaesthetized rats subject to chronic ganglionic blockade with chlorisondamine or hexamethonium and to treatment with 6-hydroxydopamine.
• 2 Isoprenaline produced ventricular fibrillation and death in rats pretreated for 6 days wirh DOCA (20 mg pellet, s.c.) and 0.9% saline. The incidence of mortality was dose dependent and was 76.6% with 150 pgfkg of isoprenaline.
• 3 Adrenalectomy did not alter the mortality rate. The combination of adrenalectomy with concurrent chronic ganglionic blockade significantly decreased the incidence of mortality and delayed the time of death.
• 4 In contrast, 6-hydroxydopamine treatment and adrenalectomy did not alter the incidence of mortality nor time of death.
• 5 No differences in myocardial noradrenaline turnover were detected in rats susceptible and non-susceptible to death in ventricular fibrillation.
• 6 We conclude that myocardial sensitization to isoprenaline induced by DOCA-salt requires a neurogenic component, which is not, however, manifested by changes in myocardial noradrenaline turnover.

     

Adrenergic-induced hyperglycemia in anaesthetized rats: involvement of peripheral alpha 2-adrenoceptors

The selective alpha 2-adrenoceptor agonist, UK 14.304, induces a time- and dose-dependent hyperglycemic response in the anaesthetized rat. This hyperglycemia seems to be mediated through the pancreas because lesions of the pancreatic beta-cells induced with streptozotocin completely abolished this hyperglycemic response to UK 14.304 while ganglionic blockade by chlorisondamine did not affect the response. The non-selective agonist, adrenaline, induces a similar hyperglycemic response which is antagonized selectivity by the alpha 2-adrenoceptor antagonist, idazoxan. Combined alpha 1- and beta-adrenoceptor blockade resulted in an increase in the alpha 2-adrenoceptor-mediated hyperglycemic response to adrenaline. Since adrenaline is known not to cross the blood-brain barrier it is concluded that alpha 2-adrenoceptor stimulation induces hyperglycemic responses through a peripheral effect that involves postsynaptic alpha 2-adrenoceptors in the pancreatic beta-cell which are linked to the inhibition of insulin release.     

Central respiratory and circulatory effects of Gymnodinium breve toxin in anaesthetized cats

1 In cats anaesthetized with pentobarbitone, observations were made on respiration, spontaneous and evoked diaphragmatic electromyograms, blood pressure, heart rate, indirectly-induced contractions of the anterior tibialis muscle and nictitating membrane, and electrical excitability of the inspiratory centre in the medulla oblongata.

2 Gymnodinium breve toxin (GBTX) was administered intravenously, intra-arterially to the brain, and intracerebroventricularly. Physiological effects were recorded while alveolar P_CO2 was controlled at a constant level except when changes in gas tension were made in order to measure CO₂-ventilatory responsiveness.

3 Adequate doses of GBTX given intravenously by bolus injection elicited a non-tachyphylactic reflex response triad of apnoea, hypotension and bradycardia mediated by the vagus nerves independently of arterial baroreceptor and chemoreceptor innervation.

4 After vagotomy, additional amounts of GBTX (i.v.) resulted in apneustic breathing, hypertension and tachycardia. The cardiovascular effects were abolished by ganglionic blockade with hexamethonium.

5 Smaller doses of GBTX were required intra-arterially and intracerebroventricularly than by the intravenous route of injection to produce respiratory irregularity and cardiovascular hyperactivity.

6 Evoked motor responses, electrical excitability of the medulla oblongata and CO₂-ventilatory responsiveness were largely spared even though GBTX caused marked disturbances in respiratory rhythmicity and cardiovascular functions.

7 It is concluded that GBTX acts reflexly on vagally innervated receptors to evoke a Bezold-Jarisch effect but that the toxin further acts centrally to cause irregular breathholding and hypertension with tachycardia, leading ultimately to respiratory and circulatory failure.

     

The Effect of a-Trinositol on Cholera Toxin-Induced Hypersecretion and Morphological Changes in Pig Jejunum

Abstract: α-Trinositol (D-myo-inositol 1,2,6-trisphosphatc, PP56) is a novel antiinflammatory drug. This study elucidates the effect of intravenous α-trinositol on basal and acute fluid transport and morphological changes following cholera toxin administration in pig jejunum in vivo. Using isolated jejunal tied-off loops, the fluid hypersecretory (accumulation) effect of different doses of cholera toxin was studied in pigs treated intravenously with saline added different doses (0, 4 , 8, 16 and 32 mg×kg^-1×hr^-1) of α-trinositol. Levels of α-trinositol, as well as stereomicroscopical, light microscopical and scanning electron microscopical morphological studies were performed. Cholera toxin evoked a dose-dependent fluid hypersecretion. Treatment with α-trinositol caused a dose-dependent inhibition of the cholera toxin-induced fluid hypersecretion and did not affect basal fluid absorption. The 16 mg×kg^-1xhr^-1 α-trinositol dose gave a maximal inhibition of 36%. Morphological studies showed only minor changes following 6 hr of exposure to 20 μg×loop^-1 cholera toxin. These changes consisted of dilation of the villus capillaries, an increase of apical membrane blebbing and a reduction of the intercellular space. Treatment with 16 mg×kg^-1+hr^-1 a-trinositol alone did not induce any morphological changes, and did not alter the morphological changes induced by cholera toxin, which caused fluid hypersecretion and only minor acute morphological changes. In conclusion, a-trinositol treatment reduced cholera toxin-induced fluid hypersecretion without altering basal fluid absorption, basal morphology, or cholera toxin-induced morphological changes in pig jejunum in vivo.     

Pharmacological evidence that the sympathetic nervous system mediates the increase in secretion of renin produced by p-chloroamphetamine

The increase in plasma renin activity produced by p-chloroamphetamine in unanesthetized rats is blocked by p-chlorophenylalanine and by lesions of the dorsal raphe nucleus and the mediobasal hypothalamus. To determine whether the pathway from these areas of the brain to the kidneys that mediates the renin response is sympathetic, the effect of beta-adrenergic blockade and ganglionic blockade on the renin response to p-chloroamphetamine was studied. The increase in plasma renin activity 60 min after the administration of p-chloroamphetamine (10 mg/kg, i.p.) was prevented by the beta-adrenergic antagonist (-)-propranolol (1 mg/kg, i.p.), but not by (+)-propranolol (1 mg/kg, i.p.), given 30 min before p-chloroamphetamine. Sotalol (30 mg/kg, i.p.) injected 30 min before p-chloroamphetamine also blocked the renin response. The ganglionic-blocking drug chlorisondamine lowered blood pressure and increased plasma renin activity by itself. However, p-chloroamphetamine administered 30 min after chlorisondamine produced no further increase in plasma renin activity. Chlorisondamine, by itself, did not produce maximal secretion of renin, since isoproterenol, 30 min after chlorisondamine, produced a large increase in plasma renin activity. The data indicate that the increase in plasma renin activity produced by p-chloroamphetamine is mediated via the sympathetic nervous system.     

Epinephrine secretion from the perfused adrenal gland produced by scorpion toxin

A purified neurotoxin obtained from the scorpion Leiurus quinquestriatus was used to stimulate epinephrine (E) secretion from the isolated, perfused bovine adrenal gland. In low concentrations values. Cholinergic blockade with hexamethonium and atropine completely blocked E secretion stimulated by either toxin or acetylcholine but not secretion stimulated by potassium chloride. The results suggest that the toxin stimulates E secretion through cholinergic mechanisms either by causing release of acetylcholine from splanchnic nerve endings or by directly activating cholinergic receptors on the adrenal chromaffin cell.