Mechanisms of the secretory response to luminal propionate in rat descending colon in vitro.

Short chain fatty acids stimulate Cl secretion in rat descending colon in vitro via an enteric reflex involving mucosa and cholinergic nerves. We used the short circuit current as the measure of Cl secretion caused by Na propionate (NaP) (0.5 mM) in luminal bath fluid and studied the mechanism of the response. The NaP response was decreased 81% by atropine and 76% by lidocaine. It was unaffected by tetrodotoxin, omega-conotoxin or by tachyphylaxis to capsaicin, CGRP, substance P, histamine or PGE2. It was not reduced by inhibitors of 5-HT2 or 5HT3 receptors or by partial tachyphylaxis to 5-HT. However, superficial mucosal injury with hypertonic Na sulfate (2 M) or xylose (4.5 M) reduced the NaP response by 90% and 86%, respectively, and mucosal concanavalin A (1 mg/ml) reduced it by 73%. Neither piroxicam (10 microM) nor nordihydroguaretic acid (10 microM) affected the NaP response. We hypothesize that NaP stimulates the superficial epithelium to release an unidentified agonist that depolarizes predominantly cholinergic nerve terminals and causes colonic secretion.

Secretory reflexes in ileum and jejunum: absence of remote effects.

We have tested the hypothesis that luminal secretagogues initiate neural reflexes that alter ion transport in small intestinal segments proximal or distal to the site of the secretory stimulus. Effects of secretagogues that act by different mechanisms were studied in vitro by measuring short circuit current (ISC) of ileum or jejunum mounted in a unique flux chamber while proximal mucosa in neural continuity with the tissue was perfused with secretagogues (Na deoxycholate. Escherichia coli STa, 5-hydroxytryptamine, theophylline) or was stimulated electrically (EFS). No proximal stimulus affected distal ISC. We also studied in vivo adjacent segments of ileum or jejunum in neural continuity but with unconnected lumens. In anesthetized rabbits, we measured transmural electrical potential difference and fluid movement (Phenol red marker). Stimulation of proximal segments of ileum or jejunum with STa, or of ileum with 5-HT or Na deoxycholate did not affect distal transport. Stimulation of distal segments of ileum and jejunum with STa or 5-HT, or of jejunum with Ha deoxycholate did not affect proximal transport. We conclude that the secretion caused by luminal secretagogues in the rabbit small intestine is limited to the area of stimulation.

Ion transport in normal and inflamed human jejunum in vitro. Changes with electric field stimulation and theophylline.

We studied ion transport in human jejunal mucosa under basal conditions and when tissues were stimulated electrically (EFS) and with theophylline 5 mM (N = 12 pairs). Tissues were mounted in a flux chamber to measure unidirectional fluxes of 22Na, 36Cl, short-circuit current, Isc (mueq/cm2/hr), electrical potential difference, PD (mV), and total ionic conductance, G (mS/cm2). Six pairs of tissues that were normal or less inflamed responded to theophylline by increasing PD and Isc and by reducing JCl(ms), the mucosal to serosal flux of Cl. In six pairs that did not respond to theophylline or to EFS, inflammation was more intense, and in the basal state, G was lower, PD was higher, and unidirectional fluxes of Na and Cl were halved. We conclude: (1) normal jejunum secretes Cl and responds to theophylline or EFS by reducing JCl(ms) thereby increasing Cl secretion; (2) unlike human ileum, neither theophylline nor EFS affect JCl(sm); (3) inflammation eliminates the response to theophylline and to EFS.

Noradrenergic influence on epithelial responses of rabbit ileum to secretagogues.

Norepinephrine is one of three neurotransmitters that may act directly on enterocytes to enhance absorption; its interaction with secretagogues is of physiological importance. We have studied the influence of norepinephrine on the short-circuit current (Isc) responses to acetylcholine (ACh; 10 microM), vasoactive intestinal polypeptide (VIP; 100 pM-10 nM), peptide histidine isoleucine (PHI; 100 pM-10 nM), histamine (0.1 mM), and to electrical field stimulation (EFS) of rabbit ileum mounted in flux chambers. Tetrodotoxin reduced the response to norepinephrine (10 microM) by 40% and to histamine by 32% but did not affect responses to VIP or PHI. Norepinephrine decreased the ACh response (EC50, 70 nM) and reduced the responses to PHI (less than or equal to 87%), to EFS (less than or equal to 75%), and to histamine (less than or equal to 42%). Norepinephrine decreased the response to VIP (500 pM) but not to higher or lower VIP concentrations. It enhanced the response to VIP (10 nM) and to theophylline (5 mM). We conclude that 1) norepinephrine increases absorption by acting on nerves and enterocytes; 2) the failure of norepinephrine to reduce the Isc response to VIP when the VIP-induced increment in Isc is comparable to that caused by EFS is evidence that VIP does not mediate the EFS response; 3) PHI might mediate the EFS response; and 4) VIP, PHI, and histamine affect enterocytes directly; histamine also affects intrinsic nerves.

The neurosecretory effect of ouabain on isolated rabbit ileal mucosa.

Ouabain, when added to fluid bathing rabbit ileal mucosa mounted in a flux chamber, transiently increases short circuit current, implying a paradoxical secretory response. To determine the cause of this change, we studied unidirectional fluxes of 36Cl and 23Na and the effects of ion substitution, of reduced Ca concentration, verapamil, tetrodotoxin and atropine. Ouabain 0.1 mM, transiently increased the serosal to mucosal flux of Cl and Na, increased Isc and PD and reduced ion conductance. The Isc response to ouabain was diminished by reducing the bath fluid concentration of Cl, of Ca, and by adding verapamil. Tetrodotoxin both delayed and reduced the maximal Isc response; atropine had no effect. We conclude that ouabain acts by releasing a neurotransmitter of unknown identity and by increasing the serosal to mucosal flux of Cl.

Ion transport in human cecum, transverse colon, and sigmoid colon in vitro. Baseline and response to electrical stimulation of intrinsic nerves.

In a flux chamber study of ion transport in human colon, we compared baseline rates with those measured during electrical stimulation of intrinsic nerves. In baseline studies, sodium was absorbed throughout, but maximally in transverse colon. In cecum, sodium absorption accounted for the short circuit current and chloride was not absorbed. Chloride was absorbed in transverse and sigmoid colon, however. Residual current was minimal in cecum and transverse colon, but increased in sigmoid colon. Neural stimulation caused chloride secretion in cecum, reduced chloride absorption in sigmoid colon, but caused no change in transverse colon; sodium absorption decreased in cecum. A neurotransmitter of unknown identity affects baseline short circuit current in sigmoid colon. Half of the increase in short circuit caused by neural stimulation in sigmoid colon is mediated by muscarinic receptors. The identity of the other transmitter(s) is not known. It is not substance P or histamine. The three divisions of the colon differ in relative rates of baseline ion transport and in their transport responses to intrinsic nerve stimulation.

Effect of neuropeptide Y on ion transport by the rabbit ileum.

Neuropeptide Y (NPY) is present in fibers extending from the submucous plexus to the epithelium of the small intestine where the liberation of NPY might affect ion transport. We sought the effects of NPY on rabbit ileal mucosa stripped of muscularis propria and mounted in a flux chamber. NPY reduced the transmural electrical potential difference and short circuit current (Isc) and increased total ionic conductance. Threshold and maximal effects were evoked at concentrations of 1 nM and 1 microM, respectively. NPY increased chloride absorption, JCl(net), by increasing the flux of Cl from mucosa to serosa, JCl(ms), and by decreasing JCl(sm). JNa(net) actually diminished because JNa(sm) rose more than JNa(ms). In the presence of NPY theophylline 5 mM caused Cl secretion, increased potential difference and Isc and reduced total ionic conductance, indicating that the tissue could respond to a secretagogue. Tetrodotoxin 0.1 microM did not diminish the Isc reduction caused by NPY, and desensitization did not alter the response of the tissue to electrical field stimulation. Like somatostatin and norepinephrine, which are also present in the submucous plexus, NPY increases Cl absorption, but unlike them, it reduces rather than augments Na absorption. The lack of effect of tetrodotoxin on the Isc response to NPY implies that NPY does not act by liberating a second neurotransmitter; the lack of effect of NPY desensitization indicates that the liberation of NPY plays no significant role in the response of the tissue to electrical field stimulation.

Intestinal nerves and ion transport: stimuli, reflexes, and responses.

The effects of extrinsic and intrinsic nerves on ion and water transport by the intestine are considered and discussed in terms of their possible physiological function. Adrenergic nerves enter the small intestine via mesenteric nerves. Adrenergic tone is usually absent in tissues in vitro but is present in vivo. The nerves increase absorption in response to homeostatic changes associated with acute depletion of extracellular fluid. Cholinergic tone that reduces fluid absorption or causes secretion has been detected in the small intestine of humans, dogs, and cats and in the colon of humans. Extrinsic cholinergic fibers generally do not affect ion transport in small intestine but probably do so in colon. Whether peptides liberated in the mucosa affect enterocytes directly is not clear. Studies on humans and rabbits suggest that the role of substance P is minor. The physiological roles of vasoactive intestinal polypeptide (VIP) and somatostatin remain to be defined. Intraluminal factors also affect ion and water transport. Mucosal rubbing, distension, and cholera toxin cause fluid secretion; acid solutions in the duodenum cause alkaline secretion; these stimuli and hypertonic glucose liberate serotonin into the lumen, the mesenteric venous blood, or both. It has been proposed that the enterochromaffin cell is an epithelial sensory cell that responds to noxious stimuli within the lumen by liberating serotonin. The serotonin initiates a neural reflex through a nicotinic ganglion to liberate a secretagogue that acts on the enterocyte. The function of VIP in this proposed reflex is unclear. The variety of intraluminal stimuli that influence epithelial function implies that there is more than one type of epithelial sensory cell (or sensory mechanism). Prostaglandins may mediate the alkaline secretion caused by acid in the duodenum. There may be other effective substances. Although it has been known for years that intraluminal stimuli affect the coordination of smooth muscle functions, it is not known whether similar stimuli also influence salt and water transport as a meal traverses the alimentary canal.

Electrical stimulus-secretion coupling in rabbit ileal mucosa.

Rabbit ileal mucosa, when mounted in a flux chamber and stimulated with a 5 Hz electrical field (EFS), secretes Cl, a change reflected in an increase in short circuit current (Iac). Because the EFS response is eliminated by agents which prevent neural transmission, the mediator is most likely a neurotransmitter present in nerves lying close to the secreting epithelium. To identify the chemical mediators of the response, we determined the effects of receptor antagonists, agonist desensitization and other agents on the Isc response to EFS. Because of the failure of antagonists or desensitization to affect the response to EFS, we eliminated the following agents as possible mediators: acetylcholine (pre- and postganglionic), norepinephrine, dopamine, 5-hydroxytryptamine, histamine, prostaglandins, adenosine triphosphate, bombesin, neurotensin and Substance P. Pyrilamine, diphenhydramine and cyproheptadine in high concentration (0.1 mM) reduced markedly the Isc response to EFS for reasons unrelated to histamine antagonism. Although acetylcholine has been shown in the ileum of humans and of guinea pigs to mediate up to half of the Isc response to EFS, the identity of the mediator(s) in rabbit ileum remains unknown.

Chymotrypsin, ileal chloride transport, and neurotransmitters.

Electrical field stimulation (EFS) depolarizes nerves and causes chloride secretion by mucosa of rabbit ileum mounted in a flux chamber. To test the hypothesis that the transmitter is a peptide, we determined whether the EFS response was prevented by the endopeptidase chymotrypsin (CT). Serosal, but not mucosal, addition of CT (200 micrograms/ml) reduced the short-circuit current (Isc) response to EFS by 90% or more. CT also reduced Cl absorption by decreasing the mucosal-to-serosal flux, but it did not affect net Na absorption. CT prevented the response to vasoactive intestinal polypeptides, but the response returned when CT activity was eliminated. The response to EFS did not return, however, implying that CT damaged cells that released transmitter or epithelial target cells. CT reduced the Isc response to serotonin by 69% and to A23187 by 10% and did not affect the theophylline response. We conclude that 1) the effects of CT on cell function limit its usefulness in identifying peptide neurotransmitters in epithelium, 2) CT irreversibly inhibits ion transport responses to EFS and to serotonin, and 3) CT reduces absorption of Cl probably by affecting a calcium pathway that modifies Cl transport.

Effects of scorpion venom on electrolyte transport by rabbit ileum.

Scorpion venom, which depolarizes nerves, was used to obtain further evidence that intramural nerves affect ion transport by the rabbit ileum. Ileal epithelium, stripped of muscularis propria, was mounted in a flux chamber modified to permit electrical field stimulation (EFS) of the tissue. Response of the short-circuit current (Isc) to venom was most rapid on the serosal surface, and the response was eliminated by tetrodotoxin. Isc response was influenced by venom batch number and by factors within the tissue. Venom (10 micrograms/ml) and EFS each caused chloride secretion by reducing mucosal-to-serosal movement and by increasing serosal-to-mucosal movement. Sodium transport and residual ion fluxes did not change. In the presence of venom, EFS caused no further changes in ion transport, but tissues still responded to glucose and to aminophylline. The early peak of Isc was reduced about 40% by atropine, implying that acetylcholine, released by venom, stimulates muscarinic receptors. The blockade of the Isc response to venom with tetrodotoxin is further evidence that venom depolarizes intramural nerves and liberates transmitters that cause chloride secretion. The identity of the other transmitters is not known.

Release of vasoactive intestinal polypeptide by electrical field stimulation of rabbit ileum.

The release of vasoactive intestinal polypeptide (VIP) induced by electrical field stimulation (EFS) of rabbit ileum was studied in vitro. EFS parallel to the muscularis propria caused a significant increase in VIP concentration in the buffer bathing the serosal surface of full-thickness ileum. This effect was blocked by 10(-7) M tetrodotoxin. When circular and longitudinal muscle was removed, the amount of measurable VIP in the tissue decreased to about one-half that of full-thickness ileum, and EFS no longer caused release of VIP into the serosal or mucosal buffers. Our data indicate that EFS of rabbit ileum causes release of VIP, presumably form VIP-containing nerves present in the tissue. These results support the idea that VIP may be a physiological neuroregulator of intestinal function.

Effect of veratrine and 50 mM K on ileal transport and electrically induced secretion.

To obtain additional evidence that the Cl secretion caused by electrical field stimulation (EFS) in rabbit ileum in vitro is mediated by nerves or amine precursor uptake and decarboxylation (APUD) cells, we determined whether the response could be prevented by agents that depolarize nerves, veratrine or 50 mM K. EFS 1) increased transmural electrical potential difference (PD) and the short-circuit current (Isc) and reduced total ion conductivity (G), 2) caused Cl secretion by reducing the mucosal-to-serosal flow of chloride (JClm leads to s) alone or in combination with an increase in the serosal-to-mucosal flux of chloride (JCls leads to m), and 3) reduced Na absorption or caused secretion by increasing JCls leads to m alone or in association with a reduction in JNam leads to s. At 10(-5) g/ml, veratrine prevented the changes in ionic fluxes caused by EFS and reduced the PD and Isc. It did not reduce the Isc response to 5 mM aminophylline, however. Fifty millimolar K decreased PD and Isc and increased G. JNam leads to s and JNas leads to m diminished in proportion to the reduction in bath Na concentration, so the net flux of sodium (JNanet) was not affected. JClnet did not change, although both JClm leads to s and JCls leads to m increased. Fifty millimolar K prevented the changes in ionic fluxes caused by EFS and reduced the Isc response to aminophylline by half. The study provides additional evidence that the effects of EFS are mediated by nerves or electrically excitable APUD cells, but the contribution of each is uncertain.

Effects of Ca2+ on ileal transport and electrically induced secretion.

[Ca2+] affects nerves and target cells in stimulus-secretion coupling. In flux-chamber studies of full-thickness rabbit ileum, we determined the effects of 1) ethylene glycol bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) 1.25 mM, 2) verapamil 0.1 mM and nifedipine 0.1 mM, and 3) trifluoperazine 0.1 mM on ion transport and its response to electrical field stimulation (EFS). EGTA increased JClm leads to s, JNam leads to s, Cl absorption and conductivity (G), and reduced Isc. In the absence of EGTA, EFS increased transmural PD and Isc and caused secretion of Na and Cl. EGTA prevented the responses to EFS, but the Isc responses to aminophylline and to glucose were normal. Verapamil reduced the response of Isc and Cl transport to EFS. Nifedipine reduced Isc but not the Isc response to EFS. Trifluoperazine reduced Isc and almost eliminated the Isc response to EFS. EFS did not increase the tissue concentration of cAMP. We conclude: 1) low extracellular [Ca2+] enhances net Cl absorption; 2) extracellular Ca2+ is required for the response of ion transport to EFS; 3) cAMP does not mediate Isc response to EFS; and 4) Isc response to EFS is blocked by trifluoperazine. The findings suggest that EFS stimulates secretion by increasing Ca entry into the epithelial cells, either directly or indirectly.

The effects of electrical field stimulation and tetrodotoxin on ion transport by the isolated rabbit ileum.

To determine whether intramural nerves affect intestinal ion transport, we studied the effect of electrical field stimulation (EFS) on the movement of ions across isolated rabbit ileum. EFS increased the transmural electrical potential difference and the short circuit current (Isc), caused C1 secretion, and reduced conductance, but did not alter fluxes of Na or the residual current (JRnet). The neurotoxin, tetrodotoxin, prevented all the changes caused by EFS but did not prevent the increase in Isc caused by theophylline (5 mM), carbachol (10 micrometer), or glucose (10 mM), or the reduction in Isc caused by norepinephrine (10 micrometer), implying that tetrodotoxin prevented responses to EFS by affecting electrically excitable cells rather than epithelial cells. Tetrodotoxin also enhanced the mucosa to serosa fluxes of Na and C1, reduced the potential difference and Isc, and increased conductance. The site of tetrodotoxin action is uncertain because it may affect the release of at least four neuro-transmitters and the release of peptides from endoctine cells. The Isc response to EFS was not affected by atropine (10 micrometer), physostigmine (10 micrometer), or by hemicholinium (1 micrometer). The mechanism by which EFS causes C1 secretion remains to be determined.