Effect of aspirin on normal and cholera toxin-stimulated intestinal electrolyte transport.

The effect of aspirin on normal and cholera toxin-stimulated electrolyte transport has been investigated in vitro, because this drug appears to inhibit cholera toxin-induced intestinal secretion in in vivo animal models. In the Ussing chamber, 10 mM aspirin decreased the control rabbit ileal potential difference and short-circuit current by 50% and increased conductance by 28%. Bidirectional electrolyte flux determinations showed that aspirin significantly increased both Na and Cl absorption and reduced flux (which probably represents HCO3 secretion) to zero. This effect of aspirin appears to be identical to that reported to others with catecholamines as determined with similar techniques. However, alpha-adrenergic blockers did not prevent the electrical effects of aspirin, suggesting that aspirin does not have its effect through release of tissue stores of catecholamines. In the presence of aspirin, cholera toxin increased the potential difference and short-circuit current, and decreased the conductance of rabbit ileum in a fashion qualitatively similar to control tissues. However, aspirin reversed cholera toxin-stimulated Na transport from secretion to absorption, inhibited cholera toxin, induced Cl secretion by 58% and partially, but not significantly, inhibited HCO3 secretion. Thus, the inhibitory effect of aspirin on cholera toxin-induced electrolyte secretion appears to be due to aspirin-stimulated Na and Cl absorption. Although aspirin reduced tissue cyclic AMP concentrations in normal and cholera toxin-stimulated ileum, it also inhibited the electrolyte secretion induced by exogenous cyclic AMP. Thus, if aspirin's stimulatory effect on sodium and anion absorption in normal tissue and its inhibitory effect on cholera toxin-stimulated sodium and anion secretion involves a cyclic AMP-mediated system, the effect must be a step distal to cyclic AMP production or degradation. The exact mechanism of aspirin's effect on normal and cholera toxin-induced electrolyte transport, and its possible usefulness in the treatment of cholera diarrhea, remains to be determined.     

Vasoactive Intestinal Peptide Stimulation of Adenylate Cyclase and Active Electrolyte Secretion in Intestinal Mucosa

Vasoactive intestinal peptide (VIP), originally isolated from hog small intestinal mucosa, has been shown to cause small intestinal secretion. More recently, this peptide has been identified in the plasma and tumors of patients with the so-called "pancreatic cholera" syndrome. In order to explore the possible role of VIP in the pathogenesis of this syndrome, we examined the effects of this peptide and other hormones on the cyclic AMP levels, adenylate cyclase activity, and ion transport in in vitro preparations of ileal mucosa. In rabbit ileal mucosa, VIP (20 mug/ml) caused a prompt fivefold increase in cyclic AMP level, whereas nine other hormones, which have been postulated to cause intestinal secretion, failed to exert such an effect. Pentagastrin and glucagon also failed to increase cyclic AMP levels in canine ileal mucosa. An increase in mucosal cyclic AMP levels was observed at a VIP concentration of 0.1 mug/ml and appeared to be nearly maximal at 2.0 mug/ml. VIP (100 mug/ml) stimulated adenylate cyclase activity in a membrane preparation from rabbit ileal mucosa. Secretin (6.0 x 10(-5) M) failed to do so. When added to the serosal side of isolated rabbit ileal mucosa clamped in an Ussing chamber, VIP (2 mug/ml) increased short-circuit current (SCC) and caused net secretion of both Cl and Na. Net Cl secretion exceeded net Na secretion. These effects of VIP on mucosal cyclic AMP metabolism and ion transport are similar to those observed with cholera enterotoxin and certain prostaglandins. VIP was also tested with normal human ileal mucosa. At a concentration of 2 mug/ml it caused a fivefold increase in cyclic AMP level and an increase in SCC of the same magnitude as that caused by 5 mM theophylline. Addition of a second 2-mug/ml dose of VIP and addition of theophylline after VIP produced no further change in SCC. We conclude the VIP stimulates adenylate cyclase and active ion secretion in both rabbit and human ileal mucosa. This may be related to the pathogenesis of diarrhea in patients with the pancreatic cholera syndrome.     

In vitro behavior of human intestinal mucosa. The influence of acetyl choline on ion transport.

The possibility that the autonomic nervous system may influence the function of intestinal mucosa was investigated by assessing the effect of acetyl choline on ion transport in human intestine. Isolated pieces of stripped ileal mucosa were mounted in Perspex flux-chambers and bathed in isotonic glucose Ringer's solution. Acetyl choline caused a rise in mean potential difference (8.8-12.3 mV, P less than 0.002) and short circuit current (287.7-417.2 muA-cm-2, P less than 0.01) (n = 12), observable at a concentration of 0.01 mM and maximal at 0.1 mM. This effect was enhanced by neostigmine and blocked by atropine. Isotopic flux determinations revealed a change from a small mean net Cl absorption (58) to a net Cl secretion (-4.3mueq-cm-2-h-1P less than 0.001) due predominantly to an increase in the serosal to mucosal unidirectional flux of Cl (10.63-14.35 mueq-cm-2-h-1P less than 0.05) and a smaller reduction in the mucosal to serosal flux (11.22 to 10.02 mueq-cm-2-h-1P less than 0.05). Unidirectional and net Na transport was unaffected. A similar electrical and ion transport response was observed in a single study of two pieces of jejunal mucosa. In the absence of glucose net chloride secretion was produced and again an insignificant effect on net sodium transport was noted. Acetyl choline did not provoke a sustained effect on mucosal cyclic adenine nucleotide levels although a short-lived cyclic adenine nucleotide response was seen in some tissues 20-30 s after drug addition. These studies demonstrate that acetyl choline does influence human intestinal ion transport by stimulating chloride secretion and suggest a possible mechanism by which the parasympathetic nervous system could be concerned in the control of ion transport.     

Effect of Cholera Enterotoxin on Ion Transport across Isolated Ileal Mucosa

The effects of cholera enterotoxin on intestinal ion transport were examined in vitro. Addition of dialyzed filtrate of Vibrio cholerae (crude toxin) to the luminal side of isolated rabbit ileal mucosa caused a delayed and gradually progressive increase in transmural electric potential difference (PD) and shortcircuit current (SCC). A similar pattern was observed upon addition of a highly purified preparation of cholera toxin, although the changes in PD and SCC were smaller. Na and Cl fluxes across the short-circuited mucosa were determined with radioisotopes 3-4 hr after addition of crude toxin or at a comparable time in control tissues. The toxin caused a net secretory flux of Cl and reduced to zero the net absorptive flux of Na. Similar flux changes were observed when either crude or purified toxin was added in vivo and tissues were mounted in vitro 3-4 hr later. Additon of D-glucose to the luminal side of toxin-treated mucosa produced a large net absorptive flux of Na without altering the net Cl and residual ion fluxes.Adenosine 3',5'-cyclic phosphate (cyclic AMP) and theophylline had previously been shown to cause a rapid increase in SCC and ion flux changes similar to those induced by cholera toxin. Pretreatment of ileal mucosa with either crude or purified cholera toxin greatly reduced the SCC response to theophylline and dibutyryl cyclic AMP, which, together with the flux data, suggest that both cyclic AMP and cholera toxin stimulate active secretion by a common pathway. Inhibition of the SCC response to theophylline was observed after luminal but not after serosal addition of toxin. In vitro effects of cholera toxin correlated closely with in vivo effects: heating toxin destroyed both; two V. cholerae filtrates which were inactive in vivo proved also to be inactive in vitro; PD and volume flow measurements in isolated, in vivo ileal loops of rabbit revealed that the PD pattern after addition of toxin is similar to that seen in vitro and also correlates closely with changes in fluid movement. The results suggest that stimulation by cholera toxin of a cyclic AMP-dependent active secretory process of the intestinal epithelial cells is a major cause of fluid loss in cholera.     

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.     

Calcium dependence of serotonin-induced changes in rabbit ileal electrolyte transport.

These studies describe the calcium dependence of the serotonin-induced changes in active electrolyte transport in rabbit ileum in vitro. In the presence of a standard calcium concentration (1.2 mM) in the serosal bathing fluid, serosal serotonin caused a transient increase in short-circuit current and a prolonged decrease in net Na and Cl fluxes. Removing calcium from the serosal (no calcium plus 1 mM EGTA) but not the mucosal bathing fluid inhibited the serotoin-induced increase in ileal short-circuit current, and also completely blocked the serotonin effects on net Na and net Cl fluxes. This inhibition was rapidly reversed by readding calcium. Removing serosal calcium did not inhibit all active electrolyte transport processes, as the effect of a maximum concentration of theophylline (10 mM) was not altered. Similarly, d,l-verapamil, a calcium channel blocker, inhibited the serotonin-induced changes in short-circuit current and in net Na and net Cl fluxes, but did not alter the theophylline effects. In contrast, d-verapamil, a stereoisomer which does not block calcium channels, did not inhibit the serotonin-induced changes. The calcium dependence of these serotonin effects was associated with increased uptake of 45Ca into rabbit ileum, including increaed 45Ca uptake from the serosal surface. Serotonin also increased the rate of 45Ca efflux from rabbit ileum into a calcium-free solution, compatible with serotonin increasing the ileal plasma membrane permeability to calcium. It is postulated that serotonin affects active intestinal electrolyte transport by a mechanism dependent on serosal but not mucosal calcium that involves an increase in the intestinal plasma membrane permeability to calcium, and perhaps an increase in intracellular calcium.     

Effect of Klebsiella pneumoniae enterotoxin on intestinal transport in the rat.

The effects on intestinal transport of either a semipurified preparation of enterotoxin elaborated by Klebsiella pneumoniae or similaryly prepared control material were tested by marker perfusion studies in the small intestine of rats. At a concentration of 2 mg/ml, the enterotoxin produced net secretion of water, Na, and Cl in both jejunal and ileal segments; HCO3 transport was not affected. Net secretion was evident within 30 min after intorduction of the toxin and was maximal after 90 min. The addition of 56 mM glucose to the enterotoxin-containing perfusion fluid resulted in reversal of water and Na transport to net absorption in both intestinal segments. The enterotoxin also produced a significant depression of xylose absorption in both the jejunum and ileum but did not affect the absorption of either glucose or L-leucine. Intestinal structure was not altered after perfusion of the toxin but insillation of approximately one-quarter of the total perfusion dose into a ligated jejunal loop for 18 h produced fluid secretion and structural abnormalities. These observations confirm the fact that other species of coliform bacteria in addition to tescherichia coli are capable of elaborating an enterotoxin. Such species commonly contaminate the small intestine of persons with tropical sprue and it is suggested that chronic exposure of the intestinal mucosa to the enterotoxin elaborated by these bacteria may be a factor in the pathogenesis of intestinal abnormalities in thid disorder.     

Interrelationships of chloride, bicarbonate, sodium, and hydrogen transport in the human ileum

Using a triple-lumen constant perfusion system, the following observations were made in normal subjects. First, chloride, bicarbonate, and sodium were found to exhibit net movement across ileal mucosa against electrochemical gradients. Second, during perfusion with a balanced electrolyte solution simulating plasma, the ileum generally absorbed, but sometimes secreted fluid. A reciprocal net movement of chloride and bicarbonate was noted when sodium movement was zero. Increasing rates of sodium absorption were associated with decreasing bicarbonate secretion rates and finally bicarbonate absorption. Even when bicarbonate was absorbed ileal contents were alkalinized (by contraction of luminal volume). Third, net chloride movement was found to be sensitive to bicarbonate concentration in ileal fluid. For instance, chloride was absorbed from solutions containing 14 or 44 mEq/liter of bicarbonate, but was secreted when ileal fluid contained 87 mEq/liter of bicarbonate. Fourth, when chloridefree (sulfate) solutions were infused, the ileum absorbed sodium bicarbonate and the ileal contents were acidified. Fifth, when plasma-like solutions were infused, the potential difference (PD) between skin and ileal lumen was near zero and did not change when chloride was replaced by sulfate in the perfusion solution.These results suggest that ileal electrolyte transport occurs via a simultaneous double exchange, Cl/HCO2 and Na/H. In this model neither the anion nor the cation exchange causes net ion movement; net movement results from the chemical reaction between hydrogen and bicarbonate. No other unitary model explains all of the following observations: (a) human ileal transport in vivo is essentially nonelectrogenic even though Na, Cl, and HCO3 are transported against electrochemical gradients, (b) the ileum can secrete as well as absorb, (c) ileal contents are alkalinized during absorption of or during secretion into a plasma-like solution, and (d) the ileum acidifies its contents when sulfate replaces chloride. Data obtained with a carbonic anhydrase inhibitor support the proposed model.     

Characterization of aldosterone-induced potassium secretion in rat distal colon.

The role of apical and basolateral membranes in aldosterone-induced active potassium (K) secretion in rat distal colon was investigated by measuring mucosal-to-serosal (Jms) and serosal-to-mucosal (Jsm) 42K fluxes (mueq.h-1.cm-2) across isolated stripped mucosa under short-circuit conditions in normal and secondary-hyperaldosterone animals. In normal colons mucosal tetraethylammonium (TEA; 30 mM) or barium (Ba; 5 mM), but not cesium (Cs; 15 mM), reduced Jsm without affecting Jms. In aldosterone animals (a) net K secretion (-0.54 +/- 0.11) was converted to net K absorption (0.63 +/- 0.15) by mucosal TEA, which produced a marked reduction in Jsm (0.82 +/- 0.07) and an increase in Jms (0.35 +/- 0.07). In contrast mucosal Ba resulted in a relatively smaller reduction in JK(sm) without altering JK(ms), whereas mucosal Cs was ineffective; (b) serosal bumetanide or the removal of serosal Na or Cl markedly inhibited JK(sm and abolished net K secretion; and (c) serosal ouabain (1 mM) produced qualitatively similar effects to those of serosal bumetanide. These results demonstrate that (a) normal rat distal colon contains apical TEA- and Ba-sensitive K channels; (b) aldosterone induces TEA-sensitive and Ba-sensitive apical K channels; (c) aldosterone-induced K secretion requires both the Na,K-pump and Na-K-2Cl cotransport for K uptake across the basolateral membrane; and (d) alteration of any of these processes results in inhibition of aldosterone-induced active K secretion simultaneously with stimulation of K absorption.     

Ricinoleic acid stimulation of active anion secretion in colonic mucosa of the rat.

Perfusion of the colon with ricinoleic acid produces fluid and electrolyte accumulation. The mechanism of these changes in water and electrolyte movement is uknown. These studies were designed to determine whether ricinoleic acid effects active ion transport across isolated rat colonic mucosa. 0.5 mM Na ricinoleate produced significant increases in potential difference (3.8 +/- 0.5 mV) and short-circuit current (Isc) (99.2 +/- 10.1 muA/cm2). The increases in Isc produced by Na ricinoleate were inhibited by both removal of bicarbonate and chloride and by the presence of theophylline. The hydroxy fatty acid also resulted in a significant decrease in net Na absorption from 4.7 +/- 0.8 to 0.1 +/- 0.7 mueq/h cm2 and reversed net Cl transport from absorption (+ 4.5 +/- 0.9) to secretion (-2.2 +/- mueq/h cm2). In parallel studies 0.5 mM Na ricinoleate increased mucosal cyclic AMP content by 58%. The concentrations of Na ricinoleate required to produce detectable and maximal increases in both Isc and cyclic AMP were the same. These results provide evidence in support of the concept that hydroxy fatty acid-induced fluid and electrolyte accumulation is driven by an active ion secretory process.     

The effects of magnesium on ion transport in short-circuited rabbit terminal ileum

The effects of MgCl2 on the electrical characteristics and sodium and chloride transport in short-circuited rabbit terminal ileum in vitro were studied. Increasing the magnesium concentration from a basal concentration of 1.1 mmol/l to either 2.5 or 10.3 mmol/l (mucosal or serosal addition) resulted in increases in the unidirectional flux of chloride from serosa to mucosa (JCl sm), short-circuit current (SCC) and transmucosal potential difference (p.d.). Increasing the magnesium concentration from a low basal concentration (0.3 mmol/l) to 10.3 mmol/l (mucosal or serosal addition) abolished net sodium absorption and converted net chloride absorption into net secretion, as a result of a decrease in the unidirectional flux of sodium from mucosa to serosa (JNa ms), and increases in the unidirectional fluxes of sodium and chloride from serosa to mucosa (JNa sm and JCl sm). Increases in SCC and p.d. occurred after mucosal, but not serosal, addition of magnesium. Incubation of stripped ileal mucosa with MgCl2 (10.3 mmol/l) did not result in increased mucosal concentrations of cAMP or cGMP. These data suggest that at least part of the cathartic effect of magnesium salts is likely to result from marked changes in ileal ion transport and provide a rational explanation for the watery diarrhoea commonly seen after the oral administration of magnesium salts to hypomagnesaemic patients.     

Cyclic adenosine monophosphate-stimulated bicarbonate secretion in rabbit cortical collecting tubules.

We studied the effects of cyclic AMP (cAMP) on HCO-3 transport by rabbit cortical collecting tubules perfused in vitro. Net HCO-3 secretion was observed in tubules from NaHCO3- loaded rabbits. 8-Bromo-cAMP-stimulated net HCO-3 secretion, whereas secretion fell with time in control tubules. Both isoproterenol and vasopressin (ADH) are known to stimulate adenylate cyclase in this epithelium; however, only isoproterenol stimulated net HCO-3 secretion. The mechanism of cAMP-stimulated HCO-3 secretion was examined. If both HCO-3 and H+ secretion were to occur simultaneously in tubules exhibiting net HCO-3 secretion, cAMP might increase the net HCO-3 secretory rate by inhibiting H+ secretion, by stimulating HCO-3 secretion, or both. These possibilities were examined using basolateral addition of the disulfonic stilbene (4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In acidifying tubules from NH4Cl-loaded rabbits, DIDS eliminated HCO-3 reabsorption, a result consistent with known effects of DIDS as an inhibitor of H+ secretion. In contrast, cAMP left acidification (H+ secretion) intact. DIDS applied to HCO-3 secretory tubules failed to increase the HCO-3 secretory rate, indicating minimal H+ secretion in HCO-3 secreting tubules. Thus, inhibition of H+ secretion by cAMP could not account for the cAMP-induced stimulation of net HCO-3 secretion. cAMP-stimulated HCO-3 secretion was reversibly eliminated by 0 Cl perfusate, whereas luminal DIDS had no effect. Bath amiloride (1 mM) failed to eliminate cAMP-stimulated HCO-3 secretion when bath [Na+] was 145 mM or 5 mM. cAMP depolarized the transepithelial voltage. The collected fluid [HCO-3] after cAMP could be accounted for by electrical driving forces, suggesting that cAMP stimulates passive HCO-3 secretion. However, cAMP did not alter HCO-3 permeability measured under conditions expected to inhibit transcellular HCO-3 movement (0 Cl- solutions and bath DIDS). This measured HCO-3 permeability was not high enough to account, by passive diffusion, for the HCO-3 fluxes observed in Cl-containing solutions. We conclude the following: cAMP increased net HCO3- secretion by stimulating HCO3- secretion and not by inhibiting H+ secretion; this HCO3- secretion may have occurred by Cl-HCO3- exchange; Na+-H+ exchange appeared not to play a role in basolateral H+ extrusion under these conditions; and the stimulation of HCO3- secretion by isoproterenol, but not ADH, suggests the existence of separate cell cAMP pools or cellular heterogeneity in this cAMP response.     

Effect of somatostatin on ion transport in the rat colon.

The effect of somatostatin (SRIF) on ion transport was determined in the rat colon in vitro. SRIF produced a sustained decrease in the short circuit current (Isc) (-0.8 +/- 0.1 mueq/h x cm2) and increased net Cl absorption (0.9 +/- 0.3 mueq/h x cm2). The threshold effect of SRIF on Isc was observed at 6 nM. 10 microM serotonin decreased net Na absorption (-2.6 +/- 0.4 mueq/h x cm2), net Cl absorption (-3.6 +/- 0.5 mueq/h x cm2) and increased Isc (0.7 +/- 0.1 mueq/h x cm2); these changes were totally blocked by 0.1 microM SRIF. SRIF completely blocked net Cl secretion induced by 10 mM theophylline (-2.5 +/- 0.7 to +4.1 +/- 2.0 muq/h x cm2) and partially blocked theophylline-induced inhibition of net Na absorption (0.7 +/- 0.5 to 2.1 +/- 0.4 mueq/h x cm2). SRIF also blocked prostaglandin E1 (PGE1) induced increase in potential difference and Isc (P < 0.001). Mucosal cyclic AMP levels were increased by theophylline and PGE1 but not by serotonin. SRIF had no effect on basal or theophylline- and PGE1-stimulated cyclic AMP levels. These results indicate that SRIF blocks both cyclic AMP and noncyclic AMP mediated changes in ion secretion and suggest that SRIF is acting at a step in the secretory process beyond the formation of cyclic AMP.     

Sodium chloride absorption by the urinary bladder of the winter flounder. A thiazide-sensitive, electrically neutral transport system

The urinary bladder of the winter flounder absorbs NaCl by a process independent of the transepithelial voltage. In contrast to most other epithelia which have a neutral NaCl-absorptive system, the flounder bladder has a high transepithelial resistance. This feature simplifies analysis of the cellular transport system because the rate of ion transfer through the paracellular pathway is rather low. Experiments were designed to distinguish among three possible mechanisms of neutral NaCl absorption: (a) Na/K/2Cl cotransport; (b) parallel Na/H and Cl/OH exchange; (c) and simple NaCl cotransport. A clear interdependency of Na and Cl for net absorption was demonstrated. NaCl absorption was not dependent on mucosal K and was minimally sensitive to loop diuretics. Thus a Na/K/2Cl transport system was unlikely. The mechanism was not parallel exchange as evidenced by insensitivity to amiloride and to 4,4'-diisothiocyano-2,2'-disulfonic stilbene, an inhibitor of anion exchange. In addition, inhibitors of carbonic anhydrase had no effect. Net absorption was almost completely abolished by hydrochlorothiazide (0.1 mM). Its action was rapid, reversible, and effective only from the mucosal surface. Metolazone, a structurally dissimilar diuretic in the benzothiadiazide class had qualitatively similar actions. The mechanism of NaCl absorption in this tissue appears to be a simple interdependent process. Its inhibition by thiazide diuretics appears to be a unique feature. The flounder bladder may be a model for NaCl absorption in the distal renal tubule.     

Physiologic and Pharmacologic Effects of Glucocorticoids on Ion Transport across Rabbit Ileal Mucosa In Vitro

Physiologic and pharmacologic effects of glucocorticoids on ileal ion transport were examined in vitro. Tissues were obtained from the three following groups of rabbits: (a) normal; (b) glucocorticoid deficient, which were treated with aminoglutethimide (AG), 100 mg twice daily for 3 d, with a resulting marked reduction in urinary cortisol excretion but no decrease in urinary aldosterone; and (c) methylprednisolone-treated (MP), 40 mg daily for 2 d. Transileal NaCl fluxes were measured with radioisotopes under short-circuit conditions, and the net HCO₃ flux was assumed equal to that portion of the short-circuit current (I_sc) not accounted for by Na and Cl. In NaCl Ringer's solution containing 25 mM HCO₃ (pH 7.4), normals absorbed both Na and Cl and secreted HCO₃; the I_sc was greater in both AG and MP groups than in normals; in the AG group, no Na was absorbed, and Cl as well as HCO₃ was secreted; in the MP group, more Na was absorbed and more HCO₃ secreted than in normals. Addition of glucose to the luminal side caused similar increments in I_sc in all three groups, suggesting similar rates of Na-coupled glucose absorption. Secretory response was assessed with a maximal secretory simulus (8-Br-cAMP) and also a submaximal, cGMP-related secretory stimulus (Escherichia coli heat-stable enterotoxin). After addition of 8-Br-cAMP, the rates of net Cl secretion were similar in all three groups, suggesting no effect of glucocorticoids on maximal secretory capacity. Because the AG group was already secreting Cl, however, the cAMP-induced change in net Cl flux was least in this group. After addition of heat-stable enterotoxin, there were similar changes in net Cl flux in all three groups. To examine specifically Cl-independent, electrogenic Na transport, we used a 10 mM HCO₃, Cl-free SO₄-Ringer (ph 7.2) in which net Na absorption was previously shown to be equal to the I_sc. Under these conditions, I_sc was greatest in the MP group and least in the AG group. In vitro addition of hydrocortisone, 50 μg/ml, to AG tissues had no effect on Cl fluxes or I_sc over a 3.5-h period. No differences among groups were observed with respect to morphology, electrical resistance, or cGMP concentration. We conclude that (a) the effect of glucocorticoid deficiency is similar to that of a submaximal secretory stimulus in that Na absorption is inhibited and some Cl secretion develops; (b) electrogenic Na absorption is depressed in glucocorticoid deficiency and enhanced in glucocorticoid excess; (c) glucocorticoid excess increases HCO₃ secretion; and (d) glucocorticoid status does not affect maximal secretory capacity.     

Intestinal type 2 proteinase-activated receptors: expression in opioid-sensitive secretomotor neural circuits that mediate epithelial ion transport

Trypsin and mast cell tryptase cleave within the extracellular N terminus of proteinase-activated receptor-2 (PAR-2), exposing a tethered ligand (SLIGRL) that binds and activates the cleaved receptor. We examined the neuronal expression of PAR-2 and its role in intestinal ion transport. Short-circuit current elevations in response to trypsin or the receptor-activating peptide SLIGRL-NH(2) were measured in sheets of mucosa-submucosa from porcine ileum. SLIGRL-NH(2) or trypsin rapidly elevated short-circuit current after their contraluminal application with respective 50% effective concentrations of 184 and 769 nM. Their actions were attenuated after contraluminal administration of the neuronal conduction blocker saxitoxin (0.1 microM); the cyclooxygenase inhibitor indomethacin (10 microM); or the Na(+)/K(+)/Cl(-) cotransport inhibitor furosemide (10 microM), but not by atropine (0.1 microM), a muscarinic cholinergic antagonist. In addition, soybean trypsin inhibitor (5 microgram/ml) reduced mucosal responses to trypsin. The delta-opioid agonist [D-Pen(2,5)]-enkephalin (0.1 microM) inhibited trypsin action, an effect that was prevented by naltrindole (0.1 microM), a delta-opioid antagonist. PAR-2 immunofluorescence was localized in the mucosa using a receptor-specific antibody. PAR-2-like immunoreactivity was detected in myenteric and submucosal neurons, nerve fibers innervating ileal smooth muscle and mucosa, and in enteroendocrine cells. Some neurons coexpressed PAR-2- and choline acetyltransferase-like immunoreactivity. These results indicate that PAR-2 is expressed on cholinergic and noncholinergic submucosal neurons in porcine ileum. PAR-2 agonists stimulate active anion secretion by a neurogenic mechanism that is modulated by prostanoids and opioids. These receptors may have a potentially important role in intestinal neuroimmunomodulation.     

Elevated intracellular Ca2+ acts through protein kinase C to regulate rabbit ileal NaCl absorption. Evidence for sequential control by Ca2+/calmodulin and protein kinase C

Calcium/calmodulin is involved in the regulation of basal rabbit ileal active Na and Cl absorption, but the mechanism by which elevated intracellular Ca2+ affects Na and Cl transport is unknown. To investigate the roles of the Ca2+/calmodulin and protein kinase C systems in ileal NaCl transport, two drugs, the isoquinolenesulfonamide, H-7, and the naphthalenesulfonamide, W13, were used in concentrations that conferred specificity in the antagonism of protein kinase C (60 microM H-7) and Ca2+/calmodulin (45 microM W13), respectively, as determined using phosphorylation assays in ileal villus cells. W13 but not H-7 stimulated basal active NaCl absorption. H-7 inhibited changes in Na and Cl absorption caused by maximal concentrations of Ca2+ ionophore A23187 and carbachol and serotonin, secretagogues that act by increasing cytosol Ca2+, while W13 had no effect. In contrast, neither H-7 nor W13 altered the change in NaCl transport caused by the cyclic nucleotides 8-Br-cAMP and 8-Br-cGMP. These data suggest that: (a) basal rabbit ileal NaCl absorption is regulated by the Ca2+/calmodulin complex and not by protein kinase C; (b) the effect of elevating intracellular Ca2+ to decrease NaCl absorption is mediated via protein kinase C but not by Ca2+/calmodulin; (c) the effects of protein kinase C are not overlapping or synergistic with those of Ca2+/calmodulin on either basal absorption or on the effects of increased Ca2+; and (d) neither Ca2+/calmodulin nor protein kinase C are involved in the effects of cAMP and cGMP on ileal active NaCl transport.     

Sodium transport in the diseased human gallbladder and the effects of indomethacin

1. Sodium ion (Na+) transport, a principal function of the gallbladder epithelium, was studied by measuring the flux of 22Na across isolated, inflamed human gallbladder mucosa maintained in a modified 'Ussing' flux chamber. Tissue was obtained from cholecystectomy specimens in symptomatic patients with cholelithiasis. 2. In 30 gallbladders studied, 57% had a net Na+ flux from mucosa to serosa (Na+ absorption), while 23% had a net Na+ flux from serosa to mucosa (Na+ secretion). The remaining 20% showed no overall net Na+ flux. 3. Indomethacin added to the serosal fluid reversed the direction of net Na+ flux in secreting gallbladders and caused an absorption of Na+. In Na+-absorbing gallbladders, indomethacin caused a slight reduction in Na+ absorption. No change in Na+ flux was induced in gallbladders with no initial net Na+ flux. 4. These results demonstrate that instead of absorbing Na+, some inflamed human gallbladders may secrete Na+. As this secretion can be reversed to the more usual absorption by indomethacin, it is likely that this secretion is mediated by prostaglandins.     

Cyclic adenosine monophosphate-stimulated anion transport in rabbit cortical collecting duct. Kinetics, stoichiometry, and conductive pathways.

Cyclic AMP stimulates HCO3 secretion and Cl self-exchange in rabbit cortical collecting tubule. We found that varying peritubular [Cl] changed the Cl self-exchange rate with saturation kinetics (Km, 3-4 mM). HCO3 secretion also showed saturation kinetics as a function of mean luminal [Cl] (Km, 4-11 mM). Both Cl self-exchange and Cl-HCO3 exchange thus appear to be carrier-mediated. Addition/removal of basolateral HCO3 qualitatively changed Cl and HCO3 transport as expected for Cl-HCO3 exchange, but quantitatively changed Cl absorption more than HCO3 secretion. The diffusive Cl permeability and the transepithelial conductance in the presence of HCO3/CO2 and cAMP were higher than in their absence suggesting that HCO3/CO2 and cAMP together increase a conductive Cl pathway parallel to a 1:1 Cl-HCO3 exchanger. Thus, cAMP not only stimulates the overall process of anion exchange (probably by increasing an electroneutral exchanger and/or a series Cl conductance), but also stimulates a Cl conductance parallel to the exchange process.     

Effects of acid-base variables on ion transport in rat colon.

Alterations in arterial acid-base variables have important effects on colonic electrolyte transport in vivo. To confirm the relative effects of these variables and to characterize the transport processes involved, we measured unidirectional 22Na and 36Cl fluxes across short-circuited, distal colonic mucosa of Sprague-Dawley rats. Stripped tissues were studied in Hepes buffer and in Ringer's solutions at HCO3 concentrations of 11, 21, and 39 mM, and CO2 tensions between 0 and 69.6 mmHg. Increases in PCO2, but not in either pH or HCO3 concentration, caused similar increases in JNanet and JClnet (net flux of sodium and chloride, respectively) from -0.2 +/- 0.3 and -1.5 +/- 0.4 mu eq/cm2 per h at PCO2 = 0 to 6.8 +/- 0.6 and 7.6 +/- 0.7 mu eq/cm2 per h, respectively, at PCO2 = 69.6 mmHg. These increases were accounted for by changes in Jms and were accompanied by small decreases in Isc. 1 mM acetazolamide decreased both JNanet and JClnet and their responses to increases in CO2. 0.75 mM luminal amiloride prevented the increase in sodium absorption, but did not affect the CO2-induced increase in chloride absorption. In the presence of amiloride, CO2 increased JR (residual flux). 0.1 mM luminal furosemide did not affect the CO2-induced increases in JNanet in the absence or presence of amiloride. Changes in HCO3 concentration did not alter JR. We conclude that ambient CO2 effects active, electroneutral sodium absorption in the rat distal colon. The process stimulated by CO2 is dependent on mucosal carbonic anhydrase activity and most likely represents Na/H and Cl/HCO3 ion exchange.