Rise In Short-circuit Current Research Articles

Prostaglandin D2 regulates human colonic ion transport via the DP1 receptor

AimsProstaglandin D2 is released by mast cells and is important in allergies. Its role in gastrointestinal function is not clearly defined. This study aimed to determine the effect of exogenous PGD2 on ion transport in ex vivo normal human colonic mucosa. Materials and methodsMucosal sheets were mounted in Ussing chambers and voltage clamped to zero electric potential. Ion transport was quantified as changes in short-circuit current. In separate experiments epithelial monolayers or colonic crypts, isolated by calcium chelation, were treated with PGD2 and cAMP levels determined by ELISA or calcium levels were determined by fluorimetry. Key findingsPGD2 caused a sustained, concentration-dependent rise in short-circuit current by increasing chloride secretion (EC50=376nM). This effect of PGD2 is mediated by the DP1 receptor, as the selective DP1 receptor antagonist BW A686C inhibited PGD2-induced but not PGE2-induced rise in short-circuit current. PGD2 also increased intracellular cAMP in isolated colonic crypts with no measurable influence on cytosolic calcium. PGD2 induces chloride secretion in isolated human colonic mucosa in a concentration-dependent manner with concomitant elevation of cytoplasmic cAMP in epithelial cells. SignificanceThe involvement of DP2 receptor subtypes has not previously been considered in regulation of ion transport in human intestine. Since inflammatory stimuli may induce production of eicosanoids, selective regulation of these pathways may be pivotal in determining therapeutic strategies and in understanding disease.

Activation of AMPK Inhibits Cholera Toxin Stimulated Chloride Secretion in Human and Murine Intestine

Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea.Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops.CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments.The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.

Duodenal epithelial transport in functional dyspepsia: Role of serotonin

To investigate functional duodenal abnormalities in functional dyspepsia (FD) and the role of serotonin (5-hydroxytryptamine, 5-HT) in mucosal ion transport and signalling. Duodenal mucosal biopsies were obtained from 15 patients with FD and 18 healthy controls. Immunohistochemistry was used to study the number of 5-HT-containing cells and real-time polymerase chain reaction for expression of 5-HT receptors 1A, 1B, 2A, 2B, 3A, 3B, 3C, 3D, 3E, 4 and 7, as well as expression of the serotonin re-uptake transporter (SERT) gene SLC6A4 and tryptophan hydroxylase 1 (TPH1). Biopsies were mounted in Ussing chambers for evaluation of basal and 5-HT-stimulated short-circuit current (SCC). Conductance was lower in FD [42.4 ± 4.7 mS/cm(2) (n = 15) vs 62.5 ± 4.5 mS/cm(2) (n = 18), P = 0.005]. 5-HT induced a dose dependent rise in SCC in both FD (n = 8) and controls (n = 9), the rise was lower in FD (P < 0.001). Mean number of 5-HT stained cells per high power field was the same [34.4 ± 8.4 in FD (n = 15) and 30.4 ± 3.7 in controls (n = 18), P = 0.647]. The following genes were highly expressed: 5-HT receptor HTR3E, HTR4, HTR7, SERT gene (SLC6A4) and TPH1. Differences in expression levels were observed for HTR3E (higher expression in FD, P = 0.008), HTR7 (lower expression in FD, P = 0.027), SLC6A4 (higher expression in FD, P = 0.033) and TPH1 (lower expression in FD, P = 0.031). Duodenal ion transport in response to exogenous 5-HT is abnormal in FD patients and associated with high expression of the HTR3E receptor and the serotonin transporter.

Involvement of prostaglandins in histamine-induced fluid and electrolyte secretion by rat colon.

Histamine increased the transmural potential difference across rat colon in-vivo and induced a net secretion of fluid. Both effects were inhibited by indomethacin. Histamine increased the potential difference and short-circuit current, and reduced tissue resistance in colonic sheets in-vitro. This response was reduced in the absence of chloride in the bathing medium or in the presence of serosal frusemide, suggesting that histamine stimulated electrogenic chloride secretion by the colon. The rise in short-circuit current induced by histamine was calcium-dependent since it was reduced in the absence of serosal calcium or in the presence of serosal verapamil. Indomethacin, a cyclo-oxygenase inhibitor, and mepacrine, a phospholipase inhibitor, both caused a dose-dependent inhibition of the electrical response of colonic sheets to histamine, without affecting the rise in short-circuit current induced by prostaglandin E2. The stimulation of chloride secretion induced by histamine in rat colon therefore appears to be mediated by an increased production of prostaglandins.

Calcium-calmodulin-dependent activation of adenylate cyclase in prostaglandin-induced electrically-monitored intestinal secretion in the rat.

The calcium-calmodulin antagonist 5-iodo-C8-W7 inhibited the PGE2-induced stimulation of cAMP production by isolated enterocytes from rat small intestine. It also reduced the secretory response of intestinal sheets to PGE2, measured as a rise in short-circuit current. It did not however, inhibit the electrical responses to forskolin and dibutyryl cAMP, nor to acetylcholine, a secretagogue whose effect is not mediated by cAMP. It is concluded that the receptor-mediated activation of adenylate cyclase and the subsequent secretory response are dependent upon calcium-calmodulin.

Small intestinal glucose absorption in cystic fibrosis: a study in human and transgenic ΔF508 cystic fibrosis mouse tissues

Intestinal transport is disturbed in cystic fibrosis (CF), with both defective Cl- secretion and changes in absorption being reported. We have examined the effects of the disease on Na(+)-dependent glucose absorption by the small intestine. Active glucose absorption was monitored as changes in short-circuit current (SCC) in intact and stripped intestinal sheets from normal (Swiss) and transgenic CF (Cftr(tm1Eur) and Cftr(tm2Cam)) mice with the DeltaF508 mutation, and in jejunal biopsies from children with CF and normal controls. Na(+)-dependent glucose uptake at the luminal membrane was measured in brush-border membrane vesicles (BBMVs). Intact and stripped sheets of jejunum and midintestine from Swiss mice exhibited a concentration-dependent increase in SCC with glucose. Apparent Km values were similar in the two preparations, but the apparent Vmax was greater in stripped sheets. This difference was not due to a loss of neural activity in stripped sheets as tetrodotoxin did not influence the glucose-induced SCC in intact sheets. Similar results were observed in stripped sheets of jejunum and mid-intestine from wild-type Cftr(tm1Eur) mice, but in tissues from CF mice the apparent Vmax value was reduced significantly. A lower Vmax was also obtained in intact sheets of mid-intestine from CF (Cftr(tm2Cam)) mice. Jejunal biopsies from CF patients however, exhibited an enhanced glucose-dependent rise in SCC. Na(+)-dependent uptake by BBMVs from CF (Cftr(tm1Eur)) mice was not reduced compared with wild-type and Swiss BBMVs. It was concluded that, in contrast to human intestine, intestinal glucose absorption was reduced in transgenic mouse models of CF with the DeltaF508 mutation, but that this could not be detected in an isolated preparation of brush-border membranes. Transgenic mouse models of CF may not accurately reflect all aspects of intestinal dysfunction in the human disease.

Gossypol induces chloride secretion in rat proximal colon

The effect of gossypol on electrolyte transport was investigated in rat colon mounted in Ussing chambers. The addition of gossypol to the mucosal or serosal side led to an increase in mucus secretion, which we did not quantify. Mucosally or serosally added gossypol also induced a rise in short circuit current ( I sc) and tissue conductance ( G t). Part of the mucosally added gossypol seemed to be bound to the mucus because the effects on I sc and G t were smaller when gossypol was added to the mucosal side. Serosally added gossypol had an effect on I sc at a concentration of 10 μmol l −1. Mucus secretion was reduced in low Ca 2+ buffer. The increase in I sc was diminished by blockers of Cl − channels, K + channels, of the Na +/K + ATPase and of the Na +/K +/2 Cl − cotransporter. Measurements of unidirectional ion fluxes showed that gossypol added to the mucosal side had no effect on net Na + transport, but increased Cl − secretion. The effect of mucosally added gossypol was significantly reduced by the use of low Cl − buffers and abolished when the buffer was additionally depleted of HCO 3 −. Calmodulin antagonists inhibited the effect on secretion. These findings indicate that gossypol induces chloride secretion via a calmodulin-dependent mechanism. High concentrations of gossypol induced a strong increase in G t that could be blocked by W7, a blocker of calmodulin-dependent myosin light chain kinase. This indicates that the rise in G t is not due to an unspecific toxic effect, but instead, to specific opening of tight junctions.

THE EFFECTS OF STROMELYSIN OR MATRIX METALLOPROTEINASE-3 (MMP-3) ON INTESTINAL SECRETORY FUNCTION

33 In inflammatory bowel disease there is extensive damage to the extracellular matrix (ECM) and loss of the transepithelial sodium gradient and hence absorption. Stromelysin (MMP-3) is produced in intestinal inflammatory states and is important in tissue degradation. We have previously used human fetal intestine to explore inflammatory mechanisms and here we study the effect of MMP-3 on its secretory function. 3 cm loops of 15 wk gestation gut or T84 cell monolayers were incubated with MMP-3 50 nM or saline (C) on the apical surface then mounted in Using Chambers. MMP-3 after 1, 2 and 3 hrs caused a rise in short circuit current (Isc) of 105±33, 125±5.8 and 145±99 μA/cm2 (p<0.005). Resistance fell from 22±8 to 9±7 ohms/cm2 and PD rose from 1.4±0.8 to 2.6±0.8 mV at 3 hrs. In Cl- free conditions Isc fell by 83%. In contrast in T84 cells the rise in Isc was insignificant (47 vs 53μA/cm2). Thus MMP-3 induces Cl- dependent secretion in intact intestine but not in T84 monolayers suggesting that this is not due to a direct effect on enterocytes. We speculate that disruption of the ECM by MMP-3 also induces secretion and that MMP inhibitors may have a therapeutic role in diarrhoeal states.

Effect of shigella enterotoxin 1 (ShET1) on rabbit intestine in vitro and in vivo.

Shigella enterotoxin 1 is a novel enterotoxin elaborated by Shigella flexneri 2a that causes fluid accumulation in rabbit ileal loops and a rise in short circuit current in Ussing chambers. To gain insights into the mechanism of action of shigella enterotoxin 1. Supernatants from genetically engineered clones either overexpressing shigella enterotoxin 1 or producing deletion mutants of the toxin were tested in rabbit ileum both in vitro and in vivo. In rabbit ileum shigella enterotoxin 1 induced an irreversible rise in short circuit current that was not mediated by any of the recognised intracellular mediators of secretion. Deletion of 90% of the A subunit of the holotoxin ablated its enterotoxicity. In the in vivo perfusion model, the toxin induced a time dependent decrease in water absorption, whereas no changes were detected in the segment perfused with supernatants obtained from the deletion mutant. Finally, partially purified toxin induced a dose dependent increment in short circuit current that reached its plateau at a toxin concentration of 4 x 10(-6) M. Shigella enterotoxin 1 induces a time and dose dependent intestinal secretion in the rabbit animal model, suggesting that it may be responsible for the watery phase of Shigella flexneri 2a infection.

The role of the 5-HT 4 receptor in Cl − secretion in human jejunal mucosa

5-Hydroxytryptamine (5-HT) is a mediator of chloride ion (Cl −) secretion in the intestine which can be seen as a rise in short circuit current ( I SC) in the Ussing chamber model. We investigated the 5-HT receptor mediating 5-HT-induced Cl − secretion in the human jejunum in vitro. Jejunal segments obtained from patients having gastric bypass surgery for obesity, were stripped of muscularis and mounted in Ussing chambers and short-circuited. The 5-HT receptor agonist-induced change (Δ) in I SC was recorded in the presence and absence of 5-HT receptor antagonists. The rank order of agonist potency was: 5-HT > 5-methoxytryptamine > renzapride (BRL 24924) > α-methyl-5-HT > > 2-methyl-5-HT. In the presence of Cl −-free media or 100 μM furosemide, 5-HT-induced Δ I SC was significantly reduced. It was also antagonized by ≥ 1 μM tropisetron (a 5-HT 3/5-HT 4 receptor antagonist) and ≥ 10 nM GR 113808 (a selective 5-HT 4 receptor antagonist) with pA 2 values of 6.5 and 7.9, respectively. Another 5-HT 4 receptor antagonist, SC 53606 (0.1 μM), antagonized the 5-HT-induced response with a pA 2 of 7.3. 5-HT 1-like/5-HT 2 (methysergide), 5-HT 1P [N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP)], 5-HT 2A (ketanserin) and 5-HT 3 (ondansetron) receptor antagonists and tetrodotoxin, had no significant effect on the EC 50 for 5-HT. In conclusion, this study demonstrates that in the human muscle-stripped jejunum in vitro, 5-HT induced change in short circuit current is mediated by a 5-HT 4 receptor via a non-neural pathway.

5-Hydroxytryptamine-induced Cl − transport is mediated by 5-HT 3 and 5-HT 4 receptors in the rat distal colon

In the rat distal colon, 5-hydroxytryptamine (5-HT)-induced Cl − secretion is seen as a rise in short circuit current ( I sc). We investigated the 5-HT receptor mediating 5-HT-induced Cl − secretion in the rat distal colon. Rat distal colon was prepared either by stripping away the muscularis propria with the neural ganglia, or by leaving it intact. The tissue was mounted in Ussing chambers and short circuited. 5-HT receptor agonist-induced changes (Δ) in I sc were recorded in the presence and absence of 5-HT receptor antagonists. In stripped preparations, the rank order of potency of agonists was: 5-HT > 5-methoxytryptamine > α-methyl-5-HT ⪢ 2-methyl-5-HT. 5-HT and 5-methoxytryptamine-induced changes in I sc were antagonized by ≥ 0.3 μM tropisetron with pA 2 values 6.5 and 6.4, respectively. The 5-HT 4 antagonist, SC 53606, antagonized the 5-HT-induced response with a pA 2 of 7.2. 5-HT 1-like (methysergide), 5-HT 1P ( N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP)), 5-HT 2A (ketanserin) and 5-HT 3 (ondansetron) receptor antagonists had no significant effect on the 5-HT response in stripped tissue. 3 μM forskolin, or 10 μM 3-isobutyl-1-methyl-xanthine (IBMX), decreased the EC 50 and increased the maximum 5-HT response. The 2-methyl-5-HT and 5-HT-induced ΔI sc in the unstripped colon preparation were antagonized by the 5-HT 3 antagonist, ondansetron (0.3 nM), and 2-methyl-5-HT activity was abolished by pretreatment with tetrodotoxin. In conclusion, 5-HT-induced ΔI sc is neurally mediated via a 5-HT 3 receptor, and non-neurally mediated via a 5-HT 4 receptor in the rat distal colon.

Evidence for a 5-HT4 Receptor Pathway Mediating Chloride Secretion in the Rat Distal Colon

Serotonin (5-HT) is a potent secretagogue of chloride ion (Cl-) and a mediator of diarrhea in the carcinoid syndrome. We investigated the role of the 5-HT4 receptor pathway in mediating 5-HT-induced Cl- secretion that is seen as a rise in short circuit current (ISC) in the Ussing chamber. Rat distal colon was stripped of the muscularis, mounted in Ussing chambers, and short circuited. By cumulative concentration responses, 5-HT-induced rise in ISC was measured in the presence and absence of 0.1 μM of the nerve conduction blocker, tetrodotoxin (TTX). TTX was later added routinely to remove any effects of residual nerve tissue. 5-HT concentration response was measured in the presence and absence of the following 5-HT receptor antagonists: 10 μM 5-HTP-DP (5-HT1p), 1 μM methysergide (5-HT1-like and 5-HT2), 0.1 μM ketanserin (5-HT2), 0.3 μM ondansetron (5-HT3) 0.1, 0.5, 1 and 3 μM ICS 205-930 (5-HT3 and 5-HT4), and 0.1, 0.03, and 0.01 μM of the new selective 5-HT4 antagonist, SC 53606. Data were analyzed by ANOVA. TTX had no effect on EC50 for 5-HT. ICS 205-930 and SC 53606 produced dextral shifts in 5-HT concentration-response curves and at all concentrations a significant shift in the EC50 for 5-HT, except at 0.1 μM ICS 205-930. The Schild plot slopes for ICS 205-930 (0.8) and SC 53606 (0.7) were not significantly different from unity. The pA2 values were 6.5 and 7.2, respectively. Methysergide, ketanserin, ondansetron, and 5-HTP-DP had no significant effect on the EC50 for 5-HT. These results suggest that the 5-HT4 receptor pathway mediates 5-HT-induced Cl- secretion.

Loperamide Inhibits the Enhanced Intestinal Glucose Absorption of Cystic Fibrosis In Vitro

Enhanced Na(+)-linked nutrient absorption has been demonstrated in the cystic fibrosis bowel and may contribute to the dehydration of the luminal contents. The ability of loperamide to inhibit glucose transport was therefore assessed in jejunal biopsy specimens from children with cystic fibrosis by measuring the increased short-circuit current associated with active glucose absorption using a mini-Ussing chamber technique. The presence of loperamide in the luminal solution reduced the rise in short-circuit current induced by glucose over the range of concentrations tested (2.5 to 40 mmol.L-1). This finding suggests that the enhanced Na+ absorption, which is a feature of cystic fibrosis, is amenable to treatment and can be restored to normal levels. This result may have therapeutic implications for gastrointestinal function and may be applicable to other tissues where similar therapeutic approaches to reduce Na+ and thus water absorption are being pursued.

A 5-HT 2 Receptor Mediates Serotonin-Induced Electrolyte Transport in Rat Left Colon

Serotonin (5-hydroxytryptamine, 5-HT) is a potent mediator of diarrhea in the carcinoid syndrome. However, the mechanisms of serotonin-induced intestinal secretion remain unclear. The purpose of this study was to determine whether 5-HT stimulates electrolyte transport by a direct action at a mucosal receptor. Serotonin-stimulated electrolyte transport was studied in flat sheet preparations of rat left colon using flux chambers. 5-HT stimulated a concentration-dependent rise in short-circuit current in sheets stripped of muscularis propria. The half-maximal concentration required to produce this effect was 50 μ M. The preferential 5-HT 2 antagonist ketanserin produced a rightward shift of the serotonin concentration-response curve with a pA 2 value of 7.7. Serotonin stimulated electrolyte transport by inhibiting mucosa to serosa movement of both sodium and chloride. The 5-HT 1 antagonist N -acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide and the 5-HT 3 antagonist 3-tropanyl-indole-3-carboxylate methiodide had no effect on these actions of serotonin on electrolyte transport. In contrast, ketanserin inhibited the actions of serotonin on both sodium and chloride movement and inhibited the rise in short-circuit current induced by 100 μ M 5-HT. This study demonstrates that a 5-HT 2 receptor located in the vicinity of the mucosa is involved in the regulation of serotonin-stimulated intestinal electrolyte transport.

Characterization of adrenoceptors involved in the electrogenic chloride secretion by cultured rat epididymal epithelium.

1. Short-circuit current (SCC) technique was used to study the adrenoceptors involved in the electrogenic chloride secretion by cultured cauda epididymal epithelium of rats. Stimulation of the epithelium with noradrenaline (primarily beta 1-adrenoceptor selective agonist), salbutamol (beta 2-adrenoceptor selective agonist) and adrenaline (non-selective beta-adrenoceptor agonist) led to a rise in SCC. At a low chart-speed (2 mm min-1), the response profile to these agonists consisted of a peak followed by a sustained response considerably higher than the basal SCC. 2. The EC50s (doses of agonist producing 50% maximum response) of noradrenaline, salbutamol and adrenaline were 300, 115 and 10 nM respectively. Pretreating the tissues with 1 microM atenolol (beta 1-selective antagonist) and 10 microM butoxamine (beta 2-selective antagonist) shifted the dose-response curves of noradrenaline (shifted EC50 = 4000 nM) and salbutamol (shifted EC50 = 1050 nM) to the right. Atenolol (1 microM) and butoxamine (10 microM) shifted the dose-response curve of adrenaline to the right with new EC50s of 30 nM and 115 nM, respectively. 3. The rapidly rising phase of the SCC response to noradrenaline and adrenaline observed at low chart-speed consisted of a brief and transient retraction followed by a rebound increase in SCC. At a high chart-speed (1 mm s-1), the retraction and rebound phenomenon manifested as a fast initial spike which could be blocked by phentolamine (non-specific alpha-adrenoceptor antagonist) in a dose-dependent fashion. Similar initial spikes were observed when the tissues were stimulated with phenylephrine (alpha-selective agonist) but not with isoprenaline (non-selective beta-agonist) or forskolin (activator of adenylate cyclase). The response of the initial spike triggered by noradrenaline was dose-dependent and the EC50 was 2000 nM.4. The present study showed that the electrogenic chloride secretion by rat epididymis could be stimulated by (alphaxi-, beta131- and beta2-adrenoceptor agonists. The al-mediated response had a faster onset and more transient action than the 3-counterpart. It is postulated that epididymal chloride secretion might be regulated by neural (noradrenaline-mediated) and humoral (adrenaline-mediated) controls and that the stimulus-secretion coupling mechanisms might involve both Ca2+(alpha-mediated response) and adenosine 3':5'-cyclic monophosphate (beta-mediated response) as intracellular second messengers.

Regional differences in the response to platelet-activating factor in rabbit colon.

1. Platelet-activating factor is an inflammatory mediator related to eicosanoids which is known to stimulate anion secretion in the distal colon. Since there are regional differences in ion transport within the colon, the influence of platelet-activating factors on ion transport and epithelial permeability has been studied in rabbit caecum and distal colon mounted in Ussing chambers. 2. The effect of platelet-activating factor (1-50 nmol/l) on net electrogenic ion transport was to stimulate a biphasic increase in short-circuit current in the distal colon but not in the caecum. The platelet-activating factor-induced rise in short-circuit current was shown by ion replacement and pharmacological inhibitor studies to be consistent with chloride and bicarbonate secretion in the early phase, but with chloride secretion alone in the later phase. The effect on ion transport was specific and reversible and was enhanced by 0.25% BSA. 3. Colonic permeability, assessed by transmucosal resistance and mannitol flux, was increased by platelet-activating factor in both the distal colon and the caecum. This was consistent with an effect on platelet-activating factor on the paracellular pathway, because resistance decreased even when transcellular chloride transport was inhibited by frusemide or ion replacement. A specific platelet-activating factor antagonist (U66985) inhibited the effects of platelet-activating factor in both the distal colon and the caecum. 4. The results show that platelet-activating factor stimulates anion secretion only in the distal colon, but increases permeability in both the caecum and the distal colon.

Functional Study and Immunocytochemical Identification of Endothelin in Cultured Epididymal Cells and Intact Epididymis of the Rat

Endothelin, a novel potent vasoconstrictor peptide produced by vascular endothelial cells, stimulated anion secretion by a cultured secretory epithelium derived from the rat epididymis as measured by changes in short-circuit current (SCC). Stimulation of the SCC was observed when endothelin was added to the basolateral or the apical side of the epithelium. The response to basolateral application was greater than that to apical application. The EC50 values were found to be 1.3 and 3.0 nM for basolateral and apical application, respectively. These values were about one-half to one order of magnitude higher than that required for its vasoconstrictor action. The stimulation of SCC by endothelin was likely to be due to an increase to anion secretion as removal of Cl from the incubation medium markedly reduced the SCC response to endothelin. Diphenylamine-2-carboxylate (DPC, 0.1 mM), a Cl-channel blocker, added to the apical side also inhibited the endothelin-induced rise in SCC. The stimulation of SCC by endothelin was accompanied by a rise in the intracellular cyclic AMP content in epididymal monolayers. Immunofluorescence staining has shown the presence of immunoreactive endothelin-like compound in the interstitium and epithelial cells of the rat epididymis. It is speculated that endogenous endothelin plays an important role in the control of water and electrolyte transport in the epididymis.

Enhanced intestinal glucose and alanine transport in cystic fibrosis.

The rise in short-circuit current associated with the active transport of glucose and alanine was measured in intestinal biopsy samples from children with cystic fibrosis. The glucose-induced increase in the short-circuit current was greater in the cystic fibrosis tissues than in control samples over the whole range of concentrations tested (2.5-35 mM), a reflection of an increased maximum rate of transport. Similar results were obtained with alanine. These findings suggest that active Na(+)-linked nutrient transport is enhanced in cystic fibrosis.

DESCRIPTION OF A PUTATIVE ENTEROTOXIN PRODUCED BY ENTEROAGGREGAT1VE ESCHERICHIA COLI (EAggEC)

EAggEC have been associated with persistent diarrhea in children, yet little is known about virulence factors. Aim. We assayed for enterotoxic activity in culture filtrates(CF) of EAggEC strains. Methods. CF from 17-2 (a prototype Chilean EAggEC), 8 other EAggEC strains and E. coli HB101 were added to stripped rabbit ileal mucosa(RIM) mounted in Ussing chambers(UCs). CFs were also tested by E. coli STa ELISA and suckling mouse assay (SMA). EAggEC colonies were hybridized under low stringency to STh and STp DNA probes. Results. Addition of 17-2 CF to the RIM in UCs caused a greater rise in short circuit current (ΔIsc=55.2 ± 5.3 uAmp/cm2)(Mean ± SE) than that seen in HB101 control (ΔIsc=16.8 ± 3.0 uAmp/cm2) (p < 0.001). This activity on Isc was 1)65% stable after heating (15 m at 65°C); 2)localized to the 2-5 kDa MW size range; and 3) reproduced with CF from HB101 transformed with 17-2 plasmid. Compared to ΔIsc response to 8-Br-cGMP on negative control tissue ( ΔIsc=123.4 ± 7.9 uAmp/cm2), the ΔIsc to 8-Br-cGMP added to tissue pretreated with 17-2 CF (87.55 ± 8.2 uAmp/cm2)was significantly less (p < 0.01), while the additive effect of 17-2 CF and 8-Br-cGMP (ΔIsc=142.8 ± 9.1 uAmp/cm2) was not significantly different (p>0.05). <10 kDa CF fractions from 6/8 other EAggEC strains screened in UCs gave ΔIsc responses similar to 17-2. The 17-2 CF activity was 1)non-neutralizable with polyclonal anti-STa in UCs; 2)non-reactive by STa ELISA; and 3)negative in SMA. Also, EAggEC colonies did not hybridize with STh or STp DNA probes. Conclusions. EAggEC strain 17-2 produces an extracellular, low MW, partially heat stable moiety which gives an electrical response in vitro consistent with an enterotoxic effect. This putative EAggEC heat stable enterotoxin (EAST) appears to be plasmid-mediated, not genetically or immunologically cross-reactive with E. coli STa, and may act via cGMP as intracellular mediator.

Effect of carbenoxolone on glucocorticoid metabolism and Na transport in toad bladder

In humans, diminished 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) enzyme activity has been associated with sodium retention and hypertension. These studies show that the toad bladder, another target tissue epithelium displaying steroid-induced sodium transport, possesses the enzyme 11 beta-OHSD. The toad urinary bladder rapidly transformed corticosterone (3 x 10(-8) M) (50% by 10 min and 90% by 180 min) with 11-dehydrocorticosterone being the major metabolite. The 11-dehydrocorticosterone produced reached an apparent plateau when the tissue incubations were repeated with higher concentrations of corticosterone (10(-7) and 10(-6) M). Carbenoxolone sodium (2.5 x 10(-5) M), a water soluble derivative of glycyrrhetinic acid, markedly inhibited the metabolism of corticosterone (3 x 10(-8) M) to 11-dehydrocorticosterone similar to previous observations in the mammalian kidney. Carbenoxolone sodium (2.5 x 10(-5) M) did not significantly affect short-circuit current (SCC) in toad bladders when added to either the serosal or mucosal bath. However, when carbenoxolone sodium was added to the mucosal bath and 60 min later corticosterone 10(-6) M was placed in the serosal bath, bladders generated a SCC 2.07 +/- 0.17 (mean +/- SE) times above base line at 360 min compared with 1.48 +/- 0.11 in bladders exposed to corticosterone alone (P less than 0.02). In parallel experiments, carbenoxolone sodium in the mucosal bath enhanced the rise in SCC induced by cortisol 10(-6) M; 1.66 +/- 0.16 times above base line at 360 min compared with 1.07 +/- 0.14 with cortisol alone (P less than 0.02). We conclude that the toad bladder contains 11 beta-OHSD and inhibition of this enzyme with carbenoxolone sodium is associated with amplification of glucocorticoid-induced transepithelial sodium transport in this tissue. However, since the quantity of 11-dehydro-product produced appears to be limited, other factors in addition to inhibition of 11 beta-OHSD may play a role in this amplification of sodium transport.