The sigma ligand igmesine (JO 1784) has been shown to act on nerves to inhibit intestinal ion transport in the isolated mouse jejunumL The present study evaluates the ability of igmesine to inhibit the VIP-induced jejunal hypersecretion in rats and investigates the possibility of the involvement of endogenous somatostatin in the response to igmesine.Methods: In anesthetized (pentobarbital, 60 mg/kg i.p.) fasted Sprague-Dawley rats (160-180 g), a jejunal loop was isolated by two ligations at 5 and 25 cm distal to the ligament of Treitz.The loop was filled with saline (2 ml, 37°C).Jejunal secretion was stimulated by a 30 min intraarterial infusion of VIP.At the end of the VIP infusion, the loop was collected, measured and weighed before and after fluid removal to determine water net flux (mg/cm).Intravenous (i.v.) bolus injections of igmesine or octreotide were performed 15 min before starting VIP infusion.Tetrodotoxin (TTX) at 5 mg/kg, the somatosatin antagonist, cyclosomatostatin (CSS) at 1 mg/kg, or the sigma antagonist, BMY-14802 (BMY) at 1 mg/kg were given by i.v.route 5 min before igmesine or octreotide.Results: In the basal state the net water flux was positive (+3.13 ±3.9 mg/cm).VIP (0.03-0.33 mg.min -1) induced a dose-related inversion of net flux.The submaximal effect (-34.1 -+ 7.1 mg/cm) was obtained at the dose of 0.1 mg.min -1.VIP (0.1 mg.min -1) induced jejunal hypersecretion was inhibited in a doserelated manner by igmesine and octreotide (EDso: 178 and 0.132 mg/kg i.v., respectively).Igmesine (1 mg/kg) and octreotide (1 mg/kg) responses were inhibited by TTX (-96% and -45%, respectively) and CSS (-88% and -100%, respectively).BMY abolished the igmesine response but did not alter the octreotide response.TI'X, CSS and BMY did not alterper se the VIP response.Conclusion: The VIP response was blocked by Igmesine in a TTX-, CSS-and BMY-sensitive manner, suggesting an indirect action on the enterocyte through sigma receptors, nerve-and somatostatin-pathways.Octreotide response was BMY-insensitive indicating that somatostatin receptors activated during the Igmesine response are likely to be distal to the sigma receptors.

Previous data indicate that adenosine 3',5'-cyclic monophosphate activates the epithelial basolateral Na(+)-K(+)-Cl- cotransporter in microfilament-dependent fashion in part by direct action but also in response to apical Cl- loss (due to cell shrinkage or decreased intracellular Cl-). To further address the actin dependence of Na(+)-K(+)-Cl- cotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed. Na(+)-K(+)-Cl- cotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellular Cl- concentration and also to activation of K+ and Cl- efflux pathways. Stabilization of actin with phalloidin blunted cotransporter activation by hypotonicity and abolished hypotonic activation of K+ and Cl- efflux. However, phalloidin did not prevent activation of cotransport by hypertonicity or isosmotic reduction of extracellular Cl-. Conversely, hypertonic but not hypotonic activation was attenuated by the microfilament disassembler cytochalasin D. The results emphasize the complex interrelationship among intracellular Cl- activity, cell volume, and the actin cytoskeleton in the regulation of epithelial Cl- transport.

Previous studies showed that cAMP-dependent transepithelial Cl- secretion of the intestinal cell line T84 is reduced by the F-actin stabilizer phalloidin, an effect in part attributable to inhibition of basolateral Na-K-2Cl cotransport. However, secretory responses are preserved in cells treated with the microfilament disrupter cytochalasin D. We explored the effects of cytochalasin D and two novel compounds derived from marine sponges on the Cl- secretory apparatus of T84 cells. Jasplakinolide (which stabilizes F-actin inhibited cAMP-dependent secretion and Na-K-2Cl cotransport. Latrunculin A (which sequesters G-actin monomers) profoundly altered the distribution of F-actin and reduced basal transepithelial resistance with minimal effect on secretion. Cytochalasin D, but not latrunculin A, activated Na-K-2Cl cotransport. The results provide further evidence that vectorial ion transport is influenced by the cytoskeleton and support a model in which disassembly of F-actin by specific pharmacological means or in response to secretory agonists favors activation of Na-K-2Cl cotransport.

We recently showed that ammonia profoundly inhibits cyclic nucleotide-regulated Cl- secretion in model human T84 intestinal epithelia but does not impair the secretory response to the Ca2+ agonist carbachol. Using transepithelial transport, fura 2 fluorescence, and radioisotopic efflux techniques, we further explored this dichotomy and arrived at a preliminary explanation for the inhibitory action of ammonia. The secretory response to the Ca(2+)-adenosinetriphosphatase inhibitor thapsigargin is unaffected by ammonia, which suggests that an increase in intracellular Ca2+ stimulates secretory pathways that are insensitive to ammonia. Surprisingly, Cl- secretion elicited by the Ca2+ ionophores ionomycin and A23187 is markedly blunted in monolayers pretreated with ammonia. However, ammonia posttreatment does not inhibit the secretory response to ionophore, which suggests that ammonia may interfere with the ability of these ionophores to increase intracellular [Ca2+]. This hypothesis is directly supported by fura 2 experiments. The inhibitory action of ammonia parallels the behavior of the K+ channel blocker Ba2+, and ammonia reduces the basolateral 86Rb+ efflux rate constant in forskolin- but not in carbachol-treated monolayers. Ammonia, which is present in high concentrations in the normal gastro-intestinal tract, may serve as a novel endogenous regulator of epithelial electrolyte transport by interfering with a Ba(2+)-sensitive basolateral K+ conductance distinct from the Ca(2+)-activated basolateral K+ conductance.

BACKGROUND:Adenosine released by cells during ischemia typically serves as a feedback inhibitor of further organ work. However, in ischemic intestine, adenosine appears to act via stimulatory A2b receptors to increase work in the form of chloride ion (Cl-) secretion. This unusual response may contribute to luminal fluid sequestration in intestinal ischemia. In nonischemic cells feed-forward activation of Cl- secretion does not occur despite the fact that adenosine may be continuously generated during normal cell metabolism. Thus we postulated that intestinal epithelia normally control the disposition of adenosine to prevent inappropriate activation of secretion.METHODS:Model T84 intestinal epithelia were studied by means of electrophysiologic and isotopic techniques.RESULTS:Dipyridamole and nitrobenzylthioinosine (inhibitors of nucleoside transport) and iodotubercidin (an inhibitor of adenosine kinase) caused adenosine to accumulate extracellularly and induced a Cl- secretory response that was prevented by adenosine receptor blockade. Uptake of exogenous adenosine was restricted to the basolateral compartment and was blocked by nucleoside transport inhibitors.CONCLUSIONS:Adenosine released from nonischemic intestinal epithelial cells is scavenged by a basolaterally restricted adenosine transporter. This system maintains extracellular adenosine levels below the prosecretory threshold and thus limits adenosine-elicited activation of Cl- secretion (and hence diarrhea) under normal conditions).

Intestinal ischemia is characterized by rapid early inhibition of absorptive function and the appearance of net secretion, although why active secretion persists in the setting of a mucosal energy deficit is unknown. The cryptlike epithelial line T84, a well-characterized model of intestinal Cl- secretion, develops a prominent increase in short-circuit current (Isc, indicative of active Cl- transport) in response to "hypoxia" induced by metabolic inhibitors. The increased Isc is associated with the initial decrease in monolayer ATP content. The Isc is transient and disappears with progressive energy depletion, although graded degrees of ATP depletion induce a more sustained Isc response. Chromatographic analysis and secretory bioassays show that the Isc response to metabolic inhibitors is related to the endogenous release of adenosine into the extracellular space in quantities sufficient to interact locally with stimulatory adenosine receptors. Unlike its classical role as a metabolic feedback inhibitor, adenosine appears to function as an autocrine "feed-forward" activator of active intestinal Cl- secretion. These studies suggest a novel role for adenosine in the conversion of the gut from an absorptive to a secretory organ during ischemic stress, thus contributing to the initial diarrheal manifestation of intestinal ischemia.

BACKGROUND: Although cyclic adenosine monophosphate (cAMP)-dependent intestinal chloride ion (Cl-) secretion is regulated primarily at the level of apical Cl- channels, cAMP also elicits basolateral microfilament remodeling and activates basolateral sodium-potassium-2 chloride (Na-K-2Cl) cotransport. Without these additional events, secretion is inhibited. However, it is unclear whether microfilament-dependent activation of Na-K-2Cl cotransport is a direct effect of cAMP or a secondary response to the opening of apical Cl- channels.METHODS: Using the human intestinal epithelial cell line T84, we examined Cl- secretion elicited by 5'-adenosine monophosphate (5'-AMP), a novel agonist that activates apical Cl- channels without elevation of intracellular cAMP.RESULTS: 5'-AMP was found to activate basolateral Na-K-2Cl cotransport, but such regulation was abolished by the actin stabilizer, phalloidin.CONCLUSIONS: Basolateral Na-K-2Cl cotransport appears to be regulated, at least in part, as an indirect response to activation of apical Cl- channels, a pathway of regulation which map require cytoskeletal remodeling.

The colon, unlike most organs, is normally exposed to high concentrations of ammonia, a weak base which exerts profound and diverse biological effects on mammalian cells. The impact of ammonia on intestinal cell function is largely unknown despite its concentration of 4-70 mM in the colonic lumen. The human intestinal epithelial cell line T84 was used to model electrogenic Cl- secretion, the transport event which hydrates mucosal surfaces and accounts for secretory diarrhea. Transepithelial transport and isotopic flux analysis indicated that physiologically-relevant concentrations of ammonia (as NH4Cl) markedly inhibit cyclic nucleotide-regulated Cl- secretion but not the response to the Ca2+ agonist carbachol. Inhibition by ammonia was 25-fold more potent with basolateral compared to apical exposure. Ion substitution indicated that the effect of NH4Cl was not due to altered cation composition or membrane potential. The site of action of ammonia is distal to cAMP generation and is not due simply to cytoplasmic alkalization. The results support a novel role for ammonia as an inhibitory modulator of intestinal epithelial Cl- secretion. Secretory responsiveness may be dampened in pathological conditions associated with increased mucosal permeability due to enhanced access of lumenal ammonia to the basolateral epithelial compartment.

BACKGROUND:The intestinal epithelial tight junction restricts the paracellular permeation of ions and nonelectrolytes. We hypothesized that this function could be altered or disrupted during cellular adenosine triphosphate (ATP) depletion (chemical hypoxia).METHODS:T84 monolayers grown on permeable supports were studied by electrophysiologic and flux techniques. Mitochondrial and glycolytic inhibitors were used to deplete cellular ATP.RESULTS:Transepithelial resistance to passive ion flow (R) rapidly decreased to 36% of control values with chemical hypoxia, an effect that was reversible if control conditions were restored within 1 hour. As ATP levels declined, a transient Cl- secretory current developed but disappeared as ATP levels reached 5% of control values. Both the secretory current and fall in R were abolished when ambient Cl- was replaced with gluconate but not with Br- or NO3-, or when N-methylglucamine replaced Na+. Transepithelial flux of mannitol but not inulin was increased during ATP depletion. Dual Na(+)-mannitol flux analysis confirmed that the decrease in R was due to an increase in paracellular, not transcellular, permeability. Dilution potentials indicated altered charge selectivity of the junctional pathway.CONCLUSIONS:Chemical hypoxia in intestinal epithelial monolayers alters but does not disrupt the permselectivity properties of the junctional complex.

The importance of microfilaments in the regulation of chloride (Cl-) secretion by the human intestinal cell line T84 was investigated using the cytoskeletal probe phalloidin to bind and stabilize F-actin. Phalloidin was found to inhibit secretion mediated by cyclic adenosine monophosphate (cAMP) and the sustained secretory response to the calcium (Ca+2) ionophore ionomycin but not to affect the transient Ca+2-mediated response to carbachol and histamine. Fluorescent microscopic examination of F-actin revealed regionally restricted microfilament remodeling in cAMP- and ionomycin-treated cells. Normal regulation of apical Cl- and basolateral potassium (K+) channel functions was evident in phalloidin-loaded cells. It is concluded that prevention of cytoskeletal remodeling by actin stabilization inhibits the generation of a sustained Cl- secretory response by a mechanism that does not involve Cl- or K+ channels. Depolymerization of F-actin plays an integral role in the regulation of intestinal Cl- secretion.

Although cAMP-dependent epithelial chloride secretion is largely regulated via apical membrane chloride channels, cAMP also remodels basolateral F-actin and activates basolateral Na-K-2Cl cotransport. Whether activation of cotransport is a primary event or secondary to activation of chloride efflux is not established, and the basis for the cytoskeletal dependence is unknown. We studied cotransport in the intestinal line HT29 (which lacks cAMP-regulated chloride efflux) and in its subclone Cl.19A (in which this pathway is present). Cotransporter activity was enhanced by forskolin in both lines but to a considerably greater extent in subclone Cl.19A, in which the number of bumetanide binding sites was also observed to increase. The F-actin stabilizer phalloidin markedly attenuated cAMP-stimulated cotransport in Cl.19A monolayers, but the increase in bumetanide binding was preserved. These studies identify two mechanisms for activation of Na-K-2Cl cotransport by cAMP: components independent and dependent of cAMP-elicited chloride efflux. Additional Na-K-2Cl cotransporters become accessible to the cell surface coincident with the salt efflux-dependent activation of cotransport. While F-actin rearrangements influence salt efflux-dependent up-regulation of the cotransporter, this influence occurs independently of increases in bumetanide-accessible cotransporters.

Vectorial ion transport and barrier function are two fundamental and defining properties of all epithelial cells. To define the role of F-actin in these dual properties, we studied the effects of two probes of cytoskeletal function on the well-differentiated human intestinal epithelial cell line T84: cytochalasin D, a fungal metabolite widely used to disrupt microfilament function by an uncertain mechanism, and phalloidin, a more specific agent which binds and stabilizes F-actin with high affinity, thus preventing actin depolymerization. T84 cells grown on collagen-coated permeable supports were studied by dual voltage-current clamping; transepithelial resistance to passive ion flow (R, an indirect measure of junctional integrity) and short-circuit current (a measure of net electrogenic ion transport response to the adenylate cyclase activator forskolin was preserved. In contrast, stabilization of F-actin by incubation with phalloidin for up to 24 hr produced no change in R but caused a profound inhibition of cAMP-stimulated short-circuit current. Thus, cytochalasin D was found to perturb barrier but not transport function of T84 monolayers; conversely, stabilization of F-actin by phalloidin was found not to affect barrier function but markedly attenuated electrogenic ion transport. The results suggest that the dual properties of barrier function and ion transport in intestinal epithelia may be differentially influenced by dynamic alterations in F-actin.