Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells

ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein-protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.     

Calpains are involved in Entamoeba histolytica-induced death of HT-29 colonic epithelial cells

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and μ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and μ-calpain may be involved in colon epithelial cell death induced by E. histolytica.     

Inactivation by Ca2+ of Ca2+-stimulated ATPase from rat red cells

Summary The effect of Ca²⁺ on the stability of the Ca²⁺-stimulated ATPase has been investigated. Our results showed that the preincubation of the rat red cell membranes in presence of Ca²⁺ causes an irreversible inhibition of the enzyme. The same effect was obtained with Ba²⁺ instead of Ca²⁺. Once initiated the inactivation of the enzyme could be halted by the addition of ethylene glycol bis (B-amino ethyl ether) N,N-tetra acitic acid (EGTA), but inactivation was irreversible. The presence of ATP in the preincubation with Ca²⁺ prevented the inactivation but calmodulin did not.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Extracellular ATP activates Ca2(+)-dependent K+ conductance via Ca2+ influx in mouse macrophages

1. Using the perforated patch recording, the effects of ATP on membrane current were investigated in mouse peritoneal macrophages. 2. Extracellularly applied ATP induced a biphasic current consisting of a initial inward current [Ii(ATP)] followed by an outward current [Io(ATP)]. These currents were associated with a marked increase in conductance at their peaks. 3. Ii(ATP) reversed close to 0 mV and was attenuated by removal of external Na+. 4. Io(ATP) reversed near -80 mV and was increased by decreasing the external concentration of K+. 5. Io(ATP) was completely abolished by removal of external Ca2+, treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (BAPTA-AM) and bath applied quinidine but not tetraethylammonium (TEA) or apamin. 6. These results suggest that Ii(ATP) and Io(ATP) are due to an activation of nonspecific cationic and Ca2(+)-dependent K+ conductances, respectively, and raise the possibility that the putative ATP receptor may be important in regulating macrophage functions, motility, phagocytosis and cytokines secretion.     

Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.     

Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line

Summary A human intestinal epithelial cell line (Intestine 407) is known to retain receptors for intestinal secretagogues such as acetylcholine (ACh), histamine, serotonin (5-HT) and vasoactive intestinal peptide (VIP). The cells were also found to possess separate receptors for secretin and ATP, the stimulation of which elicited transient hyperpolarizations coupled to decreased membrane resistances. These responses were reversed in polarity at the K⁺ equilibrium potential. The hyperpolarizing responses to six agonists were reversibly inhibited by quinine or quinidine. By means of Ca²⁺-selective microelectrodes, increases in the cytosolic free Ca²⁺ concentration were observed in response to individual secretagogues. The time course of Ca²⁺ responses coincided with that of hyperpolarizing responses. The responses to ACh and 5-HT were abolished by a reduction in the extracellular Ca²⁺ concentration down to pCa 7 or by application of Co²⁺. Thus, in Intestine 407 cells, not only the intestinal secretagogues, which are believed to act via increased cytosolic Ca²⁺ (ACh, 5-HT and histamine), but also those which elevate cyclic AMP (VIP, secretin and ATP) induce increases in cytosolic Ca²⁺, thereby activating the K⁺ conductance. It is likely that the origin of increased cytosolic Ca²⁺ is mainly extracellular for ACh- and 5-HT-induced responses, whereas histamine, VIP, secretin and ATP mobilize Ca²⁺ from the internal compartment.     

CAS/CSE 1 stimulates E-cadhrin-dependent cell polarity in HT-29 human colon epithelial cells

The establishment and maintenance of epithelial polarity are crucial for tissue organization and function in mammals. Epithelial cadherin (E-cadherin) is expressed in epithelial cell membrane and is important for cell-cell adhesion, intercellular junctions formation, as well as epithelial cell polarization. We report herein that CAS (CAS/CSE 1), the human cellular apoptosis susceptibility protein, interacts with E-cadherin and stimulates polarization of HT-29 human colon epithelial cells. CAS binds with E-cadherin but not with beta-catenin in the immunoprecipitation assays. Interaction of CAS with E-cadherin enhances the formation of E-cadherin/beta-catenin cell-cell adhesive complex. Electron microscopic study demonstrated that CAS overexpression in cells stimulates intercellular junction complex formation. The disorganization of cellular cytoskeleton by cytochalasin D, colchicine, or acrylamide treatment disrupts CAS-stimulated HT-29 cell polarization. CAS-mediated HT-29 cell polarity is also inhibited by antisense E-cadherin DNA expression. Our results indicate that CAS cooperates with E-cadherin and plays a role in the establishment of epithelial cell polarity.     

Alpha 2-adrenergic receptors and the Na+/H+ exchanger in the intestinal epithelial cell line, HT-29

alpha 2-Adrenergic receptors (alpha 2-AR) are negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to alpha 2-AR agonists, suggesting that the receptor can couple to other signal transduction pathways. One potential pathway may be the stimulation of Na+/H+ exchange elicited by alpha 2-AR activation in renal proximal tubule cells, platelets, and the NG-10815 cell line. To determine whether the various receptor-effector coupling mechanisms operate in a tissue-specific manner, we studied the effect of alpha 2-AR activation on basal and stimulated Na+/H+ exchange in epithelial cells isolated from human colon (HT-29 adenocarcinoma cells). Na+/H+ exchange was measured by quantitation of intracellular hydrogen ion concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and 22Na+ uptake. HT-29 cells expressed an amiloride-sensitive Na+/H+ exchanger that was activated by reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observed after acid loading (0.57 +/- 0.07 pH units/min/10(4) cells) was dependent on external sodium and was blocked by amiloride (Ki approximately 2.1 microM). Although epinephrine and the selective alpha 2-AR agonists clonidine and UK-14304 inhibited forskolin-activated adenylylcyclase, these compounds did not alter basal Na+/H+ exchange. Stimulated Na+/H+ exchange was similarly unaffected by epinephrine. In contrast, stimulated Na+/H+ exchanger activity was completely inhibited by the selective alpha 2-agonists clonidine, UK-14304, and guanabenz. This inhibitory effect was not blocked by the alpha 2-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchanger molecule itself. Structure/activity studies indicated that the compounds inhibiting exchanger activity possess either an imidazoline or guanidinium moiety. Although these molecules bear structural similarity to amiloride, they did not inhibit the amiloride-sensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as "amiloride-like" ligands selective for the Na+/H+ exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, alpha 2-adrenergic receptors are not coupled to the Na+/H+ exchanger, suggesting that the cell-signaling mechanisms utilized by the alpha 2-AR are tissue specific.     

Ca2+- and voltage-dependent K+ conductance in dispersed parathyroid cells

The membrane ionic conductances of dispersed parathyroid cells kept in primary culture were studied using the "whole-cell" and "inside-out excised patch" variants of the patch-clamp technique. The major component of the total current was a voltage-dependent outward K+ current without an appreciable inward current. The amplitude of the K+ current was markedly reduced when free internal Ca2+ was buffered by addition of 10 mM EGTA. Recordings of single-channel current in excised membrane patches revealed the presence of K+ channels with large unitary conductance (200 pS in symmetrical 130 mM K+ solutions) which were also activated by depolarization when internal Ca2+ concentration was about 10(-5)-10(-6) M. At any membrane voltage these channels were closed most of the time at internal Ca2+ concentrations lower than 10(-10) M. These results demonstrate the existence of a Ca2+- and voltage-dependent K+ permeability in parathyroid cells which may participate in the unusual membrane potential changes induced by alterations of external Ca2+ and, possibly, in the regulation of parathormone secretion.     

Ca(2)+-stimulated exocytosis in maize coleoptile cells

Changes in membrane capacitance (C(m)) after photolysis of the caged Ca(2)+ compound dimethoxynitrophenamine were studied in protoplasts from maize coleoptiles. Changes in C(m) values resulting from increased concentrations of free Ca(2)+ in the cytoplasm ([Ca(2)+](cyt)) were interpreted as representing changes in [Ca(2)+](cyt)-sensitive exocytosis and endocytosis. A continuous increase in [Ca(2)+](cyt) resulted in a sigmoidal increase in C(m) values with a half-maximal concentration at approximately 1 microM. The steep increase in C(m) values was followed by a variable slow phase in changing C(m) values. When [Ca(2)+](cyt) increased at a rate of 0.6 micromol L(-)(1) sec(-)(1), the initial steep increase in C(m) values lasted approximately 5 to 10 sec. During this time, protoplasts increased in surface area by approximately 2.5%. The biphasic dynamics of [Ca(2)+](cyt)-stimulated increases in C(m) values can be described by a kinetic model containing two pools of vesicles with two [Ca(2)+](cyt)-sensitive steps in the exocytotic pathway.     

Basolateral store-operated Ca(2+)-entry in polarized human bronchial and colonic epithelial cells

Bronchial epithelial cells respond to extracellular nucleotides from the luminal and basolateral side activating Cl- secretion via [Ca2+]i increase. In this study we investigated the differences of apically (ap) and basolaterally (bl) stimulated [Ca2+]i signals in polarized human bronchial epithelial cells (16HBE14o-). Specifically we investigated the localization of 'capacitative Ca2+ entry' (CCE). 16HBE14o- cells grown on permeable filters were mounted into an Ussing chamber built for the simultaneous measurement of Fura-2 fluorescence and electrical properties. Application of ATP from both sides induced a rapid [Ca2+]i increase and subsequent sustained [Ca2+]i plateau due to transmembraneous Ca(2+)-influx. The use of different nucleotides revealed the following rank order or potency which was very similar for addition from the apical or basolateral side: UTP (EC50 ap: 4 microM, bl: 5 microM) > ATP (EC50 ap: 4 microM, bl: 10 microM) > ADP (n = 4-7 from both sides). 2-MeS-ATP, AMP, adenosine and beta gamma-methylene ATP were ineffective (n = 3 from both sides). The ATP- (ap and bl) induced Ca2+ influx was only abolished by removal of basolateral Ca2+. This was also true for receptor-independent activation of Ca(2+)-influx by intracellular Ca(2+)-store depletion with 2,5 Di-(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 microM). Also in polarized T84 cells the basolateral carbachol and BHQ activated Ca2+ plateau was exclusively sensitive to removal of basolateral Ca2+. We propose that in all polarized epithelial cells the CCE entry pathway is located in the basolateral membrane. We furthermore suggest that Ca2+[i elevating agonists acting from the apical side of the epithelium lead to the opening of a basolateral CCE pathway.