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Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K- conductance, we measured short-circuit current (I(SC)) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I(SC) from 3 +/- 0.4 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), reduced I(SC) by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (E(Cl-) = -2 mV), and activated inward current (179 +/- 28 pA, n = 15) when membrane potential was held at the K+ equilibrium potential (E(K+) = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+ , 5 mM), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K + current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2 +]i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+]i. These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.

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The stimulation of epithelial chloride secretion by hormones and neurotransmitters involves the activation of apical membrane chloride channels. The regulation of chloride current by acetylcholine in the T-84 colonic cell line was investigated using single-channel patch-clamp techniques. Treatment with carbachol resulted in the stimulation of transient chloride currents in 18 of 32 previously quiescent patches. Lack of resolvable single-channel openings suggests that single-channel conductance is less than 5-pS. Of 18 responsive patches, 4 showed multiple current oscillations. Treatment of the cells with AlF4- activated sustained chloride currents, suggesting that G proteins are involved. In excised patches, chloride current was markedly sensitive to free Ca2+ concentrations between 500 and 1000 nM. Time-dependent activation and inactivation of chloride current occurred at +60 and -60 mV. These results indicate that the chloride channels responsible for cholinergic activation of chloride conductance in the T-84 colonic cell line are members of the very low conductance family of chloride channels.

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Fecal constituents such as bile acids and increased sialylation of membrane glycoproteins by alpha-2,6-sialyltransferase (HST6N-1) may contribute to colorectal tumorigenesis. We hypothesized that bile acids and phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] would upregulate HST6N-1 in colonic cells. However, deoxycholate (DOC) (300 mumol/l), a secondary bile acid, and TPA (20 ng/ml) decreased expression of an approximately 100-kDa glycoprotein bearing alpha-2,6-linked sialic acid in a colon cancer cell line (T84) in vitro. HST6N-1 mRNA levels were reduced approximately 80% by treatment (< or = 24 h) with DOC or TPA but not by cholate, a primary bile acid. Treatment (24 h) with DOC or TPA decreased activity of this enzyme to 30% and 13% of control, respectively. These effects of DOC and TPA were transcriptional and were mediated by Ca2+ and protein kinase C, respectively. Thus DOC and TPA both downregulated, and did not upregulate, alpha-2,6-sialyltransferase expression in vitro, but by different transduction pathways. As colorectal tumors grow, their progressive removal from the fecal milieu that normally downregulates this enzyme may favor invasion and metastasis.

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We examined the role of G proteins in activation of ionic conductances in isolated T84 cells during cholinergic stimulation. When cells were whole cell voltage clamped to the K+ equilibrium potential (E(K)) or Cl- equilibrium potential (E(Cl)) under standard conditions, the cholinergic agonist, carbachol, induced a large oscillating K+ current but only a small inward current. Addition of the GDP analogue, guanosine 5'-O-(2-thiodiphosphate), to pipettes blocked the ability of carbachol to activate the K+ current. Addition of the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), to pipettes stimulated large oscillating K+ and inward currents. This occurred even when Ca2+ was absent from the bath but not when the Ca2+ chelator, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, was added to pipettes. When all pipette and bath K+ was replaced with Na+ and cells were voltage clamped between E(Na) and E(Cl), GTPgammaS activated oscillating Na+ and Cl- currents. Finally, addition of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to pipettes activated large oscillating K+ currents but only small inward currents. These results suggest that a carbachol-induced release of Ca2+ from intracellular stores is activated by a G protein through the phospholipase C-Ins(1,4,5)P3 signaling pathway. In addition, this or another G protein activates Cl- current by directly gating Cl- channels to increase their sensitivity to Ca2+.

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Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores.

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We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.

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Mechanisms for the assimilation of glucose polymers have been inferred from perfusion studies. To further define these mechanisms, the results of measurements of unidirectional glucose fluxes across short-circuited rabbit jejunal segments in vitro are reported. Glucose polymer-stimulated short-circuit current was similar to that of glucose [19 +/- 6.0 microA/cm2 (n = 7) and 26 +/- 5.7 microA/cm2 (n = 13), respectively] and was inhibited by both acarbose and phlorizin. Acarbose, an alpha-glucosidase inhibitor with no effects of glucose transport, was used to uncouple digestion from absorption. Mucosal-to-serosal flux of glucose polymer-derived glucose was lower than that of an equal weight/volume of glucose [124 +/- 62 nmol.h-1.cm-2 (n = 4) vs. 452 +/- 121 nmol.h-1.cm-2 (n = 6); P less than 0.05] and was inhibited by both phlorizin and acarbose. No glucose polymers were detected in the serosal bath solutions by thin-layer chromatography. It is concluded that glucose polymer-derived glucose is transported by a phlorizin-inhibitable process at a rate slower than that of free glucose, a finding that suggests that hydrolysis limits glucose polymer assimilation.

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The effects of carbamylcholine (carbachol) on intracellular Ca2+ concentration ([Ca2+]c) of T84 cells were examined using the fluorescent Ca2+ indicator fura-2 and microfluorometric techniques. In single isolated cells, carbachol (100 microM) caused a rapid increase in [Ca2+]c of 184 +/- 15 nM (SE, n = 44) from a resting value of 56 +/- 7 nM. This initial transient was followed by a series of oscillations in 68% of the cells. Atropine (10 microM) blocked this response. Removal of bath Ca2+ did not inhibit the rise in [Ca2+]c or oscillations, but the response duration was shortened in 47% of the cells. The amplitude and latency of the initial Ca2+ rise, frequency of oscillations, and number of responding cells varied with the agonist concentration. We have previously shown that carbachol induces an oscillating K+ conductance in T84 cells [D. Devor, S. Simasko, and M. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990]. Simultaneous measurement of membrane K+ current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca2+]c and increase in K+ current. These results show that a rise in [Ca2+]c and oscillations is likely to underlie the membrane K+ current responses to carbachol in T84 cells. Responses from a single cell within a subconfluent monolayer were different from those of isolated cells. In cells of a monolayer the initial [Ca2+]c rise (111 +/- 8 nM; n = 41) was followed by a decline to a stable plateau, and oscillations were not seen. Removal of bath Ca2+ both reduced the initial transient and eliminated the plateau phase of the response. These results suggest that cell-to-cell contact or differentiation during monolayer formation influences the Ca2+ handling mechanisms of T84 cells.

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We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.

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Intracellular pH (pHc) was measured in the short-circuited epithelium of rabbit distal colon using H(+)-selective microelectrodes. pHc was 6.91 +/- 0.02 (SE) when the bath pH was 7.4. Intracellular HCO3- activity (acHCO3-) was estimated from these measurements to be 8 +/- 0 mM. When we replaced all Cl- in the tissue bathing solutions with the impermeant anion gluconate, pHc rose to 7.44 +/- 0.08 and acHCO3- increased to 30 +/- 6 mM. These results demonstrate that this tissue contains a Cl(-)-HCO3- exchange mechanism. During the Cl- replacement the apical membrane electrical potential difference hyperpolarized from -55 +/- 1 to -74 +/- 3 mV, suggesting that membrane ionic conductance had changed. Elevation of either the apical or basolateral membrane bathing solution K+ concentration produced a greater depolarization of membrane potential during Cl- replacement than when tissues were bathed in normal electrolyte solutions. In additional experiments, pHc was raised by lowering the bath CO2 concentration while the bath Cl- concentration was kept normal. Under these conditions, membrane potential hyperpolarized and was more sensitive to the elevation of bath K+ concentration than when pHc was normal. These results suggest that membrane K+ conductance in this tissue is increased by intracellular alkalinization.

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Effects of carbachol on membrane potential and current in T84 cells were determined using whole cell patch-clamp techniques. When the pipettes contained a standard KCl solution and the bath contained a standard NaCl solution, carbachol (100 microM) caused a rapid hyperpolarization to the K+ equilibrium potential (EK+), followed by potential oscillations. When membrane potential was clamped to 0 mV, carbachol induced an outwardly directed K+ current in 31 of 37 cells, with a peak value of 618 +/- 51 (SE) pA. In 77% of these cells the current oscillated and gradually declined to base line. Atropine (20 microM) blocked this response. In symmetric KCl solutions the carbachol-induced current reversed at 0 mV with no rectification. Ba2+ or Cs+ did not block the current, but tetraethylammonium ion (TEA) reduced the number of responding cells. Although a Cl- conductance was found in resting cells, carbachol did not cause an increase in Cl- current when the cells were voltage-clamped to EK+, or when voltage-clamped to +/- 60 mV while bathed in symmetric NaCl solutions. When the Ca2(+)-buffering capacity of the pipette solution was increased, 80% of the cells responded to carbachol, but only 10% oscillated; however, no K+ current was induced by carbachol when the pipette was made nominally Ca2+ free. The current was not affected by removal of Ca2+ from the bath. These results show that carbachol induces an oscillating Ca2(+)-activated K+ conductance in T84 cells, but no Cl- conductance. This K+ conductance is dependent on the mechanisms that regulate intracellular Ca2+.

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The effect of hyperbaric oxygen (HBO) on Na+ transport across the isolated toad (Bufo marinus) skin was studied by measuring the transepithelial short-circuit current (ISC) and resistance (R) at 5, 8, and 10 ATA PO2 and 15 ATA normoxia during steady state conditions. The imposition of 5, 8, and 10 ATA PO2 for 2 h resulted in 45, 52, and 85% decrease in ISC, respectively. This decrease in ISC was always accompanied by an increase in R. When amiloride (10(-4) M) was added to the bathing medium, ISC decreased to zero within 15 min regardless of the PO2 level, indicating that the HBO-induced decrease in ISC is caused by an inhibition of amiloride-sensitive Na+ transport. Addition of both superoxide dismutase (SOD) and catalase to the medium bathing both sides of the skin markedly attenuated the HBO effect on ISC and R. Applying HBO to the serosal or mucosal surface independently produced similar effects on ISC. However, the presence of antioxidant enzymes (SOD and catalase) with 10 ATA PO2 prevented the toxic HBO effect only from the serosal side; no protection by these antioxidant enzymes was observed from the mucosal side. These findings are consistent with a view that free radicals are involved in the HBO-induced inhibition of ISC. However, further studies involving the site(s) of radical generation as well as site(s) of toxic action are needed to understand the cellular and molecular mechanism of HBO toxicity.

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The purpose of this study was to quantify the effects of extracellularly generated partially reduced oxygen species on active sodium (Na+) transport across the ventral toad skin, a well-studied epithelium. Sections of skin from decapitated toads were mounted in an Ussing chamber, bathed on both sides with electrolyte solution containing 500 microM xanthine and bubbled continuously with room air. The tissues were short-circuited, and short-circuit current (Isc) and tissue resistance (Rt) were monitored continuously with an automatic voltage clamp apparatus. Fifteen mU/ml of xanthine oxidase (XO), either purchased from Calbiochem or purified from cream, were instilled in either the apical (mucosal) or basolateral (serosal) baths at t = 0 and t = 10 min. Hydrogen peroxide (H2O2) concentrations increased to 200 microM within the first 20 min and then decreased, reaching a value of 40 microM by 60 min. Mean [H2O2] was 90 microM. Instillation of XO in the apical bath resulted in a large decrease in Isc and an increase in Rt, their values being 43% and 160% of their corresponding controls 85 min after the first instillation. Addition of superoxide dismutase and catalase completely prevented these changes. Instillation of XO in the basolateral bath had no effect. Similar physiological responses were obtained using the Calbiochem XO or the purified XO, which contained no measurable protease activity. It was concluded that extracellularly generated partially reduced oxygen species may interfere with active Na+ transport by possibly damaging apical Na+ channel proteins.

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Replacement of external chloride has been known to reduce Na+ transport across whole frog skin. However, the sidedness and mechanism of the phenomenon have been unclear. In the present study, transepithelial current (IT), transepithelial resistance (RT), and basolateral membrane potential measured both with reference micropipettes (psi sc) and pH-selective microelectrodes (EscH) were monitored in isolated epithelial sheets from frog skin; removal of the underlying dermis facilitates ionic exchange across the basolateral membranes. The intracellular hydronium ion activity (acH) was 58 +/- 4 nM (means +/- SE) when the extracellular hydronium activity was 25 +/- 1 nM under base-line conditions. This measurement is equivalent to an intracellular pH (pHc) of 7.24 +/- 0.03 at an extracellular pH of 7.60 +/- 0.01, in reasonable agreement with estimates obtained by 31P- and 19F-nuclear magnetic resonance (NMR) analyses of frog skin. Complete replacement of mucosal Cl- by gluconate had variable effects on tissue current and resistance from preparation to preparation. The same ionic substitution on the serosal side uniformly produced a prompt reversible decrease in IT, increase in RT, and a substantial membrane depolarization of the short-circuited skins. In most of the preparations, the depolarization was preceded by a small hyperpolarization of 0.5-3.5 mV. The replacement of serosal Cl- also produced a fall in intracellular hydronium ion activity of 33 +/- 10 nM. The present date are consistent with the concept that serosal replacement of Cl- alkalinizes the cells by either favoring HCO3- entry or blocking HCO3- exit through a Cl- HCO3 antiport at the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

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The toad skin and urinary bladder are widely used for the study of water and Na+ transport under high pressure. These tissues can be mounted in Ussing type chambers and ion transport can be measured by evaluating electrical properties of the preparation, e.g., short-circuit current (Isc). The tacit assumption in these experiments is that the preparation behaves in the same manner at high pressure as at 1 ATA; namely, that net Na+ flux is equivalent to Isc. The purpose of the experiments described here was to test that assumption. Toad skins were mounted in an Ussing chamber and Isc was measured as an index of active net Na+ transport under hydrostatic pressures up to 100 ATA. The chamber was modified so that isotopic Na+ flux from the mucosal to serosal compartments could be measured in conjunction with Isc, without decompression. A linear regression of JNa+ms on Isc was computed and found to be described by the equation, JNa+ms = 3.83 + 0.83 Isc; n = 18; r = 0.92. The slope of the line was not significantly different from unity. No correlation was made for JNa+sm because of the difficulty in measuring JNa+sm and JNa+ms in the same skin simultaneously. Independent measurement of JNa+sm demonstrated that this flux accounted for less than 2% of JNa+net. In a second set of experiments, the influence of amiloride on Isc with and without pressure was tested. 10(-4) M amiloride abolished Isc under both circumstances. It is concluded that Isc can be wholly accounted for by net Na+ flux under pressures up to 100 ATA.

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In order to investigate the cellular mechanisms of action of thiazide diuretics, the effect of diuretic and nondiuretic thiazide compounds on Cl- absorption across rabbit distal colon was assessed in tissues mounted in Ussing chambers. This epithelium absorbs Cl- via an active electroneutral transport process. Net 36Cl- absorption across short-circuited tissues was decreased 53, 36 and 20% after addition of 10(-4) M trichlormethiazide, bendroflumethiazide or hydrochlorothiazide, respectively, to the mucosal bathing solution. This inhibitory effect was a result of a decrease in the mucosa-to-serosa unidirectional Cl- flux (P less than .02). Neither the serosa-to-mucosa Cl- flux nor Isc was affected by the thiazides. Thiazide diuretics may exert their effect on Cl- transport across rabbit distal colon through inhibition of a Cl(-)-HCO-3 exchange mechanism. The nondiuretic thiazide, diazoxide, had no effect on Cl- transport. The similarity between the diuretic potency of these compounds and their potency as inhibitors of Cl- absorption by rabbit colon suggests that the thiazides have a similar mechanism of action in renal epithelia.

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The substituted aromatic compound anthracene-9-carboxylic acid (A-9-C) was used to inhibit active Cl- secretion by the epithelium of short-circuited rabbit distal colon. Tissues were mounted in Ussing chambers and stimulated to secrete Cl- by the addition of 1 mM dibutyryl adenosine 3',5'-cyclic monophosphate to the serosal bath. Results of 36Cl-flux measurements showed that the addition of 0.1 mM A-9-C to the mucosal bath inhibited Cl- secretion by 48%. The site of Cl- secretion was determined by using conventional micro-electrodes to show that the cells of the crypt regions, and not the surface epithelial cells, responded to A-9-C by an increase in apical membrane fractional resistance from 0.75 to 0.80 and a hyperpolarization of the apical membrane from -64 to -68 mV (P less than 0.05). The sulfhydryl reagent dithiothreitol was added to the mucosal tissue bath to remove the mucus produced by goblet cells of the crypt regions of these tissues. The time for maximal inhibition of Cl- secretion by A-9-C was decreased from 30 to 15 min by removal of the mucus barrier. The effects of A-9-C on the crypt cells, as well as the effect of mucus on the inhibitory action of this compound, demonstrate that the crypt region is the site of Cl- secretion.

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The short-circuited epithelium of rabbit colon is thought to actively absorb chloride ion by a mechanism in the mucosal cell membrane that exchanges chloride for bicarbonate ion. If this model is correct bicarbonate may be accumulated above electrochemical equilibrium across the mucosal cell membrane. To test this model intracellular pH was measured using a new hydrogen ion-selective liquid membrane microelectrode that is fast, highly selective, and easy to fabricate with a very small tip diameter. These measurements show that the average intracellular pH in this epithelium is 6.9 +/- 0.1. The mucosal cell membrane electrical potential difference, measured by conventional open-tipped microelectrodes, averaged -52 +/- 3 mV. Intracellular pH is above a value predicted for an equilibrium distribution of hydrogen ion across both cell membranes, implying that a mechanism exists for "uphill" extrusion of this ion from the cell. Intracellular bicarbonate activity calculated from these measurements averaged 8 +/- 1 mM. The electrochemical potential gradient for bicarbonate across the mucosal membrane averaged -28 +/- 2 mV, demonstrating that intracellular bicarbonate is concentrated above an equilibrium distribution across the mucosal membrane. Thus energy in the "downhill" electrochemical potential gradient for bicarbonate exit from the cell may drive the entrance of chloride into this epithelium and energize transepithelial chloride absorption.

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The short-circuit current (Isc) was measured as an index of active net sodium transport across the isolated toad skin under various hydrostatic pressures up to 300 ATA. Upon compression, the base-line Isc increased transiently during the first 10 min by 10-15 microA/cm2 (congruent to 20%) and then decreased continuously, leveling off at 40 min under pressure. The latter decrease in base-line Isc (P less than 0.05 at all pressures) was pressure dependent, and its magnitude was 30 microA/cm2 (60% inhibition) at 300 ATA. Similarly, the transepithelial electric potential difference (PD) tended to increase slightly during the early phase of compression and decreased during the steady-state phase of compression. The transepithelial resistance (R), calculated from PD/Isc ratios, generally increased under pressure. The addition of vasotocin to the inside bathing medium resulted in an increase in Isc and PD and a reduction in R at all pressures. The magnitude of peak Isc response to vasotocin was 50-60 microA/cm2 at pressures up to 100 ATA, but decreased to 30 microA/cm2 at 200-300 ATA (0.05 less than P less than 0.10 as compared to the response at 1 ATA). On the other hand the stimulatory effect of 1 mM cyclic adenosine monophosphate (cAMP), added to the inside bathing medium, on Isc was not affected by pressure between 1 and 300 ATA. From these results it is postulated that the inhibition of base-line Na transport under high hydrostatic pressure may be primarily due to a decrease in the outer membrane permeability to Na rather than an inhibition of the Na-K-ATPase.

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