Reevaluation of Cl-/HCO3- exchange in cultured bovine corneal endothelial cells

PURPOSE: To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport. METHODS: BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2). RESULTS: Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response. CONCLUSIONS: Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused primarily by depolarization of Em, which drives HCO3- influx through the basolateral electrogenic Na+:nHCO3- cotransporter. In contrast with cultured BCECs, AE2 may be present in fresh cells.     

Effect of SCFA on intracellular pH and intracellular pH regulation of guinea-pig caecal and colonic enterocytes and of HT29-19a monolayers

The response of the intracellular pH (pHi, measured with BCECF) of the caecal and distal colonic epithelium of guinea pig and of monolayers of HT29 clone 19a cells on the addition of short-chain fatty acids (SCFA) was assessed. Addition of SCFA to the luminal side of these cells had no major effect on pHi, independent of whether the apical Na+/H+ exchange or the apical K+/H+ ATPase was inhibited or not. Addition of SCFA to the serosal side, on the other hand, caused a marked decrease of pHi, followed by an effective regulation back to basal values, and after removal of the acid, the cells became alkalinized. Intracellular pH is mainly regulated by mechanisms in the basolateral membrane. The basolateral Na+/H+ exchanger and the Cl-/HCO3- exchanger were mainly responsible for pHi regulation. Inhibition studies are consistent with a NHE-1 type Na+/H+ exchanger in the basolateral membranes. The apical Na+/H+ exchanger of caecal enterocytes and in HT29 cells, and the apical K+/H+ ATPase in the apical membrane of the distal colon have no or little influence on pHi regulation. The comparison shows that the HT29-19a cell line is an adequate model for studying pHi phenomena of hind gut epithelial cells.     

Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro

It has long been accepted that marginal cells of stria vascularis are involved in the generation of the endocochlear potential and the secretion of K+. The present study was designed to provide evidence for this hypothesis and for a cell model proposed to explain K+ secretion and the generation of the endocochlear potential. Stria vascularis from the cochlea of the gerbil was isolated and mounted into a micro-Ussing chamber such that the apical and basolateral membrane of marginal cells could be perfused independently. In this preparation, the transepithelial voltage (Vt) and resistance (Rt) were measured across marginal cells and the resulting equivalent short circuit current (Isc) was calculated (Isc = Vt/Rt). Further, K+ secretion (JK+,probe) was measured with a K(+)-selective vibrating probe in the vicinity of the apical membrane. In the absence of extrinsic chemical driving forces, when both sides of the marginal cell epithelium were bathed with a perilymph-like solution, Vt was 8 mV (apical side positive), Rt was 10 ohm-cm2 and Isc was 850 microA/cm2 (N = 27). JK+,probe was outwardly directed from the apical membrane and reversibly inhibited by basolateral bumetanide, a blocker of the Na+/Cl-/K+ cotransporter. On the basolateral but not apical side, oubain and bumetanide each caused a decline of Vt and an increase of Rt suggesting the presence of the Na,K-ATPase and the Na+/Cl-/K+ cotransporter in the basolateral membrane. The responses to [Cl-] steps demonstrated a significant Cl- conductance in the basolateral membrane and a small Cl- conductance in the paracellular pathway or the apical membrane. The responses to [Na+] steps demonstrated no significant Na+ conductance in the basolateral membrane and a small Na+ or nonselective cation conductance in the apical membrane or paracellular pathway. The responses to [K+] steps demonstrated a large K+ conductance in the apical membrane. Apical application of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and basolateral elevation of K+ caused an increase in Vt and a decrease in Rt consistent with stimulation of the apical K+ conductance. Similar observations have been made in vestibular dark cells, which suggest that strial marginal cells and vestibular dark cells are homologous and transport ions by the same pathways. Taken together, these observations are incompatible with a model for the generation of the endocochlear potential which ascribes the entire potential to the strial marginal cells [Offner et al. (1987) Hear. Res. 29, 117-124].(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolated perfused rabbit colon crypts: stimulation of Cl- secretion by forskolin

The aim of this study was to characterize ion conductances and carrier mechanisms of isolated in vitro perfused rabbit colonic crypts. Crypts were isolated from rabbit colon mucosa and mounted on a pipette system which allowed controlled perfusion of the lumen. In non-stimulated conditions basolateral membrane voltage (Vbl) was -65 +/- 1 mV (n = 240). Bath Ba2+ (1 mmol/l) and verapamil (0.1 mmol/l) depolarized Vbl by 21 +/- 2 mV (n = 7) and 31 +/- 1 (n = 4), respectively. Lowering of bath Cl- concentration hyperpolarized Vbl from -69 +/- 3 to -75 +/- 3 mV (n = 9). Lowering of luminal Cl- concentration did not change Vbl. Basolateral application of loop diuretics (furosemide, piretanide, bumetanide) had no influence on Vbl in non-stimulated crypts. Forskolin (10(-6) mol/l) in the bath depolarized Vbl by 29 +/- 2 mV (n = 54) and decreased luminal membrane resistance. In one-third of the experiments a spontaneous partial repolarization of Vbl was seen in the presence of forskolin. During forskolin-induced depolarization basolateral application of loop diuretics hyperpolarized Vbl significantly and concentration dependently with a potency sequence of bumetanide > piretanide > or = furosemide. Lowering bath Cl- concentration hyperpolarized Vbl. Lowering of luminal Cl- concentration from 120 to 32 mmol/l during forskolin-induced depolarization led to a further depolarization of Vbl by 7 +/- 2 mV (n = 10). We conclude that Vbl of rabbit colonic crypt cells is dominated by a K+ conductance. Stimulation of the cells by forskolin opens a luminal Cl- conductance. Basolateral uptake of Cl- occurs via a basolateral Na+:2Cl-:K+ cotransport system.     

Ion channels in isolated mouse jejunal crypts

The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K+ channel (BK) (conductance, g at 0 mV = 92 +/- 6 pS), which was highly selective for K+ [PK+ (1) > PRb+ (0.6) > PCs+ (0.09) approximately PNa+ (0.07) > PLi+ (0.04)], had a low, voltage-independent open probability (Po) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 +/- 3 pS), Po was voltage dependent and it was blocked by cytoplasmic Ba2+ (5 mmol/l). An intermediate K+ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 +/- 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl- channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 +/- 2 pS), its Po was independent of voltage and unaffected by variations in cytoplasmic Ca2+ (100 nmol/l to 1 mmol/l) or ATP (0-1 mmol/l). The NSCC had a linear conductance (20 +/- 1 pS), its Po increased with depolarisation and elevation of cytoplasmic [Ca2+] (> or = 10 micromol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl- conductance was activated. This cAMP-dependent Cl- conductance was distinct from the basolateral Cl- channel and thus is most likely located in the apical membrane.     

Na(+)-dependent and -independent Cl-/HCO-3 exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.     

Cyclic AMP-dependent anion secretion in human small and large intestine

Cyclic AMP-dependent Cl- secretion is the major secretion pathway in human intestine. The aim of the present study was to examine mechanisms involved in cAMP-dependent anion secretion in human small and large intestine. Surgical resection specimens from both jejunum and distal colon were studied under short circuited conditions. Addition of the phosphodiesterase inhibitor IBMX induced an increase in the short-circuit current (Isc) equivalent to the net increase in Cl- secretion. The Isc was inhibited by diphenylamine decarboxylate (DPC; Cl- channel blocker), bumetanide (basolateral Na+/K+/2Cl- cotransporter), BaCl2 (basolateral K+ channel) and Cl- free buffer in both segments and indomethacin (cyclo-oxygenase inhibitor) in colon alone. Diphenylamine decarboxylate appears to directly inhibit secretion in jejunum, although its inhibitory effect is possibly mediated by inhibition of cyclo-oxygenase in the colon. A small component of IBMX-stimulated Isc was inhibited by acetazolamide. Cyclic AMP-dependent secretion is largely apical Cl- secretion, although a small component appears to be HCO3. Secretion is dependent on basolateral K+ channels and Na+/K+/2Cl- cotransporters and, in the colon, is inhibited by indomethacin, implying a role for cyclo-oxygenase metabolites. The chloride channel blocker DPC inhibits secretion in both areas. This class of compounds may have potential for treatment of secretory diarrhoea.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of -62 +/- 3 mV (n = 8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na(+)-free bath and were reversibly blocked by 4 mM Co2+. They lasted 0.9-3.0s with 64 mM Cl- in the pipette, were shortened 67 +/- 8% by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 20 microM), and lengthened to 8.7 +/- 2.2 when the Cl- equilibrium potential (Vcl) was changed from -20 mV to -2 mV by using 134 mM Cl- in the pipette. With conventional whole-cell voltage-clamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35-1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of -60 mV and -50 mV and reached a maximum inward amplitude at about -40 mV. Changing the Cl- concentration in the pipette (VCl of -2MV or -20 mV) or bath (VCl of -2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl- electrode. They were inhibited by the Cl- channel antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM), NPPB (20 microM), and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca2+ deprivation, or by CO2+ (4 mM), or by replacing external Ca2+ with Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of maternal uninephrectomy on the development of fetal rat kidney: numerical and volumetric changes of the glomerulus and formation of the anionic site in the glomerular basement membrane

1. K+ and Cl- conductances and their putative regulation have been characterized in the rat colonic epithelium by Ussing-chamber experiments, whole-cell and single-channel patch-clamp recordings. 2. The apical Cl- conductance is under the control of intracellular cAMP. An increase in the concentration of this second messenger induces transepithelial Cl- secretion due to the activation of an apical 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)- and glibenclamide-sensitive Cl- conductance. 3. In addition to the apical Cl- conductance, the basolateral membrane is equipped with Cl- channels. They are stimulated by cell swelling and play a role in cell volume regulation and transepithelial Cl- absorption. 4. The basolateral K+ conductance is under the dominant control of intracellular Ca2+. An increase in the cytosolic Ca2+ concentration leads to the opening of basolateral K+ channels, which causes a hyperpolarization of the cell membrane, indirectly supporting Cl- secretion owing to an increase in the driving force for Cl- exit. The predominant effect of cAMP on the basolateral K+ conductance is an inhibitory one, probably due to a decrease in the intracellular Ca2+ concentration. 5. The apical K+ conductance, which is involved in transepithelial K+ secretion, is stimulated by an increase in the intracellular Ca2+ concentration. 6. The differential regulation of apical and basolateral ion conductances in the epithelium of the rat distal colon provides an interesting example for the mechanisms underlying vectorial transport of ions across polarized cells.     

Ion exchangers mediating NaCl transport in the proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. We identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanism's of uphill Cl- entry across the apical membrane of proximal tubule cells. For steady state Cl- absorption to occur by these mechanisms, formate and oxalate must recycle from lumen to cell. Recycling of formate from lumen to cell may occur by H(+)-coupled formate transport and nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. Oxalate recycling from lumen to cell may take place by oxalate-sulfate exchange in parallel with Na(+)-sulfate cotransport. Cl- exit across the basolateral membrane is most likely mediated by Cl- channels. To identify the Na(+)-H+ exchanger (NHE) isoform(s) expressed on the brush border membrane of proximal tubule cells, we developed isoform-specific monoclonal and polyclonal antibodies. We found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the thin and thick limbs of the loop of Henle. NHE3 is also present in a population of subapical intracellular vesicles, suggesting possible regulation by membrane trafficking. The inhibitor sensitivity of Na(+)-H+ exchange in renal brush border vesicles indicates that it is mediated by NHE3 under baseline conditions and during up-regulation by metabolic acidosis. Increased apical membrane Na(+)-H+ exchange activity in response to metabolic acidosis and during renal maturation is associated with increased NHE3 protein expression. Finally, we found that the organic anion-dependent absorption of Cl- is markedly down-regulated in metabolic acidosis in parallel with the up-regulation of brush border membrane Na(+)-H+ exchange. Thus, differential regulation of apical membrane ion exchangers may provide a mechanism to regulate the relative rates of NaHCO3 and NaCl reabsorption.     

Conformational state-dependent effects of halothane on cardiac Na+ current

BACKGROUND: The Na+ channel is voltage gated and characterized by three distinct states: closed, open, and inactivated. To identify the effects of halothane on the cardiac Na+ current (I(Na)) at various membrane potentials, the effects of 1.2 mM halothane at different holding potentials (V(H)) on I(Na) were examined in single, enzymatically isolated guinea pig ventricular myocytes. METHODS: The I(Na) was recorded using the whole-cell configuration of the patch-clamp technique. Currents were generated from resting V(H)s of -110, -80, or -65 mV. State-dependent block was characterized by monitoring frequency dependence, tonic block, and removal of inactivation by veratridine. RESULTS: Halothane produced significant (P < 0.05) V(H)-dependent depressions of peak I(Na) (mean +/- SEM): 24.4 +/- 4.1% (V(H) = -110 mV), 42.1 +/- 3.4% (V(H) = -80 mV), and 75.2 +/- 1.5% (V(H) = -65 mV). Recovery from inactivation was significantly increased when cells were held at -80 mV (control, tau = 6.0 +/- 0.3 ms; halothane, tau = 7.1 +/- 0.4 ms), but not at -110 mV. When using a V(H) of -80 mV, halothane exhibited a use-dependent block, with block of I(Na) increasing from 8.6 +/- 1.4% to 30.7 +/- 3.5% at test pulse rates of 2 and 11 Hz, respectively. Use-dependent inhibition was not apparent at V(H) of -110 mV. When inactivation of I(Na) was removed by exposure to 100 microM veratridine, no significant difference was observed in the depressant effect of halothane at both V(H)s: 26.6 +/- 4.5% (V(H) = -80 mV) and 26.4 +/- 5.6% (V(H) = -110 mV). CONCLUSIONS: The present findings indicate that the depressant action of halothane on cardiac I(Na) depends on the conformational state of the channel. As more channels are in the inactivated state, the more potent is the effect of halothane. Removal of channel inactivation by veratridine abolished the dependence of the halothane effect on V(H), but depression of the current was still evident. These results indicate a complex interaction between halothane and the various conformational states of the Na+ channel.     

Membrane function and vascular reactivity

The resting potential and monovalent ions in the marginal cells and scala media were measured before and 20 min after the onset of anoxia using ion-sensitive microelectrodes. The resting potential of the marginal cells decreased from 62.7 to -2.4 mV. The K+ activity decreased from 77.7 to 53.2 mEq/l, while the Na+ activity increased from 2.6 to 24.7 mEq/l. The Cl- activity did not change significantly. In the scala media, the endocochlear potential decreased rapidly from 80.9 to -28.0 mV after the onset of anoxia. The K+ activity decreased from 119.0 to 96.5 mEq/l, the Na+ activity increased from 1.3 to 9.5 mEq/l and that of Cl- decreased from 127.0 to 115.1 mEq/l. The electrochemical gradients determined for each ion based on the ionic changes in the scala media and marginal cells, suggested the existence of an Na/K pump and Na-K-2Cl cotransport at the basolateral membrane of the marginal cells, and a rheogenic K pump and Na-K-2Cl transport at the luminal membrane of the marginal cells. The Na+ and K+ must be recycled at the basolateral membrane and luminal membrane of marginal cells, respectively.     

Effect of lung volume reduction surgery on gas exchange and pulmonary hemodynamics at rest and during exercise

The present study was an investigation of the regulation of anion secretion across cultured mouse endometrial epithelium by prostaglandin E2 (PGE2) using the short-circuit current (ISC) technique. The cultured endometrial monolayers responded to both apical and basolateral application of PGE2 with a sustained rise in ISC in a concentration-dependent manner. However, the potencies of apical and basolateral addition of PGE2 were different, with apparent EC50 of 200 and 4 nM, respectively. Replacement of Cl- or HCO3- in the bathing solution significantly reduced the ISC responses to both apical and basolateral addition of PGE2; however, the apical response exhibited greater dependence on HCO3- . Pretreatment with diphenylamine 2,2'-dicarboxylic acid, a Cl- channel blocker, significantly reduced both PGE2-induced ISC responses, while pretreatment with amiloride, a Na+ channel blocker, did not exert any effect. Forskolin, an adenylate cyclase activator, and 3-isobutyl-dihydro-testosterone-1-methyl-xanthine, a cAMP phosphodiesterase inhibitor, mimicked the ISC response to PGE2 while MDL12330A, an adenylate cyclase inhibitor, completely abolished the PGE2-induced ISC. The results of the present study indicate that the anion secretion across the mouse endometrial epithelium may be regulated by PGE2 involving a cAMP-dependent mechanism predominantly. The differential responses to apical and basolateral challenge with PGE2 also suggest that PGE2 of different origins may play different roles in uterine function.     

A micromechanical model of lung tissue rheology

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.     

Effects of HCO3- on cell composition of rabbit ciliary epithelium: a new model for aqueous humor secretion

PURPOSE: To determine whether the Na+-K+-2Cl- symport or the parallel Na+/H+ and Cl-/HCO3- antiports provide the dominant pathway for NaCl uptake into the ciliary epithelium. Both pathways are known to support NaCl entry from the stroma into the pigmented ciliary epithelial (PE) cells, after which Na+ and Cl- diffuse across the gap junctions into the nonpigmented ciliary epithelial (NPE) cells and are released into the aqueous humor. METHODS: Rabbit iris ciliary bodies were preincubated in HCO3-/CO2-containing or HCO3-/CO2-free solutions before quick freezing, cryosectioning, dehydration, and electron probe x-ray microanalysis. RESULTS: The NPE and the PE cells contained more K and Cl when incubated with bicarbonate. Inhibition of carbonic anhydrase with 0.5 mM acetazolamide had little effect in HCO3--free medium but prevented the increase in Cl in both cell types in HCO3-/CO2 solution. Inhibition of the Na+-K+-2Cl- symport with 10 to 500 microM bumetanide caused Cl loss from both cell types in HCO3--free solution, but bumetanide produced a paradoxical increase in Cl and Na in HCO3-/CO2 solution. Together, acetazolamide and bumetanide resulted in significant Cl loss in HCO3--free solution and prevented the gains of Cl and Na in HCO3-/CO2 solution. CONCLUSIONS: The present results indicate that the dominant entry pathway of NaCl from the stroma into the ciliary epithelial syncytium is through an acetazolamide-inhibitable Cl-/HCO3 and a parallel Na+/H+ antiport. The dominant release pathways into the aqueous humor appear to be a Na+-K+-2Cl-symport, which can be outwardly directed under physiological conditions, together with the Na+/K+-exchange pumps and Cl- channels.     

Membrane polarity of the Na(+)-K+ pump in primary cultures of Xenopus retinal pigment epithelium

The retinal pigment epithelium is a transporting epithelium that helps regulate the volume and composition of the subretinal space surrounding photoreceptor outer segments. The capacity of the RPE to actively transport Na+ and K+ between the retina and the blood supply depends on the localization of the Na+, K(+)-ATPase to the apical membrane, but in culture this polar distribution can be lost. Using primary cultures of Xenopus RPE, we examined the anatomical and functional polarity of this electrogenic pump. Confluent monolayers were established on Matrigel-coated microporous filters and cultured for 2-4 weeks in serum-free defined medium. Electrogenic pump activity at the apical and basolateral membranes was assayed by mounting the monolayer and filter in an Ussing chamber and exposing one or the other surface to ouabain while recording the apical (Vap) and basolateral (Vba) membrane potentials with an intracellular microelectrode. The addition of 0.2 mM ouabain to the apical bath caused Vap to rapidly depolarize by about 4 mV, consistent with the inhibition of a hyperpolarizing pump current at that membrane. When ouabain was added to the basal bath, however, it had no effect on Vba, suggesting the absence of a functional Na(+)-K+ pump on the basolateral membrane. To confirm these electrophysiological results, we examined the distribution of the Na+, K(+)-ATPase catalytic component using an antiserum specific for the bovine kidney alpha subunit. Antibody labeling of cultures was highly polarized, with strong reaction present on the apical microvilli, but not the basolateral cell surfaces. The findings of this study indicate that the Na(+)-K+ pump in monolayers of Xenopus RPE, as in native RPE, is located mainly in the apical membrane, providing evidence of a functionally intact transport pathway in these primary cultures.     

Kinetics of chloride-bicarbonate exchange across the human red blood cell membrane

We use a fluorescent probe of [Cl-], 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), to study Cl-/HCO-3 exchange in human erythrocyte ghosts in a stopped-flow apparatus at 4 degrees C. The quench constant of SPQ in our Cl-/HCO-3/HPO=4 system at pH 7.4 is 0.065 +/- 0.005 mM-1. The time course of Cl-/HCO-3 exchange does not follow a single exponential function at 4 degrees C and we propose an extended ping-pong model in which slippage is explicitly considered in order to account for this phenomenon. The solution of the system of equations generated by our model is a double exponential function which fits the time course of Cl-/HCO-3 exchange. Our results confirm the predictions of the model concerning the functional dependence of the two rate constants. One rate constant (k1) is independent of medium composition; it is determined by the sum of the two slippage rate constants and its value is 1.04 +/- 0.14 sec-1. The other rate constant (k2) varies inversely with [Cl-]; the regression line is 1/k2 = 18.8 sec - 0.095 mM-1sec [Cl-].     

Hepatorenal reflex plays an important role in natriuresis after high-NaCl food intake in conscious dogs

Responses of renal nerve activity and urinary Na+ and Cl- excretion were examined in chronically instrumented conscious dogs through feedings of boiled rice with or without NaCl. The boiled rice (20 g/kg body wt) without NaCl did not influence plasma Na+ and Cl- concentrations, renal nerve activity, or urinary Na+ excretion but decreased urinary Cl- excretion. On the other hand, boiled rice containing NaCl (0.4 g/kg body wt) increased plasma Na+ (+3.8 +/- 0.7 meq/l) and Cl- (+3.0 +/- 1.5 meq/l) concentrations, then decreased renal nerve activity by 61 +/- 4%, and increased urinary Na+ and Cl- excretions. In dogs with hepatic denervation, a decrease in renal nerve activity, which was observed in intact dogs in response to the high-NaCl food intake, was completely abolished along with significant attenuation of postprandial natriuresis. That is, only 9 +/- 5% of the loaded Na+ and 7 +/- 3% of the loaded Cl- were excreted during 4 consecutive hours in hepatic-denervated dogs, whereas 36 +/- 5% of the loaded Na+ and 36 +/- 4% of the loaded Cl- were excreted in intact dogs. In dogs with renal denervation, postprandial natriuresis was also attenuated. These results indicate that the high-NaCl food intake elicits a decrease in renal nerve activity, the decrease is predominantly mediated by the hepatic nerves, and the decrease in renal nerve activity plays an important role in augmentation of urinary Na+ and Cl- excretion. Thus, the hepatorenal reflex may play an important role in controlling extracellular fluid homeostasis during food intake.     

Mutations in the EXT1 and EXT2 genes in hereditary multiple exostoses

In an in-vitro preparation of gastric mucosae of Rana pipiens, the effect of adding melittin to a concentration of 5x10-6 M in the secretory solution on the transepithelial potential difference (PD), resistance (R) and short-circuit current (Isc) was studied. In 20 min, melittin decreased the PD by 9.3 mV and R by 148 ohm cm2. These changes can be explained by a decrease in the resistance, RP, of the paracellular pathway. To determine whether specific-ion pathways were responsible for the decrease in R, the effect of melittin on the partial conductances of Cl-, K+ and Na+ was also studied using the ion substitution method. Melittin decreased the PD response to changes in nutrient Na+, K+ and Cl- and the PD response to changes in secretory Cl-, but did not affect PD responses to changes in secretory Na+ or K+. Therefore, melittin decreased the nutrient membrane partial conductances of Cl-, K+ and Na+ and secretory membrane partial conductance of Cl-, without affecting the secretory partial conductances of Na+ or K+. Initially, melittin increased Isc in regular and Cl--free but not in Na+-free solutions. There was a delayed decrease in Isc. The results indicate that melittin decreases RP, increases the Na+ conductance of the secretory membrane and inhibits, eventually, the Na+/K+-ATPase pump.