Normal and anomalous potential responses due to K+ changes in bullfrog antrum

The effect of changing the K+ concentration in the bathing media was studied in the bullfrog antrum. Usually increasing K+ on the nutrient side in standard Cl- -containing and Cl- -free solutions decreased the transmucosal potential difference (nutrient became more negative) - a normal effect. Similar results were obtained on the secretory side. Moreover, for K+ changes on the nutrient side in Cl- media, a plot of magnitude of delta V vs. log [K+] was linear for [K+] greater than 20 mM with slope of 27 mV per 10-fold change in [K+]. However, after bathing the mucosa in Cl- media with zero K+ for about 20 min, elevating the nutrient [K+] to 4 mM increased the potential difference (V) by 4.8 mV in 5 min and repeating the same sequence increased V by 6.9 mV in 5 min - both anomalous effects. Beyond 20 mM K+ the response was normal. In SO2-4 media, an anomalous potential difference of about 1 mV was obtained for changes from 0 to 3 or 6 mM nutrient K+. Ouabain (1 X 10(-3) M) in the nutrient solution abolished the anomalous response in Cl- and SO2-4 media. The normal response is attributed to passive, conductance pathways and the anomalous response because of the effect of ouabain, to a (Na+ + K+)-ATPase pump on the nutrient-facing membrane in which more Na+ than K+ ions are transported per cycle.     

Intracellular activities of chloride, potassium and sodium ions in rabbit corneal epithelium

The mechanism of ion transport in the epithelium of rabbit cornea was studied by determining the intracellular ion activity of Cl-, Na+ and K+ under various conditions. Ionic activities were measured by means of microelectrodes containing liquid ion-exchangers selective for Cl-, Na+ or K+. The Cl- activity in basal cells of the epithelium in Na+ containing bathing solutions amounts to 28 +/- 2 mM (n = 11). This value is 1.9-times greater than expected on the basis of passive distribution across the tear side membrane. This finding suggests the existence of a Cl- accumulating process. Replacement of Na+ in the aqueous bathing solution by choline or tetraethylammonium results in a reversible decrease in Cl- activity to 22 +/- 1 mM (n = 11, P less than 0.025). The ratio of observed and predicted Cl- activity decreased significantly from 1.9 to 1.4 (P less than 0.05). The decrease in Cl- activity due to Na+ replacement was rather slow. In contrast, after readmittance of Na+ to the aqueous bathing solution, Cl- activity rose to a stable level within 30 min. These results indicate involvement of Na+ in Cl- accumulation into the basal cells of the epithelium. The K+ and Na+ activities of the basal cells of rabbit corneal epithelium in control bathing solutions were 75 +/- 4 mM (n = 13) and 24 +/- 3 mM (n = 12), respectively. The results can be summarized in the following model for Cl- transport across corneal epithelium. Cl- is accumulated in the basal cells across the aqueous side membrane, energized by a favourable Na+ gradient. Cl- will subsequently leak out across the tear side membranes. Na+ is extruded again across the aqueous side membrane of the epithelium by the (Na+ + K+)-ATPase.     

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.     

Anomalous potential response and (Na+ + K+)-ATPase in in vitro frog gastric mucosa

In general, increasing K+ on the nutrient side decreases the transmucosal PD (nutrient becomes more negative) but after bathing the mucosa in zero K+ media for about 30 min, or longer, elevation of K+ on the nutrient side increases the PD, an anomalous effect. In Cl- media, increasing nutrient K+ from zero to 4 mM produces an increase in PD (an anomalous response) of 3.1 and 5.3 mV in 2 and 5 min, respectively. Ouabain (10(-3) M) to the nutrient side abolished the anomalous response as did removal of Na+ (choline for Na+) from bathing media. In SO4(2-) media (SO4(2-) for Cl-), a significant anomalous PD response was observed when K+ on the nutrient side was increased from zero to 1, 2 or 3 mM but not to higher K+ concentrations. In this case, ouabain also abolished the anomalous response. It is postulated, on the basis of the effects of ouabain and the use of choline media, that an electrogenic (Na+ + K+)-ATPase pump is present on the nutrient-facing membrane in which more Na+ than K+ are transported per cycle.     

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla

Effect of changing [K+], [Na+] and [Cl-] in nutrient solution on potential difference (PD) and resistance was studied in bullfrog antrum with and without nutrient HCO3(-) but with 95% O2/5% CO2 in both cases. In both cases, changing from 4 to 40 mM K+ gave about the same initial PD maximum (anomalous response) which was followed by a decrease below control level. Latter effect was much less with zero than with 25 mM HCO3(-). Changing from 102 to 8 mM Na+ gave initial normal PD response about the same in both cases. However, 10 min later the change in PD with zero HCO3(-) was insignificant but with 25 mM HCO3(-) the PD decreased (anomalous response of electrogenic NaCl symport). PD maxima due to K+ and Na+ were largely related to (Na+ + K+)-ATPase pump. Changes in nutrient Cl- from 81 to 8.1 mM gave only a decrease in PD (normal response). Initial PD increases are explained by relative increases in resistance of simple conductance pathways and of parallel pathways of (Na+ + K+)-ATPase pump and Na+/Cl- symport. Removal of HCO3(-) and concurrent reduction of pH modify resistance of these pathways.     

Potential difference responses due to K+, Na+ and Cl- changes in bullfrog antrum with and without HCO3-

The effect of changing [K+], [Na+] and [Cl-] in nutrient solution was studied in bullfrog antrum with and without HCO3- in nutrient. In 25 mM HCO3- (95% O2/5% CO2) and in zero HCO3- (100% O2), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K+ or from 81 to 8.1 mM Cl- gave a decrease 10 min later in transmucosal PD (nutrient became more negative)--a normal response. These responses were less in zero than in 25 mM HCO3-. A decrease from 102 to 8 mM Na+ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO3-. In contrast, change from 4 to 40 mM K+ gave initial anomalous PD response and change from 102 to 8 mM Na+, initial normal PD response with either zero or 25 mM HCO3-. Both responses were associated with (Na+ + K+)-ATPase pump and were greater in zero than in 25 mM HCO3-. Initial PD increases in zero HCO3- are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO3- modifies conductance pathways of nutrient membrane.     

Ammonium effects on colonic Cl- secretion: anomalous mole fraction behavior

A significant amount of ammonium (NH4+) is absorbed by the colon. The nature of NH4+ effects on transport and NH4+ transport itself in colonic epithelium is poorly understood. The goal of this study was to elucidate the effects of NH4+ on cAMP-stimulated Cl- secretion in the colonic cell line T84. In HEPES-buffered solutions, application of basolateral NH4+ resulted in a reduced level of Cl- secretory current. The effect of NH4+ appears to occur by at least three mechanisms: 1) basolateral membrane depolarization, 2) a competitive effect with K+, and 3) a long-term (>20 min) increase in transepithelial resistance (TER). The competitive effect with K+ exhibits anomalous mole fraction behavior. Transepithelial current relative to that in 10 mM basolateral K+ was inhibited 15% by 10 mM NH4+ alone and by 30% with a mixture of 2 mM K+ and 8 mM NH4+. A mole fraction mix of 2 mM K+:8 mM NH4+ produced a greater inhibition of basolateral membrane K+ current than pure K+ or NH4+ alone. Similar anomalous behavior was also observed for inhibition of bumetanide-sensitive 36Cl- uptake, e.g., Na+-K+-2Cl- -cotransporter (NKCC-1). No anomalous effect was observed on Na+-K+-ATPase current. Both NKCC-1 and Na+-K+-ATPase activity were elevated in 10 mM NH4+ with respect to 10 mM K+. The effect on TER did not exhibit anomalous mole fraction behavior. The overall effect of basolateral NH4+ on cAMP-stimulated transport is dependent on the [K+]o /[NH4+]o ratio at the basolateral membrane, where o is outside of the cell.     

Sodium-pump potentials and currents in guinea-pig ventricular muscles and myocytes.

When guinea-pig papillary muscles were depolarized to ca. -30 mV by superfusion with K+-free Tyrode's solution supplemented with Ba2+, Ni2+, and D600, addition of Cs+ transiently hyperpolarized the membrane in a reproducible manner. The size of the hyperpolarization (pump potential) depended on the duration of the preceding K+-free exposure; peak amplitudes (Epmax) elicited by 10 mM Cs+ after 5-, 10-, and 15-min K+-free exposures were 12.9, 17.7, and 23.2 mV, respectively. Pump potentials were unaffected by external Cl- but suppressed by cardiac glycosides, hyperosmotic conditions, and low-Na+ solution. Using Epmax as an indicator of Na+ pump activation, the half-maximal concentration for activation by Cs+ was 12-16.3 mM. At 6 mM, Cs+ was three times less potent than Rb+ or K+ and five times more potent than Li+. From these findings, and correlative voltage-clamp data from myocytes, we calculate that (i) a pump current of 7.8 nA/cm2 generates an Epmax of 1 mV and (ii) resting pump current in normally polarized muscle (approximately 0.16 microA/cm2) is five times smaller than previously estimated.     

Microelectrode studies of potential difference responses to changes in stromal K+ in bullfrog cornea

The effects of changing stromal K+ were studied using microelectrodes in an in vitro preparation of frog cornea. The intracellular potential (V0) responded in two opposite ways under short-circuit conditions: (1) depolarization (normal response) when stromal K+ was increased from 4 to 20 or to 79 mM, about 30 mV per 10-fold K+ concn. change; (2) a hyperpolarization (anomalous response) of 10 mV maximum when stromal K+ was increased from 0 to 4 mM. The increase from 4 to 20 or 79 mM decreased or even reversed the short-circuit current (Isc). The transepithelial conductance (gt) increased when K+ was increased to 79 mM but no change occurred in the apical membrane fractional resistance (fRo). Increase of stromal K+ from 0 to 4 mM increased Isc and minimally changed gt and fRo. Ouabain (10(-3) M) abolished the anomalous responses, that is, the increases in V0 and Isc when stromal K+ was increased from 0 to 4 mM. These results are interpreted in terms of two K+ conductive pathways in the basolateral membrane of the corneal epithelium, a Nernstian conductance and an electrogenic (Na+ + K+)-ATPase pump transporting more Na+ than K+ ions per cycle. The normal or anomalous potential difference responses to changes in stromal K+ appear to depend on the relative resistance of the two pathways at the time stromal K+ is changed.     

Effect of ibuprofen and rofecoxib transport parameters in the frog corneal epithelium

Effects of cyclooxygenase (COX) inhibitors on transport parameters of the frog corneal epithelium were studied. Epithelial cells of the intact cornea were impaled with microelectrodes. Under short-circuit current (I(sc)) conditions, 10(-4) M ibuprofen (IBU) (non-specific COX inhibitor) or 5 x 10(-5) M rofecoxib (COX-2 inhibitor) were added to the tear solution. With ibuprofen, I(sc) decreased by 1.0 from 3.1 microA/cm2; intracellular potential, V(o), depolarized by 14.2 from -56.9 mV; IBU did not affect the transepithelial conductance, g(t), or the apical membrane fractional resistance, fR(o). With rofecoxib, I(sc) decreased by 0.9 from 4.3 microA/cm2; V(o) depolarized by 18 from -62.4 mV; g(t) significantly increased by 0.03 from 0.37 ms/cm2; and fR(o) decreased by 12 from 50. Basolateral membrane K+ and apical membrane Cl- partial conductances were studied by the ion substitution method. Depolarization of V(o) by an increase in stromal K+ from 4 to 79 mM was smaller with IBU (17.5 mV) or rofecoxib (19.2 mV) than without the inhibitors (29.1 and 29.3 mV, respectively). Depolarization of V(o), by a decrease in tear Cl- from 81 to 8.1 mM, was abolished by the COX inhibitors. Decrease in I(sc) and V(o) can be explained by a decrease in the K+ and Cl-? conductances. Experiments with amphotericin B ruled out a major effect of the inhibitors on the Na+/K+ ATPase pump.     

Acid pH and weak acids induce Na−Cl contransport in the rabbit urinary bladder

We have described a coupled Na--Cl entry step at the apical membrane of a tight epithelium, the rabbit urinary bladder. Mucosal pH values, more acid than 4.6, stimulate a 20 to 40-fold increase in mucosal-to-serosal Na+ and Cl- flux. The flux increase is almost completely blocked by low concentrations of of bumetanide. The transepithelial movement of Na+ and Cl- is normally electroneutral; however, when weak acids (such as acetate) are present in the mucosal solution, the acid-induced increase in flux is accompanied by a large increase in short-circuit current. Besides blockage by bumetanide, both the increase in flux and short-circuit current are blocked by: (1) Na+-free solutions on the mucosa; (2) Cl--free solutions on the mucosa; (3) phosphodiesterase inhibitors; (4) ouabain in the serosal solution; (5) K+-free solutions on the serosa; and (6) HCO3--free solutions on the serosa. The increase in the fluxes and the short-circuit current is unaffected by: (1) amiloride application in the mucosal solution; (2) mucosally applied stilbene derivatives which block Cl-/HCO3- exchange (SITS); and (3) Cl--free solutions applied to the serosa. We interpret these results to imply a coupled Na--Cl uptake step at the apical membrane which is stimulated by intracellular acetate (or (pH). The uptake step leads to a movement of Na+ and Cl- across the basolateral membrane, which is mediated by the Na+, K+-ATPase and a Na/Cl/HCO3- exchange mechanism. Our results demonstrate that "tight" epithelia may, under appropriate circumstances, demonstrate mechanisms of ion movement which are similar to "leaky" epithelia.     

Chloride cotransport in the membrane of earthworm body wall muscles

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).     

K+ and Cl- uptake by cultured oligodendrocytes

Cultured oligodendrocytes take up K+ triggered by an increase in [K+]o. Simultaneously [Cl-]i increases in the majority of the oligodendrocytes. This KCl uptake, which is not furosemide sensitive, can be explained by the following model. The first event is the entry of Cl- into the cell driven by the discrepancy between the membrane and Cl- equilibrium potential. As a consequence of the movement of negative charge across the membrane, K+ is driven into the cell. The prerequisites of this model, a passive Cl- distribution at resting membrane potential and a Cl- conductance of the membrane were found to exist in most cultured oligodendrocytes. The chloride equilibrium potential (-61 mV, SD +/- 10 mV) was slightly more positive than the membrane potential (-64 +/- 8 mV). Since cell input resistance determined with two independent electrodes increased by 11% (SD +/- 0.07) when [Cl-]o was reduced to 10 mM, part of the membrane conductance appears to be mediated by Cl-. Differences between membrane potential and Cl- equilibrium potential therefore will lead to Cl- fluxes across the membrane. In contrast with oligodendrocytes, [Cl-]i in astrocytes is significantly increased (from 20 to 40 mM) above the equilibrium distribution owing to the activity of an inward directed Cl- pump; this suggests a different mechanism of K+ uptake in these cells.     

Acetyl phosphate can act as a substrate for Na+ transport by (Na+ + K+)-ATPase

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.     

Effect of Ba2+ on the K+ conductance pathways in the frog cornea

Two types of transepithelial potential difference (PD) responses have been observed in the bullfrog, Rana catesbeiana, when the K+ concentration is changed in the aqueous solution. (1) A normal response, that is, a decrease in the positivity of the aqueous solution when the K+ is increased in this solution. (2) An anomalous response, that is, an increase in PD when K+ is increased from 0 to 4 mM in the aqueous solution. In present experiments 2 mM Ba2+ results in a significant decrease in transepithelial PD and an increase in resistance (R), consistent with the well-known effect of Ba2+ on the K+ conductance in other biological membranes. In the presence of Ba2+ compared to its absence the normal PD responses were decreased when K+ was increased from 4 to 20 or to 79 mM in the aqueous solution. Barium enhanced, but not significantly, the anomalous PD response (PD increase) when K+ was increased from 0 to 4 mM. An anomalous PD response (PD decrease) was obtained with Ba2+ when K+ was changed from 4 to 0 mM while in its absence the response was normal (PD increase) or did not change. These findings support the concept that anomalous PD responses as a result of the electrogenic (Na+ + K+)-ATPase may be obtained when the resistance of the simple K+ pathway is increased.     

Effects of diphenylamine-2-carboxylate on sodium and chloride transport across bovine tracheal epithelium: studies in monolayers and in the native tissue

1. The short-circuit current (I0) across monolayers of bovine tracheal epithelial cells is the sum of Na+ absorption and Cl- secretion. 2. Diphenylamine-2-carboxylate (DPC), added to the mucosal side of the native tissue or monolayers induced a rapid, dose-dependent and fully reversible reduction in I0, which reached zero with 3 mM DPC. 3. The blocking effect of DPC was examined during incubation (1) in standard, (2) in Cl(-)-free and (3) in Na+-free solution. Dose response curves revealed that the IC50 was not altered following ion substitution: it was approximately 0.7 mM DPC. 4. Thus, in bovine tracheal epithelium, DPC was an effective blocking agent of both Na+ and Cl- transport.     

Imidazole chloride and tris-chloride substitute for sodium chloride in inducing high-affinity AdoPP[NH]P binding to (Na+ + K+)-ATPase

Optimal binding of [2,8-3H]AdoPP[NH]P to (Na+ + K+)-ATPase requires 25 mM Na+ (Cl-), 50 mM imidazole+ (Cl-) or 50 mM Tris+ (Cl-). Chloride is essential as counterion. We conclude that imidazole+ and Tris+ are able to bind to the Na+ site, and recommend the use of dilute buffers for studying the partial reactions of (Na+ + K+)-ATPase. In NaCl or the substituting buffers the dissociation constant for the enzyme-AdoPP[NH]P complex at 0 degrees C and pH 7.25 is 0.4 microM, whereas in millimolar MgCl2 it is about 2 microM. These distinct levels in affinity with MgCl2 as compared to NaCl, together with the MgCl2-dependence of photolabelling of the enzyme with ATP analogues (Rempeters, G. and Schoner, W. (1981) Eur. J. Biochem. 121, 131-137), suggest significant changes within the substrate site of (Na+ + K+)-ATPase upon binding of Mg2+ (Cl-)2.     

External anions and volume-sensitive anion current in guinea-pig ventricular myocytes

The objective of this study was to determine the effects of anion replacement on volume-sensitive anion current in guinea-pig ventricular myocytes. Myocytes in the conventional whole-cell voltage-clamp configuration were superfused and dialysed with Na(+)-, K(+)-, and Ca(2+)-free solution, and exposed to external 75 mM Cl- solution of one-half normal osmolality. Prolonged exposures to hyposmotic solution promoted the development of outwardly-rectifying currents that were inactivated at high positive potentials and reversed in a Cl(-)-dependent manner (50 mV per decade pipette Cl- concentration). Replacement of external Cl- by iodide and aspartate affected the reversal potential (E(rev)) and slope conductance of the volume-sensitive current. Relative permeabilities calculated from changes in E(rev) were 1.49 +/- 0.09, 1.00, and 0.29 +/- 0.04 for iodide, Cl-, and aspartate, respectively; relative slope conductances between E(rev) and E(rev) + 40 mV were 1.21 +/- 0.09, 1.00, and 0.43 +/- 0.07, respectively. Replacement of Cl- also affected the time dependence of the volume-sensitive current; replacement by iodide reversibly enhanced the decay of outward current at positive potentials, whereas replacement by aspartate reduced it. These results are compared with earlier findings on noncardiac time- and voltage-dependent anion current activated by hyposmotic solution.     

Effect of pentachlorophenol (PCP) on frog cornea epithelium

Pentachlorophenol (PCP) is a toxic substance that affects many tissues adversely. Present experiments, using an in vitro preparation, were designed to study whether PCP affected the electrophysiological parameters of the bullfrog cornea epithelium, specifically, the Na+/K+ ATPase pump and the K+ conductance located in the basolateral membrane and the Cl- conductance located in the apical membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (Isc) conditions. Addition of PCP to a concentration of 5 x 10-5 M to the tear solution gave a marked decrease in Isc; a marked depolarization of the intracellular potential, Vo; and minimal but significant decreases in the apical membrane fractional resistance, fRo, and in the transepithelial conductance, gt. Isc experiments in Cl--free solutions with amphotericin B in the tear solution confirm results indicating that PCP inhibits the active transepithelial transport mechanism and produces a small increase in the basolateral membrane resistance due to a decrease in the K+ conductance.     

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na(+),K(+)-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na(+),K(+)-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current (I(sc)) with a Na(+),K(+)-ATPase inhibitor, ouabain (100 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). The directional apical secretion of Cl(-) was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl(-)-dependent, ouabain-sensitive AP --> BL I(sc) in response to CCh, consistent with current models for Na(+)-dependent Cl(-) secretion.