Na+ and Cl− conductances are controlled by cytosolic Cl− concentration in the intralobular duct cells of mouse mandibular glands

Our previously published whole-cell patch-clamp studies on the cells of the intralobular (granular) ducts of the mandibular glands of male mice revealed the presence of an amiloride-sensitive Na⁺ conductance in the plasma membrane. In this study we demonstrate the presence also of a Cl^– conductance and we show that the sizes of both conductances vary with the Cl^– concentration of the fluid bathing the cytosolic surface of the plasma membrane. As the cytosolic Cl^– concentration rises from 5 to 150 mmol/liter, the size of the inward Na⁺ current declines, the decline being half-maximal when the Cl^– concentration is approximately 50 mmol/liter. In contrast, as cytosolic Cl^– concentration increases, the inward Cl^– current remains at a constant low level until the Cl^– concentration exceeds 80 mmol/liter, when it begins to increase. Studies in which Cl^– in the pipette solution was replaced by other anions indicate that the Na⁺ current is suppressed by intracellular Br^-, Cl^– and NO ₃ ^- but not by intracellular I^-, glutamate or gluconate. Our studies also show that the Cl^– conductance allows passage of Cl^– and Br^- equally well, I-less well, and NO ₃ ^- , glutamate and gluconate poorly, if at all. The findings with NO ₃ ^- are of particular interest because they show that suppression of the Na⁺ current by a high intracellular concentration of a particular anion does not depend on actual passage of that anion through the Cl^– conductance. In mouse granular duct cells there is, thus, a reciprocal regulation of Na⁺ and Cl^– conductances by the cytosolic Cl^– concentration. Since the cytosolic Cl^– concentration is closely correlated with cell volume in many epithelia, this reciprocal regulation of Na⁺ and Cl^– conductances may provide a mechanism by which ductal Na⁺ and Cl transport rates are adjusted so as to maintain a stable cell volume.This project was supported by the National Health and Medical Research Council of Australia. We thank Professor P. Barry (University of New South Wales) for assistance with the junction potential measurements.     

Acetylcholine-activated Cl? Channel in the Chara Tonoplast

Acetylcholine has long been suggested to play a role in controlling physiological processes in plants, but no mechanism has been shown for its action. We show here that a chloride channel in the tonoplast (vacuolar membrane) of Chara corallina responds to acetylcholine. The channel has a conductance of 45 pS. The effect of acetylcholine is enhanced by nicotine, with the open probability increasing from 0.05 in the presence of 4 mM acetylcholine to 0.3 in the presence of 4 mM acetylcholine + 6 mM nicotine. Some effects of acetylcholine were seen at concentrations as low as 20 microM, with a maximum effect between 1 and 10 mM. In the intact cell, acetylcholine prolongs the depolarized phase of the action potential. We propose that this acetylcholine-gated channel has evolved separately from the mammalian acetylcholine-gated channel, and suggest that this represents a third form of acetylcholine signal transduction, after the nicotinic and muscarinic pathways in animal systems.     

Microscopic elements of electrical excitation in Chara: Transient activity of Cl− channels in the plasma membrane

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl^– as Ca²⁺ or Ba²⁺ (50 or 100 mm), but not as Mg²⁺ or K⁺ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl^– channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl^– channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl channel activity was favored by voltages positive of the free running membrane voltage (> –45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activtion of these channels. Neither type of Cl^– channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca²⁺ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl^– channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca²⁺ and Cl^– for triggering and time course: Ca²⁺ uptake does not activate Cl^– channels directly but first supplies a membrane-associated population of Ca²⁺ storage sites. Depolarization enhances discharge of Ca²⁺ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca²⁺ concentration ([Ca²⁺]_cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca²⁺ buffer system. In turn, the transient rise in [Ca²⁺]_cyt causes the transient activity of those Cl^– channels, which are more likely to open at an elevated Ca²⁺ concentration.The financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.     

Increased intracellular Cl− concentration mediates Trichomonas vaginalis-induced inflammation in the human vaginal epithelium

Trichomonas vaginalis is a primary urogenital parasite that causes trichomoniasis, a common sexually transmitted disease. As the first line of host defense, vaginal epithelial cells play critical roles in orchestrating vaginal innate immunity and modulate intracellular Cl⁻ homeostasis via the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that plays positive roles in regulating nuclear factor-κB (NF-κB) signalling. However, the association between T. vaginalis infection and intracellular Cl⁻ disequilibrium remains elusive. This study showed that after T. vaginalis infection, CFTR was markedly down-regulated by cysteine proteases in vaginal epithelial cells. The intracellular Cl⁻ concentration ([Cl⁻]_i) was consequently elevated, leading to NF-κB signalling activation via serum- and glucocorticoid-inducible kinase-1. Moreover, heightened [Cl⁻]_i and activated NF-κB signalling could be sustained in a positive feedback regulatory manner resulting from decreased intracellular cAMP through NF-κB-mediated up-regulation of phosphodiesterase 4. The results conclusively revealed that the intracellular Cl⁻ of the human vaginal epithelium could be dynamically modulated by T. vaginalis, which contributed to mediation of epithelial inflammation in the human vagina.     

Ca2+-dependent Cl− efflux in tonoplast-free cells ofNitellopsis obtusa

Summary In order to demonstrate the presence of a Ca²⁺-activated Cl^–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca²⁺ concentration was modified by internal perfusion. An increase in the Ca²⁺ concentration caused a large Cl^– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca²⁺ concentration was about 4.0 m. Neither the Cl^– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl^–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl^– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca²⁺ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca²⁺-dependent Cl^–-channel.     

F-actin network may regulate a Cl− channel in renal proximal tubule cells

A variety of mechanisms have been proposed for the regulation of ion channel molecules. As integral membrane proteins, ion channels may interact with the cytoskeleton. Regulation of channels by the actin network may therefore be important. In the present study we used cytochalasin D and exogenous actin to test this possibility. The Cl^– channel of the apical membrane of renal proximal epithelium was detected in its active state after prolonged depolarization. Within 6 sec after its addition, cytochalasin D (0.05 g/ml) significantly decreased the number of open channels and mean open probability (NPo) of the Cl^– channel. Colchicine (1 mm), which affects microtubules, did not influence channel activation. Cytochalasin D is known to not only disrupt the F-actin network but to inhibit polymerization of F-actin as well. The latter effect is also produced by DNaseI. Cytochalasin D, but not DNaseI, inactivated Cl^– channels in cell-free membrane patches, suggesting that cytochalasin D inactivated the channel by disrupting the actin network. Cytochalasin D appeared to specifically affect the channel, as opposed to membrane permeability, since only the activated whole-cell Cl^– currents were altered by cytochalasin D. Addition of actin polymer, but not actin monomer, reactivated the cytochalasin-D-depressed channel. Thus, repair of the disrupted F-actin network with actin polymer apparently restored the activity and number of open Cl^– channels. We therefore conclude that the F-actin network interacts with and possibly regulates the Cl^– channel of renal proximal tubule epithelia.We would like to thank T. Tamatsukuri for technical support. This study was presented to the American Society of Nephrology, Baltimore, 1991.     

A Ca2+-activated Cl− current in sheep parotid secretory cells

Previous studies have shown that the whole-cell current-voltage (I-V) relation of unstimulated sheep parotid cells is dominated by two K⁺ conductances, one outwardly and the other inwardly rectifying. We now show that once these K⁺ conductances are blocked by replacement of pipette K⁺ with Na⁺ and by the addition of 5 mmol/liter CsCl to the bath, there remains an outwardly rectifying conductance with a reversal potential of 0 mV. Replacement of 120 mmol/liter NaCl in the pipette solution with an equimolar amount of Na-glutamate shifted the reversal potential of this residual current to -55 mV, indicating that the conductance was Cl^? selective. The Cl^? current was activated by increasing the free Ca²⁺ in the pipette solution from 10 to 100 nmol/liter. When the Ca²⁺ concentration in the pipette solution was 10 nmol/liter, the relaxations observed in response to membrane depolarization could be fitted with a single exponential, whose time constant increased from 81 to 183 ms as the pipette potential was increased from -30 to +60 mV. Relaxation analysis showed that the current was activated by membrane depolarization. Reversal potential measurements in experiments in which external Cl^? was replaced with various anions, gave the following relative permeabilities: SCN^- (1.80) > I^- (1.09) > CI^- (1) > NO ₃ ^- (0.92) > Br^- (0.75). The relative conductances were: SCN^- (2.18) > I^- (1.07) > Cl^? (1.00) > Br^- (0.91) > NO ₃ ^- (0.50). The Cl^? current was blocked by NPPB (ID₅₀ ≈ 10 μm), DIDS (10 or 30 μmol/liter) and furosemide (100 μmol/liter).     

Fe homeostasis in plant cells: Does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.     

Coenzyme Q10 concentration in plasma and blood cells: what about diurnal changes?

Coenzyme Q10 (CoQ10) is used by the body as an endogenous antioxidant and performs essential functions in mitochondrial energy production. The value of CoQ10 as a biomarker for oxidative stress will be severely restricted if there are huge individual daily variations in its concentration. For analysis of diurnal changes in CoQ10 plasma and blood cell concentrations, blood was collected from nine healthy adults (at two- or three-hour intervals for plasma, and three times a day for blood cells). CoQ10 was analysed by HPLC using electrochemical detection and internal standardisation. Daytime variations in CoQ10 concentration in plasma are maintained within narrow limits and show no statistically significant difference (Kruskal-Wallis). However, a drop at night-time (0300 h) is accompanied by a drop in total cholesterol concentration. Remarkable inter-individual differences in blood cell (erythrocytes, platelets, white blood cells) content of CoQ10 occur with only slight intra-individual daily variations. A correlation (Spearman) is found for cholesterol and CoQ10 content in circulation which may be explained by the carrier capacity of blood for this highly lipophilic substance. Moreover, a diurnal change in hepatic HMG-CoA reductase activity may suggest a common diurnal regulation of synthesis of both CoQ10 and cholesterol.     

How is the cytoplasmic calcium concentration controlled in nerve terminals?

1. The ability of intraterminal organelles to sequester calcium and buffer the cytoplasmic free Ca2+ concentration ([Ca2+]i) has been investigated in isolated mammalian presynaptic nerve terminals (synaptosomes). A combination of biochemical and morphological methods has been used. 2. When the plasmalemma of synaptosomes is disrupted by osmotic shock or saponin, Ca from the medium can be sequestered by two types of intraterminal organelles in the presence of ATP. 2. Typical mitochondrial poisons (e.g., oligomycin, azide and 2,4-dinitrophenol) block the Ca uptake into one type of organelle (mitochondria); the second type of organelle, which has a higher affinity for Ca (half-saturation congruent to 0.35 microM Ca2+) is spared by the mitochondrial poisons. 4. When the "leaky" synaptosomes are incubated in media containing oxalate, and then fixed and prepared for electron microscopy, electron-dense deposits are observed in the intraterminal mitochondria and smooth endoplasmic reticulum (SER). Mitochondrial poisons block the formation of the deposits in the mitochondria, but spare the SER. 5. X-ray microprobe analysis demonstrates that these deposits contain Ca. 6. Experiments with the Ca-sensitive metallochromic indicator, arsenazo III, demonstrate that the intraterminal organelles in the "leaky" synaptosomes can buffer Ca2+ in the medium to below 5 X 10(-7) M. With small (physiological) Ca loads, the Ca2+ is effectively buffered (to < 5 X 10(-7) M) even in the presence of mitochondrial poisons. 7. The data indicate that the SER in presynaptic terminals may play an important role in helping to buffer the Ca that normally enters during neuronal activity.     

Cl− transport in basolateral renal medullary vesicles: I. Cl− transport in intact vesicles

Summary This paper provides the results of studies which characterized conductive³⁶Cl^– flux in basolaterally enriched membrane vesicles prepared from rabbit renal outer medulla. Conductive³⁶Cl^– uptake was studied under two different experimental conditions. In the first,³⁶Cl^– flux was driven by an inside positive voltage created with oppositely directed Cl^– and gluconate gradients. In the second, an inwardly direct K⁺ gradient was used to drive³⁶Cl^– uptake. By these two methods, voltage-sensitive³⁶Cl^– uptake was shown to comprise about 45 and 65%, respectively, of the initial rates of total³⁶Cl^– flux. Separate paired studies demonstrated that the conductive³⁶Cl^– uptake was inhibited by the Cl^– channel blocker diphenylamine-2-carboxylate (DPC) with an IC₅₀ for DPC of 154 m. The voltagedependent³⁶Cl^– uptake had an activation energy of 6.4 kcal/mole. This³⁶Cl^– conductance had an anion selectivity sequence of I^–>Cl^–NO ₃ ^– gluconate.     

Intracellular Na+, K+ and Cl− activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (V_m) and intracellular Na⁺, K⁺ and Cl⁻ activities were measured under steady-state conditions in normal saline medium (Na⁺ = 154, K⁺ = 6, Cl⁻ = 150 mequiv./l). Membrane potential was estimated to be −23.3 ± 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of V_m and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na⁺, K⁺ and Cl⁻ activities are 4.6 ± 0.5; 68.3 ± 8.0; and 43.6 ± 2.1 mequiv./l, respectively. The apparent activity coefficients for Na⁺ and K⁺ are 0.18 ± 0.02 and 0.41 ± 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl⁻ (0.67 ± 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl⁻ is not different from zero. The results establish that the energy available from the Na⁺ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Activation by hyperpolarization and atypical osmosensitivity of a Cl− current in rat osteoblastic cells

During whole-cell recording of rat osteoblastic cells with high-Cl^– internal solutions, 10 sec hyperpolarizing jumps from 0 mV induce a slow inward current relaxation, which is shown to be carried by hyperpolarization-activated Cl^– channels. This relaxation increases and becomes faster with stronger hyperpolarizations. It is insensitive to Cs⁺ ions but is blocked in a voltage-dependent manner by 4,4-diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS) 1 mm and is reduced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) 0.1 mm. Cd²⁺ ions are potent blockers of this current, blocking completely above 300 m. The amplitude of the Cl^– current activated by a given hyperpolarization increases during the first 10–20 min of whole-cell recording. This evolution and the fact that some recently cloned Cl^– channels have been reported to be activated both by hyperpolarization and by external hyposmolarity led us to investigate the effects of external osmolarity. Reducing the external osmolarity induces a large Cl^– current. However, this hyposmolarity-induced Cl^– current and the hyperpolarization-activated Cl^– current are shown to be distinct; 1,9-dideoxy forskolin selectively blocks the hyposmolarity-activated current. We show that the hyperpolarization-activated Cl^– current is osmosensitive, but in an unusual way: it is reduced by external hyposmolarity and is increased by external hyperosmolarity. Furthermore, these modulations are more pronounced for small hyperpolarizations. The osmosensitivity of the hyperpolarization-activated Cl^– current suggests a mechanosensitivity (activation by positive external pressure) that is likely to be physiologically important to bone cells.We wish to thank P. Ascher and B. Barbour for useful comments.     

Oxidative stress caused by pyocyanin impairs CFTR Cl− transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H₂O₂ threefold above the endogenous H₂O₂ production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 μM) oxidized the cytosol from a resting value of − 318 ± 5 mV by 48.0 ± 4.6 mV within 2 h; a comparable oxidation was induced by 100 μM H₂O₂. Whereas resting Cl⁻ secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl⁻ secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for ΔF508 CFTR failed to secrete Cl⁻ in response to pyocyanin or H₂O₂, indicating that these oxidants specifically target the CFTR and not other Cl⁻ conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H₂O₂, depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

Apical Cl− Channels in A6 Cells

Short-circuit current (I _sc ), transepithelial conductance (G _t ), electrical capacitance (C _T ) and the fluctuation in I _sc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na⁺- and Cl⁻-free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in G _t , C _T , I _sc and generated a Lorentzian I _sc -noise. The responses could be related to active, electrogenic secretion of Cl⁻. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in G _t with only moderate peaking of I _sc . Phosphodiesterase inhibitors also stimulated Cl⁻ secretion with peaking responses in G _t and I _sc . All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl⁻ channel, with almost identical corner frequency (40–50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused C _T , a measure of apical membrane area, to increase in a manner roughly synchronous with G _t . These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl⁻ channel pool. Apical flufenamate depressed Cl⁻ current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor. Received: 20 May 1998/Revised: 8 September 1998     

Calcium ionophore-induced changes in HCO3− secretion and Cl− absorption in turtle bladder: relation to action of 3-isobutyl-1-methylxanthine

The calcium ionophore A23187 stimulates luminal alkalinization and inhibits Cl⁻ absorption in short-circuited urinary bladders of postprandial or alkalotic turtles. The ionophore appears to mimic the action of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) by its similar effects on HCO₃⁻ secretion and Cl⁻ absorption and by increasing cytosolic cAMP levels of isolated bladder epithelial cells. However, only A23187 (or ionomycin), but not IMBX or cAMP, elevated cytosolic Ca²⁺ of aequorin- or quin2-loaded cells. Since A23187, but not IBMX or cAMP inhibits luminal acidification, we postulate that cytosolic Ca²⁺ (1) regulates the acidification process by a cAMP-independent mechanism and (2) controls HCO₃⁻ secretion as well as Cl⁻ absorption, at least in part, via cAMP-mediated pathways.     

Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl− secretion in MDCK cells

Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled alpha, beta, and gamma subunits of epithelial Na(+) channel (ENaC) showed that, although most of the short-circuit current (I (sc)) was amiloride sensitive (AS-I (sc)), there was also an amiloride-insensitive component (NS-I (sc)) due to Cl(-) secretion (Morris and Schafer, J Gen Physiol 120:71-85, 2002). In the present studies, we observed a progressive increase in NS-I (sc) and a corresponding decrease in AS-I (sc) during experiments. There was a significant negative correlation between AS-I (sc) and NS-I (sc) both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-I (sc) could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca(2+)-activated Cl(-) channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-I (sc) by 40-60%, with a proportional decrease in NS-I (sc). Addition of 100 muM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-I (sc) that was followed by a sustained increase of 50-60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl(-) secretion and the proportional fall in ENaC-mediated Na(+) absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl(-) secretion and Na(+) absorption.