Na+ transport and impedance properties of cultured renal (A6 and 2F3) epithelia

Previous impedance analysis studies of intact epithelia have been complicated by the presence of connective tissue or smooth muscle. We now report the first application of this method to cultured epithelial monolayers. Impedance analysis was used as a nondestructive method for deducing quantitative morphometric parameters for epithelia grown from the renal cell line A6, and its subclonal cell line 2F3. The subclonal 2F3 cell line was chosen for comparison to A6 because of its inherently higher Na+ transport rate. In agreement with previous results, 2F3 epithelia showed significantly higher amiloride-sensitive short-circuit currents (Isc) than A6 epithelia (44 +/- 2 and 27 +/- 2 microA/cm2, respectively). However, transepithelial conductances (GT) were similar for the two epithelia (0.62 +/- 0.04 mS/cm2 for 2F3 and 0.57 +/- 0.04 mS/cm2 for A6) because of reciprocal differences in cellular (Gc) and paracellular (Gj) conductances. Significantly lower Gj and higher Gc values were observed for 2F3 epithelia than A6 (Gj = 0.23 +/- 0.02 and 0.33 +/- 0.04 mS/cm2 and Gc = 0.39 +/- 0.16 and 0.26 +/- 0.10 mS/cm2, respectively). Nonetheless, the cellular driving force for Na+ transport (Ec) and the amount of transcellular Na+ current under open-circuit conditions (Ic) were similar for the two epithelia. Three different morphologically-based equivalent circuit models were derived to assess epithelial impedance properties: a distributed model which takes into account the resistance of the lateral intercellular space and two models (the "dual-layer" and "access resistance" models), which corrected for impedance of small fluid-filled projections of the basal membrane into the underlying filter support. Although the data could be fitted by the distributed model, the estimated value for the ratio of apical to basolateral membrane resistances was unreasonably large. In contrast, the other models provided statistically superior fits and reasonable estimates of the membrane resistance ratio. The dual-layer model and access resistance models also provided similar estimates of apical and basolateral membrane conductances and capacitances. In addition, both models provided new information concerning the conductance and area of the basolateral protrusions. Estimates of the apical membrane conductance were significantly higher for 2F3 (0.79 +/- 0.23 mS/cm2) than A6 epithelia (0.37 +/- 0.07 mS/cm2), but no significant difference could be detected for apical membrane capacitances (1.4 +/- 0.04 and 1.2 +/- 0.1 microF/cm2 for 2F3 and A6, respectively) or basolateral membrane conductances (3.48 +/- 1.67 and 2.95 +/- 0.40 mS/cm2). The similar basolateral membrane properties for the two epithelia may be explained by their comparable transcellular Na+ currents under open-circuit conditions.     

Transport-dependent alterations of membrane properties of mammalian colon measured using impedance analysis

Summary Direct current (DC) measurement methods have been commonly used to characterize the conductance properties of the mammalian colon. However, these methods provide no information concerning the effects of tissue morphology on the electrophysiological properties of this epithelium. For example, distribution of membrane resistances along narrow fluid-filled spaces such as the lateral intercellular spaces (LIS) or colonic crypts can influence DC measurements of apical and basolateral membrane properties. We used impedance analysis to determine the extent of such distributed resistance effects and to assess the conductance and capacitance properties of the colon. Because capacitance is proportional to membrane area, this method provides new information concerning membrane areas and specific ionic conductances for these membranes.We measured transepithelial impedance under three conditions: (1) control conditions in which the epithelium was opencircuited and bathed on both sides with NaCl–HCO₃ Ringer's solutions, (2) amiloride conditions which were similar to control except that 100 m amiloride was present in the mucosal bathing solution, and (3) mucosal NaCl-free conditions in which mucosal Na and Cl were replaced by potassium and sulfate or gluconate (K⁺ Ringer's). Three morphologically-based equivalent circuit models were used to evaluate the data: (1) a lumped model (which ignores LIS resistance), (2) a LIS distributed model (distributed basolateral membrane impedance) and (3) a crypt-distributed model (distributed apical membrane impedance). To estimate membrane impedances, an independent measurement of paracellular conductance (G _s ) was incorporated in the analysis. Although distributed models yielded improved fits of the data, the distributed and lumped models produced similar estimates of membrane parameters. The predicted effects of distributed resistances on DC microelectrode measurements were largest for the LIS-distributed model. LIS-distributed effects would cause a 12–15% underestimate of membrane resistance ratio (R _a /R _b ) for the control and amiloride conditions and a 34% underestimate for the K Ringer's condition. Distributed resistance effects arising from the crypts would produce a 1–2% overestimate ofR _a /R _b .Apical and basolateral membrane impedances differed in the three different experimental conditions. For control conditions, apical membrane capacitance averaged 21 F/cm² and the mean apical membrane specific conductance (G _a-norm) was 0.17 mS/F. The average basolateral membrane capacitance was 11 F/cm² with a mean specific conductance (G _b-norm) of 1.27 mS/F.G _a-norm was decreased by amiloride or K⁺ ringer's to 0.07 mS/gmF and 0.06 mS/F, respectively. Basolateral conductance was also reduced by amiloride, whereas capacitance was unchanged (G _b-norm=0.97 mS/F). For the K⁺ Ringer's condition, both basolateral conductance and capacitance were greatly increased such thatG _b-norm was not significantly different from the control condition.     

cAmp activation of apical membrane Cl(-) channels: theoretical considerations for impedance analysis.

Transepithelial electrical impedance analysis provides a sensitive method to evaluate the conductances and capacitances of apical and basolateral plasma membranes of epithelial cells. Impedance analysis is complicated, due not only to the anatomical arrangement of the cells and their paracellular shunt pathways, but also in particular to the existence of audio frequency-dependent capacitances or dispersions. In this paper we explore implications and consequences of anatomically related Maxwell-Wagner and Cole-Cole dielectric dispersions that impose limitations, approximations, and pitfalls of impedance analysis when tissues are studied under widely ranging spontaneous rates of transport, and in particular when apical membrane sodium and chloride channels are activated by adenosine 3',5'-cyclic monophosphate (cAMP) in A6 epithelia. We develop the thesis that capacitive relaxation processes of any origin lead not only to dependence on frequency of the impedance locus, but also to the appearance of depressed semicircles in Nyquist transepithelial impedance plots, regardless of the tightness or leakiness of the paracellular shunt pathways. Frequency dependence of capacitance precludes analysis of data in traditional ways, where capacitance is assumed constant, and is especially important when apical and/or basolateral membranes exhibit one or more dielectric dispersions.     

Membrane capacitance and conductance changes parallel mucin secretion in the human airway epithelium

Measurement of the magnitude and kinetics of exocytosis from intact epithelia has historically been difficult. Using well-differentiated cultures of human bronchial epithelial cells, we describe the use of transepithelial impedance analysis to enable the real-time quantification of mucin secretagogue-induced changes in membrane capacitance (surface area) and conductance. ATPgammaS, UTP, ionomycin, and PMA induced robust increases in total cellular capacitance that were demonstrated to be dominated by a specific increase in apical membrane surface area. The UTP-induced increase in capacitance occurred in parallel with goblet cell emptying and the secretion of mucin and was associated with decreases in apical and basolateral membrane resistances. The magnitude and kinetics of the capacitance increases were dependent on the agonist and the sidedness of the stimulation. The peak increase in capacitance induced by UTP was approximately 30 mucin granule fusions per goblet cell. Secretagogue-induced decreases in apical membrane resistance were independent of exocytosis, although each of the secretagogues induced profound reductions in basolateral membrane resistance. Transepithelial impedance analysis offers the potential to study morphological and conductance changes in cultured human bronchial epithelial cells.     

Use of AC Impedance Analysis to Study Membrane Changes Related to Acid Secretion in Amphibian Gastric Mucosa

We have applied transepithelial AC impedance techniques to gastric mucosa to reconcile ultrastructural and electrophysiological findings about gastric acid secretion and the mucosal barrier. By fitting impedance data measured at different HCl secretion rates to equivalent circuit models, we extracted capacitances and resistances (as measures of membrane area and ionic conductance, respectively) for the apical and basolateral membranes. The impedance measurements were found to be incompatible with earlier equivalent circuit models that modeled membrane electrical properties as lumped circuits based on one or two cell types. A distributed circuit model was developed that assumed only one dominant electrical pathway (i.e., one cell type), but that incorporated electrical effects arising from long and narrow membrane-lined structures present in the epithelium (e.g., gastric crypts, tubulovesicles, lateral intercellular spaces). This morphologically based model was found to represent the measured data accurately, and to yield values for membrane capacitances consistent with morphometric measurements of membrane areas. The main physiological conclusions from this analysis were as follows: (a) The dominant transepithelial current pathway may reside in the oxyntic cells. (b) The transepithelial conductance increase associated with the onset of acid secretion is entirely due to increased conductance of the apical membrane. This is in turn due entirely to increased area of this membrane, resulting from incorporation of tubulovesicular membrane. (c) When membrane conductances are normalized to actual membrane area by use of membrane capacitances, it turns out that acid secretion is not associated with a change in specific ionic conductance (change in conductance per unit area) at either the apical or basolateral membrane. (d) The puzzlingly low value of transepithelial resistance (≤400 Ω-cm²) arises because there are hundreds or thousands of square centimeters of actual membrane area per square centimeter chamber area. Apical membrane resistance is 25 kΩ-cm² (actual membrane area), implying a tight barrier to back-diffusion of protons.     

Hydraulic Properties of MDCK Cell Epithelium

The water permeability of the apical and basolateral cell membranes and the compliance of the lateral intercellular spaces (LIS) of MDCK monolayers were measured on confluent cultures grown on permeable supports. Cell membrane water permeabilities were determined, using quantitative differential interference light microscopy, from the rate of cell volume decrease after exposure to a hyperosmotic bathing solution. Both membranes exhibited osmotic water permeabilities (P_OSM) of ∼10 μm/sec, comparable to that of unmodified lipid bilayers. The compliance of the cell membranes forming the lateral intercellular space (LIS) between cells was determined from the pressure-volume relation. Confocal microscopy of fluorescent labeling of the basolateral cell membranes was used to delineate the LIS geometry as transepithelial hydrostatic pressure was varied. The LIS were poorly deformable as a function of transepithelial hydrostatic pressure until a pressure of ≥8 cm H₂O (basolateral > apical) was reached where catastrophic failure of intercellular connections occurred. The compliance of the LIS was calculated from the geometry changes at pressures <8 cm H₂O and ranged from 0.05–0.11 cm H₂O⁻¹, comparable to that previously predicted in mathematical models of the rat proximal tubule. Received: 10 January 1996/Revised: 9 May 1996     

A rapid method for the evaluation of the ionic permeabilities across epithelial cell membranes

This short note presents a recipe for the calculation of the ionic permeabilities across epithelial cell membranes. The method requires the Goldman-Hodgkin-Katz formalism as well as the consideration of the equivalent electrical circuit for an epithelial cell. The equivalent electrical circuit is solved in terms of the equivalent electromotive forces coupled in series with the ionic resistances of both cell membranes (apical and basolateral). The present procedure is feasible for any leaky epithelial cell membrane with the condition that this membrane (apical or basolateral) does not contain primary or secondary mechanisms for active transport.     

Electrical properties of rabbit corneal endothelium as determined from impedance measurements.

Alternating- and direct-current electrical characteristics of rabbit corneal endothelium were studied under varying experimental conditions. The measurements were performed by sending a 10-microA current (AC or DC) across the tissue layer. Maximal values of transendothelial potential difference and resistance were 1.3 +/- 0.1 mV and 73 +/- 6 omega . cm2, respectively. The short-circuit current was estimated from the potential and resistance values. Impedance loci were obtained for the frequency range 0.5-100 kHz. A capacitive reactance (C = 0.63 +/- 0.02 microF/cm2) was observed in the 100 Hz-100 kHz range. To relate the impedance data to the electrical parameters of the cell membranes, the voltage-divider ratio was determined by sending square pulse across the tissue and measuring voltage responses across the apical and basal membranes with an intracellular microelectrode. The intracellular potential difference was on the average -61 +/- 1 mV, and the voltage-divider ratio was found to be between 0.33 and 4. Impedance data were fit by a computer to an equivalent circuit representing a "lumped" model, and the agreement between the model and the data was satisfactory. The results are discussed in terms of both the morphological characteristics and properties of the fluid transport mechanism across the preparation.     

System for dynamic measurements of membrane capacitance in intact epithelial monolayers.

Dynamic measurements of exocytosis have been difficult to perform in intact epithelial monolayers. We have designed a system that estimates with +/-1% accuracy (99% confidence) the total membrane capacitance of monolayers represented by a lumped model. This impedance measurement and analysis system operates through a conventional transepithelial electrophysiology clamp, performing all signal measurements as frequently as every 5 s. Total membrane capacitance (the series combination of apical and basolateral membranes) is the inverse of one of three unique coefficients that describe the monolayer impedance. These coefficients are estimated using a weighted, nonlinear, least-squares algorithm. Using the estimated coefficients, solution ranges for individual membrane parameters are calculated, frequently providing results within +/-20% of true values without additional electrophysiological measurements. We determined the measurement system specifications and statistical significance of estimated parameters using 1) analytical testing with circuit simulation software and equation-generated data; 2) a system noise analysis combined with Monte Carlo simulations; and 3) analog model circuits for calibration of the electronic system and to check equation-generated results. Finally, the time course of capacitance changes associated with purinergically stimulated mucin exocytosis are quantified in monolayers of the colonic goblet cell-like cell line HT29-CI.16E.     

PGE(2) activation of apical membrane Cl(-) channels in A6 epithelia: impedance analysis.

Measurements of transepithelial electrical impedance of continuously short-circuited A6 epithelia were made at audio frequencies (0.244 Hz to 10.45 kHz) to investigate the time course and extent to which prostaglandin E(2) (PGE(2)) modulates Cl(-) transport and apical membrane capacitance in this cell-cultured model epithelium. Apical and basolateral membrane resistances were determined by nonlinear curve-fitting of the impedance vectors at relatively low frequencies (<50 Hz) to equations (P?unescu, T. G., and S. I. Helman. 2001. Biophys. J. 81:838--851) where depressed Nyquist impedance semicircles were characteristic of the membrane impedances under control Na(+)-transporting and amiloride-inhibited conditions. In all tissues (control, amiloride-blocked, and amiloride-blocked and furosemide-pretreated), PGE(2) caused relatively small (< approximately 3 microA/cm(2)) and rapid (<60 s) maximal increase of chloride current due to activation of a rather large increase of apical membrane conductance that preceded significant activation of Na(+) transport through amiloride-sensitive epithelial Na(+) channels (ENaCs). Apical membrane capacitance was frequency-dependent with a Cole-Cole dielectric dispersion whose relaxation frequency was near 150 Hz. Analysis of the time-dependent changes of the complex frequency-dependent equivalent capacitance of the cells at frequencies >1.5 kHz revealed that the mean 9.8% increase of capacitance caused by PGE(2) was not correlated in time with activation of chloride conductance, but rather correlated with activation of apical membrane Na(+) transport.     

Influence of cellular and paracellular conductance patterns on epithelial transport and metabolism

Theoretical analysis of transepithelial active Na transport is often based on equivalent electrical circuits comprising discrete parallel active and passive pathways. Recent findings show, however, that Na+ pumps are distributed over the entire basal lateral surface of epithelial cells. This suggests that Na+ that has been actively transported into paracellular channels may to some extent return to the apical (mucosal) bathing solution, depending on the relative conductances of the pathways via the tight junctions and the lateral intercellular spaces. Such circulation, as well as the relative conductance of cellular and paracellular pathways, may have an important influence on the relationships between parameters of transcellular and transepithelial active transport and metabolism. These relationships were examined by equivalent circuit analysis of active Na transport, Na conductance, the electromotive force of Na transport, the "stoichiometry" of transport, and the degree of coupling of transport to metabolism. Although the model is too crude to permit precise quantification, important qualitative differences are predicted between "loose" and "tight" epithelia in the absence and presence of circulation. In contrast, there is no effect on the free energy of metabolic reaction estimated from a linear thermodynamic formalism. Also of interest are implications concerning the experimental evaluation of passive paracellular conductance following abolition of active transport, and the use of the cellular voltage-divider ratio to estimate the relative conductances of apical and basal lateral plasma membranes.     

Stimulation of Cl- secretion via membrane-restricted Ca2+ signaling mediated by P2Y receptors in polarized epithelia.

Extracellular nucleotides such as ATP have been shown to regulate ion transport processes in a variety of epithelia. This effect is mediated by the activation of plasma membrane P2Y receptors, which leads to Ca(2+) signaling cascade. Ion transport processes (e.g. activation of apical calcium-dependent Cl(-) channels) are then stimulated via an increase in [Ca(2+)](i). Many polarized epithelia express apical and/or basolateral P2Y receptors. To test whether apical and basolateral stimulation of P2Y receptors elicit polarized Ca(2+) signaling and anion secretion, we simultaneously measured the two parameters in polarized epithelia. Although activation of P2Y receptors located at both apical and basolateral membranes evoked an increase in [Ca(2+)](i), only apical P2Y receptors-coupled Ca(2+) release stimulated an increase in anion secretion. Moreover, the calcium influx evoked by apical and basolateral P2Y receptor stimulation is predominately via the basolateral membrane domain. It appears that the apical P2Y receptor-regulated Ca(2+) release and activation of apical Cl(-) channels is compartmentalized in polarized epithelia with basolateral P2Y-stimulated Ca(2+) release failing to activate anion secretion. These data suggest that there may be two distinct ATP-releasable Ca(2+) pools, each coupled to apical and basolateral membrane receptor but linked to the same calcium influx pathway located at the basolateral membrane.     

Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10-25 mM glucose, alanine or leucine. Resistances of intercellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01-10 kHz. The segments were then fixed in situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixed in situ at 38 degrees C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Summary Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10–25mm glucose, alanine or leucine. Resistances of inter-cellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01–10 kHz. The segments were then fixedin situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixedin situ at 38°C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

Biophysical Model of Ion Transport across Human Respiratory Epithelia Allows Quantification of Ion Permeabilities

Lung health and normal mucus clearance depend on adequate hydration of airway surfaces. Because transepithelial osmotic gradients drive water flows, sufficient hydration of the airway surface liquid depends on a balance between ion secretion and absorption by respiratory epithelia. In vitro experiments using cultures of primary human nasal epithelia and human bronchial epithelia have established many of the biophysical processes involved in airway surface liquid homeostasis. Most experimental studies, however, have focused on the apical membrane, despite the fact that ion transport across respiratory epithelia involves both cellular and paracellular pathways. In fact, the ion permeabilities of the basolateral membrane and paracellular pathway remain largely unknown. Here we use a biophysical model for water and ion transport to quantify ion permeabilities of all pathways (apical, basolateral, paracellular) in human nasal epithelia cultures using experimental (Ussing Chamber and microelectrode) data reported in the literature. We derive analytical formulas for the steady-state short-circuit current and membrane potential, which are for polarized epithelia the equivalent of the Goldman-Hodgkin-Katz equation for single isolated cells. These relations allow parameter estimation to be performed efficiently. By providing a method to quantify all the ion permeabilities of respiratory epithelia, the model may aid us in understanding the physiology that regulates normal airway surface hydration.     

Current-voltage relations of the apical and basolateral membranes of the frog skin

We determined the current-voltage (I-V) relations of the apical and basolateral barriers of frog skins by impaling the cells with an intracellular microelectrode and assuming that the current across the cellular pathway was equal to the amiloride-inhibitable current. We found that: (a) The responses in transepithelial current and intracellular potential to square pulses of transepithelial potential (VT) varied markedly with time. (b) As a consequence of these transient responses, the basolateral I-V relation was markedly dependent on the time of sampling after the beginning of each pulse. The apical I-V plot was much less sensitive to the time of sampling within the pulse. (c) The I-V data for the apical barrier approximated the I-V relations calculated from the Goldman constant field equation over a relatively wide range of membrane potentials (+/- 100 mV). (d) A sudden reduction in apical bath [Na+] resulted in an increase in apical permeability and a shift in the apical barrier zero-current potential (Ea) toward less positive values. The shift in Ea was equivalent to a change of 45 mV for a 10-fold change in apical [Na+]. (e) The transient responses of the skin to square VT pulses were described by the sum of two exponentials with time constants of 114 and 1,563 ms, which are compatible with the time constants that would be produced by an RC circuit with capacitances of 65 and 1,718 microF. The larger capacitance is too large to identify it comfortably with a true dielectric membrane capacitance.     

Changes in cellular membrane and paracellular conductances by amphotericin B in the epithelium of the bullfrog cornea.

In the isolated bullfrog cornea, measurements of DC electrical parameters in conjunction with AC impedance and ultrastructural analyses were used to determine the effects of 10(-5) M amphotericin B on epithelial cellular membrane and paracellular conductances. In NaCl Ringers, amphotericin B elicited a 3.5-fold increase in the specific apical membrane conductance (Ga/Ca); where Ga and Ca are the apical membrane conductance and capacitance, respectively. The basolateral membrane conductance (Gb) and the basolateral membrane capacitance (Cb) fell by 57% and 50%, respectively. In the paracellular pathway, the tight junctional complex (Gj) was unchanged whereas the lateral intercellular space resistance (Rp) decreased by 55%. The declines in Gb and Cb were suggestive of cell volume shrinkage because these changes were consistent with a previously described decline in intracellular K+ content and reduction in exposed basolateral membrane area to current flow. Ultrastructural analysis validated that amphotericin B caused cell volume shrinkage because there was: (1) increased folding of the basolateral membrane and waviness of the basal aspects- of the plasma membrane; (2) dilatation of the lateral intercellular spaces. This agreement suggests that intracellular activity decreased following exposure to amphotericin B which resulted in cell volume shrinkage and an impairment of Cl- uptake across the basolateral membrane.     

Basolateral chloride current in human airway epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption. However, the pathways for basolateral Cl(-) exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl(-) conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl(-) conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl(-) current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl(-) > Br(-) > I(-). Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca(2+) and inducing cell swelling had no effect. The basolateral Cl(-) current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl(-) currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl(-) conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl(-) conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl(-) movement.     

Voltage dependent membrane conductances in cultured renal distal cells.

Cultured Na(+)-transporting epithelia from amphibian renal distal tubule (A6) were impaled with microelectrodes and analyzed at short-circuit and after transepithelial voltage perturbation to evaluate the influence of voltage on apical and basolateral membrane conductances. For equivalent circuit analysis, amiloride was applied at each setting of transepithelial potential. At short-circuit, apical and basolateral membrane conductances averaged 88 and 497 microS/cm2, respectively (n = 10). Apical membrane conductance, essentially due to Na(+)-specific pathways, decreased after depolarization of the apical membrane. The drop was considerably larger than predicted by the Goldman-Hodgkin-Katz (GHK) constant-field equation. This suggests decrease in permeability of the apical Na+ channels upon depolarization. Basolateral membrane conductance, preferentially determined by K+ channels, increased after hyperpolarization of the basolateral membrane. This behavior is contrary to the prediction of the GHK constant field equation and reflects inward rectification of the K+ channels. The observed rectification patterns can be valuable for maintenance of cellular homeostasis.     

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R-induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 microM CCCP and 2.5 microg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R-dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl- secretion was investigated in studies simultaneously measuring Ca2+i and Cl- secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl- secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains.