Polarity of alveolar epithelial cell acid-base permeability

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HCO[Formula: see text]-free Na+ buffer (140 mM Na+, 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked ≥80% by 100 μM dimethylamiloride, an inhibitor of Na+/H+exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is Na+/H+ exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ.

Interaction of trichlormethiazide or amiloride with PTH in stimulating Ca2+ absorption in rabbit CNT

To determine the renal tubular site and mechanism of the anticalciuric effects of trichloromethiazide (TCM) and amiloride, we studied the effects of these diuretics on net Ca2+ absorption (JCa) in isolated rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). TCM (100 microM) in the lumen increased JCa in the CNT without affecting lumen-negative transepithelial voltage (VT). This effect was dependent on the amount of parathyroid hormone (PTH, 0.1 or 1 nM) in the bath. TCM had no detectable effect on JCa in the absence of PTH. Addition of 100 microM ouabain to the bath decreased PTH-stimulated JCa and abolished the TCM-stimulated JCa. Elimination of Na+ from the lumen increased PTH-stimulated JCa. Under this condition the ability of TCM to increase JCa was abolished, suggesting that the inhibition of Na+ entry from the lumen with TCM may account for the increase in PTH-stimulated JCa. Elimination of Na+ from the bath in the presence of PTH decreased JCa and abolished the stimulatory effect of TCM on JCa in the presence of PTH. Changes in VT caused by amiloride may not account for the increase in JCa, because JCa was not changed when VT was deflected to more positive direction by increasing bath K+ from 5 to 15 mM. Neither TCM nor amiloride affected JCa in the CCD. From these observations, we speculate that the inhibition of Na+ entry across the apical cell membrane by either thiazides or amiloride may stimulate PTH-induced JCa. The intact Na(+)-Ca2+ antiporter in the basolateral cell membrane is essential for the anticalciuric effect of thiazides and amiloride.

Relative osmotic effects of raffinose, KCl, and NaCl across basolateral cell membrane

Lumen-collapsed segments of rabbit S2 proximal tubule were bathed in isotonic medium and then exposed acutely to a medium made hypertonic by the addition of raffinose, NaCl, KCl, Na gluconate, K gluconate, or choline Cl. The result was a rapid efflux of water and a shrinking of the tubule, which could be measured by video techniques within the first 0.1 s. After reequilibration in isotonic medium, each tubule was then exposed to a second hypertonic medium to provide a direct comparison between two different solutes, either NaCl vs. KCl or raffinose vs. any one of the other solutes. Because raffinose is impermeant across the basolateral cell membrane, the ratio of its effect to that of another solute is a measure of the reflection coefficient (sigma) of that other solute. The following results were obtained: sigma KCl = 0.70 +/- 0.02, sigma K gluconate = 0.97 +/- 0.07, sigma Na gluconate = 0.84 +/- 0.06, and sigma choline Cl = 0.75 +/- 0.06. We previously have reported sigma NaCl = 0.56 +/- 0.07. If sigma of each salt is considered to be the arithmetic average of its component parts, and if gluconate and choline are considered to be impermeant, we also obtain sigma Na+ = 0.68, sigma K+ = 0.94, and sigma Cl- = 0.50.

Na+ electrochemical gradient and Na+-Ca2+ exchange in rat proximal tubule

he basolateral cell membrane of the rat proximal tubule contains a Na+-Ca2+ exchanger that may participate in the regulation of cytosolic calcium (Cai) and Ca2+ transport. In this work, the activity and orientation of the Na+-Ca2+ exchanger was studied in rat proximal tubules. The experiments were based on the thermodynamic notion that the exchanger is driven by the prevalence of either of two electrochemical gradients, that for Na+ (delta mu Na+) or for Ca2+ (delta mu Ca2+). Reductions in delta mu Na+, achieved by lowering extracellular Na+ (Nao) from 150 to 15 mM, increased Cai, decreased 45Ca efflux, and increased 45Ca influx. These changes occurred concurrently. When delta mu Na+ was reduced by increasing intracellular Na+ (Nai) with 10(-3) M oubain, Cai also increased. The effect of ouabain was probably dependent on Nai accumulation because the surge in Cai was prevented by exposure of the tubules to 5 mM Nao before ouabain exposure. On the other hand, when delta mu Na+ was lowered mM Nao and then by reducing Nao to 15 mM, Cai rose in two additive stages. We conclude from these data that in the rat proximal tubule the basal state of the Na+-Ca2+ exchanger is in forward mode, Nao-Cai. Moreover, the function of the Na+-Ca2+ exchanger is in accord with predictions derived from a thermodynamic analysis of its function.

Immunoelectron microscopy of fodrin in the rat uriniferous and collecting tubular epithelium.

We examined the localization of fodrin in epithelial cells of rat uriniferous and collecting tubules by immunofluorescence and immunoelectron microscopy of frozen sections. In the uriniferous tubule, fodrin was found along the cell membrane and in the well-developed terminal web, as previously reported in other epithelial cells: in the terminal web and along the basolateral cell membrane in the proximal tubule; all around the cell surface in the thin limb of Henle; along the basolateral surface in the thick limb of Henle's thick segment and the distal tubule. In the intercalated cells of the collecting tubule, fodrin was found not only along the basolateral cell membrane but also in the apical cytoplasm. The most peculiar labeling was obtained in the principal cells of the collecting tubule. In addition to labeling in the basolateral cell membrane, fodrin was found diffusely in the cytoplasmic matrix. Association of fodrin with any particular structure could not be identified, but the Golgi area was apparently free of labeling. Cytoplasmic labeling was more conspicuous in the principal cells of the medulla than in those of the cortex. The present results show that fodrin need not always exist in association with the cell membrane or the cytoskeleton but can occur in the cytoplasmic matrix, at least in epithelial cells. We discuss the possible physiological significance of the latter distribution.

The collecting tubule of Amphiuma. I. Electrophysiological characterization

Single collecting tubules of Amphiuma kidneys were perfused in vitro to characterize their electrophysiological properties. The lumen-negative potential (-24 mV) was abolished by amiloride in the lumen and by ouabain in the bath. Ion substitution experiments in the lumen demonstrated the presence of a large sodium conductance in the apical cell membrane, but no evidence was obtained for a significant potassium or chloride conductance. Ion substitutions in the bath solution and the depolarizing effect of barium on the basolateral membrane potential demonstrated the presence of a large potassium conductance in the basolateral cell membrane. Measurements of dilution potentials in amiloride-treated tubules revealed a modest cation selectivity of the paracellular pathway. These results support a cell model in which sodium reabsorption occurs by electrodiffusion across the apical cell membrane and active transport across the basolateral cell membrane. The absence of a detectable potassium conductance in the apical cell membrane suggests that secretion of this ion cannot take place by diffusion from cell to lumen.

Morphometric analysis of distinct microanatomy near the base of proximal tubule cells

Models of cell shape in the rabbit S2 proximal renal tubule were derived from transmission electron micrographs and compared with scanning micrographs. Standard morphometric procedures were used to measure basolateral cell membrane surface density (SVt) relative to total epithelial volume in numerous zones of cell height. In the basal 20% region we also measured the volume fraction (F) of intercellular spaces and calculated new surface densities in reference only to the intercellular volume, SVi = SVt/F, or to the cellular volume, SVc = SVt/(1-F). Combined use of these surface densities then enabled us to calculate the diameter, length, and separation of effectively cylindrical microvilli at the cell base. Assuming that lateral cell membranes are radially oriented in the apical region but disposed on microvillus like structures of arbitrary orientation at the cell base, an improved cell model was developed that agreed with the scanning picture throughout the entire cell height. Basal microvillar elements contain approximately 60% of the total basolateral cell membrane surface area and possibly constitute a hydrostatic resistive region for absorbate flow. These features have interesting physiological implications.

Salinity Acclimation and Intestinal Salt Transport in the Flounder: the Role of the Basolateral Cell Membrane

1. Intestinal absorption of Na+ and Cl−, measured using bidirectional tracer fluxes, is higher in saltwater (SW)-acclimated flounders than in freshwater (FW)- acclimated ones. 2. Removal of the selective permeability of the apical cell membrane by application of amphotericin B to the mucosal solution enhances the difference in Na+ transport, whilst the difference in Cl− absorption is lost. 3. Transepithelial resistance and JsmNa were similar between the two groups of fish, whilst JsmCl, was always greatest in SW-acclimated tissues, even after mucosal application of amphotericin. 4. Analysis of the present results and previous work suggests that the principal acclimatory changes following salt adaptation occur in the basolateral cell membrane, and that both the Na+, K+-ATPase activity and the basolateral cell membrane permeability to Cl− are increased in the SW-acclimated tissues.