Recovery of cell volume and electrolytes of A6 cells after re-establishing isotonicity following hypotonic stress.

Cellular element concentrations and dry weight contents in A6 cells were determined using electron microprobe analysis to establish whether these cells exhibit a regulatory volume increase (post-RVD-RVI) when re-establishing isotonicity following a hypotonically induced regulatory volume decrease (RVD). Hypotonic stress was induced by reducing basolateral [NaCl], and hence, osmolarity fell from 260 to 140 mosmol/l. The alterations in cell volume after re-establishing isotonicity, calculated from the cellular dry weight changes, indicate within the first 2 min cell shrinkage from 120 to 76% of control, compatible with almost ideal osmometric behaviour of A6 cells, and thereafter a post-RVD-RVI to 94%. The cellular uptake of osmolytes necessary to explain the post-RVD-RVI could be accounted for solely by a gain in cellular K and Cl. The involvement of a Na-K-2Cl cotransporter in most of the KCl uptake seems plausible since basolateral bumetanide blocked KCl uptake and post-RVD-RVI. The net uptake of cations (K uptake of 185.2, Na loss of 8.2 mmol/kg dry wt) during the isotonic period exceeded the Cl uptake by 38.2 mmol/kg dry wt, suggesting the uptake of another anion and/or the alteration of cellular buffer capacity. The relatively low Na concentration maintained during the isotonic period (13.3 vs. 20.4 mmol/kg wet wt under control conditions) might favour electrolyte uptake via the Na-K-2Cl cotransporter.

Changes in element composition of A6 cells following hypotonic stress.

Cellular element concentrations and dry weight contents were determined in A6 epithelia using electron microprobe analysis. This was done to assess the quantitative contributions of Na, K and Cl to the regulatory volume decrease (RVD) and isovolumetric regulation (IVR) after decreasing the basolateral osmolality from 260 to 140 mosmol/kg in a stepwise or gradual way. Two minutes after inducing acute hypotonic stress the cells behaved almost like ideal osmometers, as indicated by a pronounced increase in cell height and decreases in the cellular dry weight and concentrations of all measured elements by about the same degree. Sixty minutes after inducing acute hypotonic stress the dry weight and concentrations of the impermeant elements P, Mg and Ca had returned approximately to control values, indicating normalized cell volume. Na, K and Cl concentrations, however, remained greatly reduced. The cellular amounts of Na, K and Cl diminished during RVD by approximately 31%, 24% and 46%, respectively. The dry weights and element concentrations measured 60 min after inducing acute hypotonic stress were similar to those obtained after a continuous reduction of basolateral osmolality. The cellular loss of Na and K following hypotonic stress exceeded that of Cl by about 40 mmol/kg wet wt., suggesting the exit of an other anion and/or the titration of fixed negative charges. The contribution of Na, K and Cl to total cellular osmolality increased from about 75% under control conditions to about 85% during RVD and IVR. Since only approximately 70% of the loss of cellular osmolytes necessary for the observed RVD and IVR is accounted for by the cellular exit of Na, K and Cl, other osmolytes, possibly amino acids, must leave the cells following hypotonic stress.

Inner-medullary organic osmolytes and inorganic electrolytes in K depletion.

The renal concentrating defect typical for chronic K depletion has been ascribed to malfunction of renomedullary cells caused by inadequate accumulation of organic osmolytes. A reduction in intracellular ionic strength, which is believed to influence decisively the accumulation of organic osmolytes, has been held responsible for insufficient osmolyte accumulation. To test this hypothesis, intra- and extracellular Na, Cl and K concentrations, the major determinants of ionic strength, were measured in the papilla by electron microprobe analysis and organic osmolytes (glycerophosphorylcholine, betaine, sorbitol, myo-inositol, free amino acids) in inner-medullary tissue by HPLC in antidiuretic rats kept on either a control (normal-K) or a K-deplete (low-K) diet and in euhydrated rats with free access to water and control diet. K depletion was associated with a reduced urine concentrating ability. Papillary interstitial ionic strength (sum of Na, Cl and K) in antidiuretic low-K rats was significantly reduced compared with antidiuretic normal-K rats (688+/-19 vs. 971+/-61 mmol/kg wet wt) but was similar to that in euhydrated normal-K rats (643+/-35 mmol/kg wet wt). The lower interstitial ionic strength in antidiuretic low-K and euhydrated normal-K rats was associated with a lower total content of organic osmolytes in the inner medulla (365+/-14 and 381+/-20, respectively, vs. 465+/-11 mmol/kg protein in antidiuretic normal-K rats). Intracellular ionic strength (sum of Na, Cl and K) of papillary collecting duct cells, however, was similar in antidiuretic normal-K and euhydrated normal-K rats (171+/-5 and 179+/-11 mmol/kg wet wt) but lower in antidiuretic low-K rats (138+/-9 mmol/kg wet wt). These results do not support the view that, in the steady state of osmotic adaptation of renomedullary cells in situ, intracellular ionic strength is the decisive factor for maintaining high levels of organic osmolytes. During chronic K depletion, reduced osmolyte accumulation by renomedullary cells may be the consequence, rather than the cause, of lower medullary interstitial tonicity.

Adaptation of Madin-Darby canine kidney cells to hypertonic medium: an electron microprobe analysis.

Madin-Darby canine kidney (MDCK) cells of confluent epithelial sheets grown on permeable supports respond to hyperosmotic stress by short- and long-term regulatory volume increase (RVI). Although short-term RVI includes the uptake of inorganic electrolytes, long-term RVI does not and seems therefore to result from accumulation of organic osmolytes.

Characteristics of sodium uptake across the basolateral membrane of oxyntic cells.

To characterize further serosal Na uptake into gastric oxyntic cells under resting conditions, cellular element concentrations were determined in isolated frog (Rana temporaria) gastric mucosae using electron microprobe analysis. The epithelia were kept short circuited in Ussing-type chambers, and element analysis was performed on freeze-dried cryosections. After ouabain (10(-4) M), the [Na] in oxyntic cells increased within 30 to 60 minutes from approximately 25 to 100 mmol/kg wet wt, and [K] decreased similarly (from 100 to 25 mmol/kg wet wt). These changes occurred regardless of whether the basolateral incubation medium contained HCO3 or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) as buffers. When, prior to the addition of ouabain, 10(-3) M amiloride was applied to the serosal side to inhibit the Na-H antiporter, the ouabain-induced increase in cellular [Na] was prevented completely in HEPES-, but not in HCO3-Ringer. The data are compatible with the notion that Na is taken up by a Na-H antiporter and a Na-HCO3 symporter. At least under these experimental conditions, these transporters seem to contribute substantially to basolateral Na uptake in oxyntic cells.

Cellular site of active K absorption in the guinea-pig distal colonic epithelium.

The mammalian distal colon, which is composed of different cell types, actively transports Na, K and Cl in absorptive and K and Cl in secretory directions. To further characterize the K absorption process and to identify the cells involved in K absorption, unidirectional Rb fluxes and luminal Rb uptake into different epithelial cell types were determined in isolated guinea-pig distal colon. Net Rb absorption (1.5-2.5 micromol.h-1.cm-2) was not influenced by inhibition of Na transport with amiloride or by incubating both sides of the epithelium with Na-free solutions, but was almost completely abolished by luminal ouabain, ethoxzolamide or by incubating both sides of the epithelium with Cl-free solutions. Luminal Rb uptake, blockable by luminal ouabain, preferentially occurred in columnar surface and neck cells, to a lesser extent in surface goblet cells and to an insignificant degree in lower crypt cells. Employing a luminal Rb-Ringer (5.4 mM Rb) the Rb concentration increased within 10 min in columnar surface and neck, surface goblet and lower crypt cells to 70, 32 and about 10 mmol. kg-1 wet weight, respectively. The presence of 5.4 mM K in the luminal incubation solution reduced Rb uptake almost completely indicating a much higher acceptance of the luminal H-K-ATPase for K than for Rb. The increase in Na and decrease in K concentrations in surface and neck cells induced by luminal ouabain might indicate inhibition of the basolateral Na-K-ATPase or drastic enhancement of cellular Na uptake by the Na-H exchanger. Bilateral Na-free incubation did not alter Rb uptake, but bilateral Cl-free incubation drastically reduced it. Inhibition of net Rb absorption by ethoxzolamide and inhibition of both Rb absorption and Rb uptake by bilateral Cl-free incubation support the notion that cellular CO2 hydration is a necessary prerequisite for K absorption and that HCO3 leaves the cell via a Cl-HCO3 exchanger. Since ouabain-inhibitable transepithelial Rb flux and luminal Rb uptake rate by surface and neck cells were about the same, Rb(K) absorption seems to be accomplished mainly by columnar surface cells.

The role of mitochondria-rich cells in sodium transport across amphibian skin.

The possible participation of mitochondria-rich cells in transepithelial Na+ transport across frog skin under "physiological conditions" (low apical [Na+], open circuited) was analysed by recording electrophysiological parameters from principal cells with intracellular microelectrodes and using measurement of Rb+ uptake into the epithelial cells from the serosal side via the Na+/K+-ATPase. It was observed that transport perturbation with amiloride induced changes in the apical potential difference and fractional apical resistance in principal cells, observations which are compatible with the notion that the essential fraction of transcellular current flow occurs across these cells. Amiloride-inhibitable uptake of Rb+ was also restricted to principal cells, the amount being about equivalent to the predicted rate of K+ recycling via the Na+/K+-ATPase. The results indicate that principal cells are responsible for transepithelial Na+ transport irrespective of the experimental conditions. Flow of Na+ across mitochondria-rich cells appears to be negligible.

Ischemia-induced changes in cell element composition and osmolyte contents of outer medulla.

The effect of 60 minutes of ischemia and subsequent reflow on cell electrolyte and water homeostasis in the rat renal outer medulla was studied by determining sodium, potassium, chloride and phosphorus concentrations and dry weights in individual tubule cells using electron microprobe analysis. HPLC was employed to measure glycerophosphorylcholine, betaine, inositol and sorbitol, as well as several free amino acids in cortical and outer medullary tissue. Ischemia caused cell sodium and chloride concentrations to rise and cell potassium and phosphorus concentrations and cell dry weights to fall. These changes were most pronounced in the proximal straight tubule (PST) cells, less in thick ascending limb (MAL) and outer medullary collecting duct (OMCD) dark cells and barely noticeable in OMCD light cells. Except for some PST cells these changes were almost completely reversed 60 minutes after reintroducing blood flow. After 24 hours of reperfusion the number of PST cells exhibiting deranged electrolyte homeostasis was greatly increased. The contents of glycerophosphorylcholine, betaine or inositol in the cortex and outer medulla were not affected immediately following ischemia. After 24 hours of reperfusion, the cortical contents of osmolytes were still normal, while outer medullary contents were reduced. Except for low glycine contents, the ischemia-induced changes in amino acid contents were reversed after 24 hours of reflow in the cortex, whereas in the outer medulla aspartate, glycine and taurine contents were diminished. These results indicate increasing manifestation of PST cell injury in the reflow period. The defective re-accumulation of organic osmolytes and free amino acids in the outer medulla during reflow may reflect reduced interstitial tonicities, or may be due to inappropriate cellular uptake, synthesis or/and release.(ABSTRACT TRUNCATED AT 250 WORDS)

Loop diuretics affect transcellular electrolyte transport in cells of the distal convoluted tubule.

Although loop diuretics act preferentially on sodium chloride absorption in the thick ascending limb of the loop of Henle in the nephron, high concentrations of some loop diuretics also impair sodium absorption in the distal convoluted tubule (DCT). To characterize further the inhibitory effect of these agents on sodium absorption in the DCT, the action of torsemide and furosemide on cell sodium, chloride and potassium concentrations was examined in individual DCT cells of the kidney cortex and also, for comparison, in proximal convoluted tubule cells. In addition, initial cell uptake rates of rubidium, an index of in vivo Na+/K(+)-ATPase activity, were studied. Both diuretics caused a significant reduction of intracellular sodium concentration and rubidium uptake in DCT cells but not in connecting tubule, principal, intercalated or proximal tubule cells. These findings are consistent with the concept that both diuretics reduce transcellular sodium absorption in DCT cells by impairing sodium entry across the apical cell membrane and, as a consequence, sodium extrusion by primary active Na+/K+ (Rb+) exchange across the basolateral membrane.

Electron microprobe analysis of electrolytes in whole cultured epithelial cells.

Microprobe analysis was used to determine electrolyte contents in whole epithelial sheets of A6 cells and to investigate the most critical points of this method. Analysis of dextran standard sections of different thickness revealed that low accelerating voltages of about 10 kV are best suited for whole freeze-dried cells on thick supports, since 5 microM thick sections are not penetrated by 10 kV electrons. Washing of A6 cells for 10 sec with distilled water led to cell swelling of about 40%, but the molar concentration ratios and the concentrations per dry weight (dw) were not altered. Washing for 60 sec with distilled water caused a further increase in cell volume (120%) and loss of cellular K and Cl (90 mmol/kg dw). Washing with isotonic NH4- acetate led to a loss of cell Cl already after 10 sec. To characterize the Na transport compartment, A6 cells cultured on permeable supports were washed for 5 sec with distilled water, freeze-dried, and analyzed. Inhibition of transepithelial Na transport by ouabain increased Na/P from 0.15 +/- 0.07 to 0.75 +/- 0.03 and Cl/P from 0.21 +/- 0.001 to 0.38 +/- 0.003 while K/P decreased from 0.83 +/- 0.08 to 0.32 +/- 0.03. The changes in cell Na and K contents can be explained by K/Na exchange; the increase in Cl content indicates some cell swelling. Since the ouabain-induced changes could be prevented by apical amiloride, the apical membrane provides the most important pathway for Na entry in A6 cells.

Transcellular sodium transport and basolateral rubidium uptake in the isolated perfused cortical collecting duct.

The relation between transcellular Na+ absorption, intracellular Na+ concentration and Na+/K(+)-ATPase activity (the last estimated by the rubidium uptake across the basolateral cell membrane) was examined in the different cell types of the rabbit cortical collecting duct (CCD). Experiments were performed on isolated perfused CCD in which Na+ absorption was varied by perfusing the tubule with solutions containing different Na+ concentrations (nominally Na(+)-free, 30 mM and 144 mM). Experiments were terminated by shock-freezing the tubules during perfusion. Precisely 30 s before shock-freezing, the K+ in the bathing solution was exchanged for Rb+. Intracellular element concentrations, including Rb+, were determined in freeze-dried cryosections of the tubules using energy-dispersive X-ray analysis. Increasing Na+ concentration in the perfusion solution caused significant rises in intracellular Na+ concentration and Rb+ uptake of principal cells. Principal cell Na+ and Rb+ concentrations were 7.8 +/- 0.9 and 7.0 +/- 0.8 mmol/kg wet weight respectively, when the perfusion solution was Na(+)-free, 10.1 +/- 0.7 and 11.6 +/- 0.6 mmol/kg wet weight with 30 mM Na+ in the perfusion solution, and 14.5 +/- 1.5 and 14.9 +/- 0.9 mmol/kg wet weight with 144 mM Na+ in the perfusion solution. In contrast, a comparable relationship between lumen Na+ concentration, intracellular Na+ concentration and basolateral Rb+ uptake was not seen in intercalated cells. These results support the notion that principal, but not intercalated, cells are involved in transepithelial Na+ absorption. In addition, the data demonstrate that apical Na+ entry and basolateral Na+/K(+)-ATPase activity are closely coupled in principal cells of the rabbit CCD.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmotic adaptation of renal medullary cells during transition from chronic diuresis to antidiuresis.

The cells of the renal medulla adapt osmotically to high extracellular tonicities by high concentrations of organic osmolytes. Intracellular accumulation of these substances is, however, relatively slow. The aim of the present study was to assess the effect of an abrupt rise in extracellular tonicity on intracellular osmotically active substances after prior reduction of medullary contents of organic osmolytes by chronic diuresis. Intra- and extracellular electrolyte concentrations at the papillary tip and the tissue contents of methylamines (glycerophosphorylcholine, betaine), polyols (myo-inositol, sorbitol), and several amino acids were determined in the different kidney zones by electron microprobe analysis and high-performance liquid chromatography in control animals, in rats infused for 6 days with furosemide via osmotic minipumps, and in rats given the vasopressin analogue [deamino-Cys1,D-Arg8]vasopressin (DDAVP) after the chronic furosemide treatment. Chronic diuresis greatly reduced interstitial tonicity and inner medullary contents of methylamines and polyols and moderately reduced inner medullary amino acid contents but did not significantly affect intracellular electrolyte concentrations. When the diuretic rats were infused with DDAVP for 2 h, interstitial tonicity more than doubled and intracellular K and Cl concentrations rose by approximately 60 and 160%, while inner medullary contents of methylamines, polyols, and amino acids were not changed significantly. These data demonstrate that after effective depletion of medullary organic osmolytes by long-term diuresis, the cells of the renal papilla adapt osmotically to an abrupt increase in extracellular tonicities by elevated cell electrolyte concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of increased distal sodium delivery on organic osmolytes and cell electrolytes in the renal outer medulla.

Sodium absorption in distal tubule segments was stimulated by increasing the distal delivery via infusion of hypertonic saline. In these animals, and in control rats, electrolyte concentrations in thick ascending limb cells, light and dark cells of the collecting duct in the outer and inner stripe of the outer medulla and in cells of the proximal straight tubule (outer stripe only) were studied. The measurements were performed by electron microprobe analysis of freeze-dried cryosections of the outer medulla. In addition, organic osmolytes (glycerophosphorylcholine, betaine and myo-inositol) were measured by high performance liquid chromatography in cortex and outer medulla. Augmented delivery of sodium chloride to the distal tubule was associated with increased sodium concentrations of thick ascending limb cells both in the outer and inner stripe and of medullary collecting duct light and dark cells in the outer stripe. While the sum of organic osmolyte concentrations was 28% higher in the outer medulla of the salt-loaded animals compared with controls, this value was unchanged in the renal cortex. These findings indicate that the primary event underlying stimulation of sodium absorption along the thick ascending limb during increased distal sodium delivery is enhanced entry of sodium across the apical cell membrane. This would be expected to lead to higher cell sodium concentrations and stimulation of basolateral active Na-K-exchange. The enhanced transport activity of outer medullary tubules may be associated with increased interstitial tonicities and intracellular retention of organic osmolytes.

Effect of loop diuretics on organic osmolytes and cell electrolytes in the renal outer medulla.

Electron microprobe analysis on freeze-dried cryosections was used to determine the effect of the loop diuretics torasemide and furosemide on intracellular electrolyte concentrations in individual cells of the outer and inner stripe of the outer medulla and on cell rubidium uptake, the latter a measure of basolateral Na-K-ATPase activity. In addition, the organic osmolytes glycerophosphorylcholine (GPC), betaine, inositol and sorbitol in cortex, outer medulla and inner medulla were measured using HPLC. Both loop diuretics significantly reduced sodium and chloride concentrations and rubidium uptake in thick ascending limb cells, but did not affect sodium concentration or rubidium uptake in the proximal straight tubule (PST) cells or in the light or dark cells of the outer medullary collecting duct (OMCD). Chloride concentrations in these cells (that is, PST cells, OMCD light and dark cells) were lowered by loop diuretics, albeit less than in thick ascending limb cells. Administration of both loop diuretics for only 20 minutes was sufficient to significantly depress tissue concentrations of GPC, betaine, and myo-inositol in the outer medulla and of GPC, betaine and sorbitol at the papillary tip. These results indicate that loop diuretics, presumably by blocking apical sodium entry, decrease thick ascending limb cellular sodium concentration and, as a consequence, reduce Na-K-ATPase activity as assessed by cell rubidium uptake. Although this has been shown previously in in vitro preparations, the present study confirms this for the first time in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Osmolytes in renal medulla during rapid changes in papillary tonicity.

The effect of acute changes in extracellular tonicity on cell electrolyte concentrations at the renal papillary tip and on organic osmolytes in different kidney zones was studied using electron microprobe analysis and high-performance liquid chromatography in four groups of rats: controls, 1- or 4-h water diuresis, and 4-h water diuresis followed by 30-min deamino-[Cys1,D-Arg8]vasopressin (ddAVP). The sum of the papillary interstitial concentrations of Na, K, and Cl was reduced from 981 mmol/kg wet wt in controls to 318 mmol/kg wet wt after 4-h diuresis and increased after ddAVP to 840 mmol/kg wet wt. In papillary collecting ducts intracellular electrolytes fell from 225 to 156 mmol/kg wet wt after 4-h diuresis and rose to 268 mmol/kg wet wt (significantly higher than control) after ddAVP. Organic osmolytes [sum of glycerophosphorylcholine (GPC), betaine, myo-inositol, and sorbitol] at the papillary tip decreased from 2,018 (control) to 1,037 mmol/kg protein after 4-h diuresis and did not increase after ddAVP. After ddAVP, cell P concentration, an index of cell GPC concentration, increased, indicating cell shrinkage. GPC concentration increased, indicating cell shrinkage. The results suggest that the concentrations of all osmoeffectors in papillary cells initially increase due to cell shrinkage in response to hypertonic stress. The higher intracellular ionic strength may be a signal for modulation of transport and metabolism of organic osmolytes.

Effect of diuretics on cell potassium transport: an electron microprobe study.

To study the short-term uptake of potassium across the basolateral membrane into individual tubule cells, rubidium was used and measured by electron microprobe analysis. Changes of rubidium uptake were interpreted to reflect altered sodium entry and basolateral Na-K-ATPase activity. The effects of hydrochlorothiazide, amiloride and furosemide were determined in saline-loaded animals. Hydrochlorothiazide inhibited rubidium uptake in proximal convoluted and distal convoluted tubule cells. The effect was largest in distal convoluted tubule cells. Amiloride reduced rubidium uptake in principal cells as well as in proximal convoluted, distal convoluted and connecting tubule cells. Furosemide depressed rubidium uptake in distal convoluted tubule cells, but increased uptake in principal cells. Rubidium uptake into intercalated cells was not affected by any of the diuretics used. Hydrochlorothiazide and amiloride altered rubidium uptake also in cells not associated with the main diuretic action. These effects of hydrochlorothiazide and amiloride may be due to interference with cell transport mechanisms of Na-H and anion exchange.

Studies of epithelial electrolyte transport by marker ions.

The paper reviews several recent studies in which marker ions, such as Rb and Br, were used to identify ion transport pathways and membrane properties in epithelia. In the frog skin epithelium, using Rb as a substitute for K, Cl transport mechanisms across the basolateral membranes of principal cells were studied. The data suggest that intracellular Cl is maintained above electrochemical equilibrium by an Na-K-2Cl cotransport system which, under non-stimulated conditions, is normally quiescent. In toad and frog skins, the route of transepithelial Cl movement was investigated. A subpopulation of mitochondria-rich cells demonstrated a ready exchange of Br with the apical and basal bathing media, consistent with the view that these cells constitute a transcellular anion shunt. Moreover, voltage-activation resulted in an increased Br uptake from the apical bath. Nevertheless, because of the very small number of these cells, it may be questioned whether the mitochondria-rich cell constitutes the only shuntpathway for Cl. In other studies, Rb uptake was employed to measure the Na/K-pump activity. In principal cells of the frog skin epithelium, amiloride inhibited Rb uptake and lowered Na concentration, supporting the view that this cell type is engaged in amiloride-sensitive Na transport. In contrast, no significant changes in the Rb, Na, and Cl concentration of mitochondria-rich cells were detectable. Studies with Rb as marker ion in the rabbit urinary bladder revealed that the epithelium behaves like a functional syncytium with regard to transepithelial ion transport.

Effect of amiloride on electrolyte concentrations and rubidium uptake in principal and mitochondria-rich cells of frog skin.

The role of mitochondria-rich cells (MR cells) in transepithelial Na transport was investigated by determining electrolyte concentrations and Rb uptake in individual cells of frog skin epithelium using electron microprobe analysis. Measurements were performed under control conditions and after blocking the transepithelial Na transport with amiloride. Under control conditions, Na and Cl concentrations of MR cells scattered much more than those of principal cells and ranged from a few up to more than 30 mmol/kg wet weight. Rb uptake from the basal side into individual MR cells also showed a large variation and was, on the average, much less pronounced than into the principal cells. In principal cells, amiloride reduced the Na concentration and Rb accumulation. In contrast, no effect was observed upon electrolyte concentration and Rb uptake of MR cells. Rb uptake was correlated to the Na concentration of MR cells both under control conditions and after amiloride. It is concluded that, in contrast to the principal cells, MR cells are not involved in amiloride-sensitive transepithelial Na transport and that their Na/K-pump activity is very low.

Analysis of anion conductance in frog skin.

Electrophysiological characteristics of transepithelial Cl-specific conductance (gCl) and intracellular element concentrations were analyzed in frog skins before and during voltage perturbation to serosa +100 mV, both under control conditions and after mucosal application of procaine. Under control conditions, gCl was often minimal and almost insensitive to voltage perturbation. Procaine stimulated gCl in many cases considerably and further activation resulted then from voltage perturbation. Microelectrode determinations indicated that conductive pathways parallel to the principal cells account for the procaine-induced increase in gCl. The responses in gCl were not related to the density of mitochondria-rich (MR) cells. Electron microprobe analysis of intracellular electrolyte concentrations showed that procaine increased the Cl content of MR cells significantly. Gain of Cl was primarily due to uptake across the basolateral membrane, as indicated by the small accumulation of Br after unilateral mucosal application. Voltage perturbation to serosa +100 mV in the presence of Br on the mucosal side led in procaine-stimulated tissues to an increase of the ratio of Br/Cl content in the majority of MR cells. It was much less than predicted for conductive transcellular anion transport. Also, intracellular Cl concentrations of MR cells were far above those expected for a highly Cl-permeable basolateral membrane. The data, although indicating finite Cl/Br transport across MR cells, are incompatible with the idea that the voltage-activated conductive Cl transport occurs though these cells. Alternatively, we suggest passage across highly Cl-specific sites of a paracellular pathway.