The osmotic behaviour of toad skin epithelium (Bufo viridis)

The effect of saline adaptation on the intracellular Na, K, Cl, P concentrations and dry weight content of the toad skin epithelium (Bufo viridis) was studied using the technique of electron microprobe analysis. The measurements were performed on isolated abdominal skins either directly after dissection or after additional incubation in Ussing-type chambers.Adaptations of the toads to increasing NaCl concentrations for 7 days resulted in increased blood plasma osmolarity and a parallel increase in the cellular electrolyte, P and dry weight concentrations of the epithelium, the K increase representing the most significant fraction of the intracellular osmolarity increase. No evidence was obtained to show that the nucleus and cytoplasm reacted differently from each other and all living epithelial cell types basically showed the same response.Incubation of the isolated skins under control conditions showed a drastic inhibition of the transepithelial Na transport after adaptation to high salinities. In spite of the large variations in the transport rate almost identical intracellular electrolyte concentrations were observed. In tap water adapted toads the average cellular concentrations were 8.8 mmole/kg wet weight for Na, 109.6 for K, 41.5 for Cl, and 135.3 for P, respectively. Incubation of the skin with Ringer's solution of different osmolarities demonstrated that the epithelial cells are in osmotic equilibrium with the inner bathing solution. The results are consistent with the view that the osmotic adaptation is mainly accomplished by the movement of water.This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk     

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.     

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. Received: 29 February 1996 / Received after revision: 23 June 1996 / Accepted 9 September 1996     

Effects of environmental conditions on mitochondrial-rich cell density and chloride transport in toad skin

Chloride flux across amphibian skin is usually passive, yet largely conductive; previous reports have suggested that aldosterone influences this pathway. The conductive Cl^– pathway and its regulation were examined further, across the abdominal skin of toads (Bufo marinus) adapted to various environments. Short-circuit current (I _sc), total conductance (G _t) and Cl^– influx (J _Cl) were measured in conditions such that there was net Cl^– movement in absence of Na⁺ transport. In salt-deprived animals compared to salt-adapted ones, there was a significant increase in J _Cl (563 vs 200 pmol cm^–2 s^–1), aldosteronaemia (4.2 vs 1.1 nmol/l), as well as MRC density (1458 vs 851 mm^–2). After adaptation to dilute Na₂SO₄ compared to MgCl₂, J _Cl (631 vs 313 pmol cm^–2 s^–1) as well as the density of mitochondria-rich cells (MRC) (1306 vs 710 mm^–2) practically doubled, while the toads' aldosteronaemia was lower (2.4 vs 10.8 nmol/1). In all groups of toads, J _Cl was matched by I _sc, and there was a close correlation between G _t and J _Cl (r=0.96), which confirms the conductive nature of transepithelial Cl^– movement. Furthermore, the relationship between J _Cl and MRC density (r=0.75) argues in favour of a role played by MRC on Cl^– conductance of epithelia such as amphibian skin. As aldosterone injected for 1 week into NaCl-adapted toads did not influence MRC density and as aldosteronaemia was not correlated with Cl^– conductance, this hormone does not emerge as the determinant of these parameters.     

Localization of transport compartments in turtle urinary bladder

To characterize different transport compartments in the urinary bladder epithelium of postabsorptive turtles, the electrolyte composition of individual cells was determined using electron microprobe analysis. After blocking the transepithelial Na transport, the short-circuit current decreased from positive to negative values (from 26.5±17.7 to –3.9±2.9 after ouabain and from 25.4±17.2 to –8.0±5.1 A/cm² after amiloride). Whereas under control conditions the Na and K concentrations were similar in all cell types and the same was true for Cl in most of the cells, some cells exhibited very low Cl concentrations. The epithelial cells were subdivided according to their electrolyte composition into ouabain-sensitive and ouabain-insensitive ones. In the ouabain-sensitive cells, which made up the majority of epithelial cells and showed a relatively high Cl concentration (about 36 mmol/kg wet weight), the Na concentration increased after ouabain by about 90 mmol/kg wet weight and the K concentration decreased by a similar amount. Since these alterations could largely be prevented when amiloride was applied before ouabain, it is suggested that the granular and basal cells form a syncytial Na transport compartment similar to that in other multilayered epithelia. The ouabain-insensitive cells, in which almost no alteration in Na and K concentrations was observed after ouabain, were subdivided into a Cl-rich (34.6±7.6 mmol/kg wet weight) and a Cl-poor (12.0±5.6 mmol/kg wet weight) population. Since in these cells no large mucin granules were detectable, they are regarded as carbonic anhydrase-rich cells involved in H and HCO₃ transport.     

Comparative roles of voltage and Cl ions upon activation of a Cl conductive pathway in toad skin

(1) Combined use of external Cl concentration pulses and apical membrane depolarization permitted to compare the roles of apical voltage and Cl ions upon the activation of a skin Cl conductance,G _Cl, which is assumed to reflect activation of the permeability of a Cl pathway. (2) Apical membrane depolarization induced by skin hyperpolarization, or by short-circuiting skins with high K Ringer's on the inner side, failed to activateG _Cl in the absence of external Cl,G _Cl remaining negligible. Under apical membrane depolarization, a step elevation of [Cl]₀ slowly activatedG _Cl as characterized by a sigmoidal current response of slow onset concomitant to a slow conductance increase. External Cl removal had the reverse effect, showly inactivatingG _Cl. (3) With the apical membrane in the normal polarized state, a step increase of [Cl]₀ slowly activatedG _Cl to submaximal values. This indicates that the interaction of Cl ions with the apical membrane partially activatesG _Cl in the absence of apical membrane depolarization. (4) Activation ofG _Cl was interpreted on the basis of a direct effect of Cl ions upon the apical membrane, having been attributed to the apical membrane voltage an indirect role. Voltage would affect the Cl distribution across the apical membrane, and, as a result, the Cl concentration at a proposed regulatory site which modulates the apical membrane permeability to Cl ions.     

Analysis of anion conductance in frog skin

Electrophysiological characteristics of transepithelial Cl-specific conductance (g _Cl) 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, g _Cl was often minimal and almost insensitive to voltage perturbation. Procaine stimulated g _Cl 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 g _Cl. The responses in g _Cl 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 Clspecific sites of a paracellular pathway.     

Intracellular electrolyte concentrations in rat sympathetic neurones measured with an electron microprobe

Intracellular element concentrations were measured in rat sympathetic neurones using energy dispersive electron microprobe analysis. The resting intracellular concentrations of sodium potassium and chloride measured in ganglia maintained for about 90 min in vitro at 25° C were 3, 155 and 25 mmol/kg total tissue wet weight respectively. Recalculated in mmol/l cell water, these values are 5, 196 and 32 respectively. There were no significant differences between the nuclear and cytoplasmic values of these ions. Incubation in either carbachol (108 mol/l, 4 min) or ouabain (1 mmol/l, 60 min) significantly increased the intracellular sodium and decreased the intracellular potassium concentrations. Neither substance materially altered the intracellular chloride concentration. The data obtained are compared and contrasted to those obtained in mammalian sympathetic neurones using chemical analysis and ion-sensitive microelectrodes.     

Sodium entry routes in principal and intercalated cells of the isolated perfused cortical collecting duct

Transmembrane sodium transport pathways were studied in principal and intercalated cells of the isolated perfused rabbit cortical collecting duct. Intracellular electrolyte concentrations in individual collecting duct cells were measured by electron microprobe analysis during blockage of basolateral Na-K-ATPase by ouabain and simultaneous inhibition of sodium entry across the apical and/or basolateral cell membrane. In principal cells the ouabain-induced rise in cell sodium concentration could only partially be blocked by amiloride (10^–4mol/l) in the perfusion fluid. Amiloride (10^–3mol/l) added to the bathing solution produced a further, significant reduction of sodium influx. In principal cells the ouabain-induced increase in sodium concentration was completely prevented by amiloride in the perfusion solution in combination with omission of sodium from the peritubular bathing solution. In intercalated cells ouabain caused a less pronounced increase in sodium concentration than in principal cells. Neither amiloride in the perfusate, nor amiloride in both bathing and perfusion solution, significantly reduced the ouabain-induced rise in intercalated cell sodium concentration. These results indicate that in principal cells amiloride-sensitive sodium channels constitute the predominant pathway for sodium entry across the apical cell membrane. In addition, substantial amounts of sodium enter principal cells across the basolateral cell membrane, probably via Na-H exchange. Finally, the data suggest that in intercalated cells sodium channels and the Na-H exchange are sparse or even absent.     

Effect of acute metabolic acidosis on transmembrane electrolyte gradients in individual renal tubule cells

We studied the effect of acute metabolic acidosis on potassium, sodium and chloride gradients across the apical membrane of proximal and distal tubule cells by determining electrolyte concentrations in individual cells and in tubule fluid employing electron microprobe analysis. Cellular measurements were performed on freeze-dried cryosections of the renal cortex, analysis of tubule fluid electrolyte concentrations on freeze-dried microdroplets of micropuncture samples obtained from proximal and from early and late distal collection sites. Acidosis (NH₄Cl i.v. and i.g.) induced a substantial rise in plasma potassium concentration without significant effects on cell potassium concentrations. Potassium concentrations along the surface distal tubule were also unaltered; thus the chemical driving force for potassium exit from cell to lumen was not affected by acidosis. In all but intercalated cells acidosis markedly increased cell phosphorus concentration and cell dry weight indicating cell shrinkage and thus diminution of cell potassium content. Because the increase in intracellular chloride concentration exceeded the increase in plasma chloride concentration, the chemical chloride gradient across the contraluminal membrane was markedly depressed by acidosis.     

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.     

The key role of the mitochondria-rich cell in Na+ and H+ transport across the frog skin epithelium

We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na⁺ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3–4 days or 7–10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na₂SO₄ (25 mmol/l) solutions modified the Na⁺ transport rate and the morphology of the epithelium. The highest Na⁺ transport rates were found for the animals adapted to the Na⁺ free solutions and were correlated with an increase in the total MR cell pit-surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO₂ increase and HCO ₃ ^– decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10^–6 M) or during acidification of the epithelium by serosally applied CO₂. Na⁺ transport was enhanced by AVT (10^–6 M) or oxytocin (100mU/ml) when the skin was bathed on its apical side with a high Na⁺ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na⁺ transport when the apical solution was low in Na⁺ (0.5mmol/l). It is concluded that MR cells play a key role in Na⁺ and H⁺ transport through the frog skin epithelium when bathed on its apical side with a low Na⁺ containing solution. Distinct pathways for sodium transport through two cell types (MR cells and granular cells) are proposed depending on the Na⁺ concentration of the solution bathing the apical side of the epithelium.     

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 μmol·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. Received: 4 August 1997 / Received after revision: 12 November 1997 / Accepted: 4 December 1997     

Correlation between chloride flux via the mitochondria-rich cells and transepithelial water movement in isolated frog skin (Rana esculenta)

The coupling between net transepithelial Cl- influx and net water flow was investigated. Experiments were performed on isolated frog skin bathed in isotonic Cl- Ringer's solution in the presence of the Na⁺ channel blocking agent amiloride in the mucosal solution. The skins were voltage-clamped at -80 or - 100 mV (with the serosal solution as reference). Under these conditions the current across the skin is carried by an influx of Cl-. In the absence of antidiuretic hormone the correlation between current and net water flow was low, but in the presence of the antidiuretic hormone, arginine vasotocin, there was a highly significant correlation between current and net water flow. The data presented here indicate that under steady state conditions about 70 molecules of water follow each Cl- ion across the skin. If the water influx is driven by electroosmosis one would expect that a change in current should result in an immediate change in the water flow. There was, however, a considerable time delay between the change in current and water flow. This indicates that the observed coupling between Cl- flux and water flow is caused by current-induced local osmosis and not electroosmosis.     

Opposite effects of indacrinone (MK-196) on sodium and chloride conductance of amphibian skin

Indacrinone, a drug chemically related to ethacrynic acid, usually stimulated reversibly short circuit current and sodium influx when applied to the epithelial surface of amphibian skin. Concomitantly, transepithelial conductance,g _t, decreased, provided chloride was the main anion in the incubation fluid. Electrophysiological analysis including microelectrode impalement indicated that the drug increased the sodium-conductance at the apical border of the impaled (most likely granular) cells. The decrease ing _t thus points at shunt conductance being reduced with indacrinone, sometimes drastically. Decrease of transepithelial chloride flux with the drug as well as lack of effect of the drug ong _t in the absence of chloride on the epithelial side demonstrate the influence of indacrinone on a chloride specific pathway. Whether this is along a paracellular route or through a cellular compartment not coupled to granular cells (mitochondria-rich cells?) cannot be decided on the basis of the present data.     

Enzymatic changes in mitochondria-rich cells of Xenopus laevis skin epithelium are induced by ionic acclimation

Morphological, biochemical and histochemical components of mitochondria-rich (MR) cells of skin epithelium of Xenopus laevis (Daudin) were investigated after acclimation in distilled water (DW) and mild solutions (50 mmol/l) of either NaCl or KCl for over 10 days. The animals readily acclimated to NaCl, but approximately 50% of the animals died in the KCl solution. Electrophysiological measurements confirmed the poor transport properties of skin in all conditions. Silver staining and exposure to methylene blue (MB) have shown that two types of MR cells can be distinguished, especially after KCl acclimation. Immunohistochemistry with the use of anti-band 3 polyclonal and anti H+-ATPase monoclonal antibodies demonstrated that band 3 and H+-ATPase enzymes were localized in MR cells in all conditions. H+-ATPase was greatly reduced during NaCl acclimation as verified with SDS gel electrophoresis. Intensity of the immunohistochemical staining differed between the various conditions of acclimation. Histochemical localization of carbonic anhydrase and alkaline phosphatase activities was more intense during NaCl acclimation. Morphological changes were also observed between the various acclimation conditions. The present findings substantiate the existence of at least two forms of MR cells in Xenopus skin epithelium but their functional significance remains to be established.     

Preparation of freeze-dried cryosections for quantitative X-ray microanalysis of electrolytes in biological soft tissues

Summary A procedure is described which allows the evaluation of wet weight concentrations of diffusible substances in biological soft tissue on a cellular level by the use of energy dispersive X-ray microanalysis. Epithelia of frog skin and toad urinary bladder were used to prepare freeze-dried cryosections without the use of chemical fixatives, cryoprotectants, floating solutions or coating materials.The specimens were shock-frozen inl-propane (–180°C), cryosectioned (–80°C), sandwiched between collodion films and freeze-dried (–80°C). The analysis was performed in a scanning electron microscope at an acceleration voltage of 15 kV, probe current of 0.5 nA, using scanning areas of 1–2 m². The spatial resolution power using 1–2 m thick sections was about 0.7 m. In a superficial layer of about 30 m the analysis was found not to be influenced by tissue damage due to ice crystal formation. The mass loss during electron bombardement was restricted to constituents of the organic matrix (30%). No changes of characteristic radiation were observed for Na, K and Cl. Albumin standards, containing electrolyte concentrations in the range of biological interest, revealed linear calibration curves. To obtain reliable wet weight concentrations, the characteristic X-rays of the tissue were compared to those of an internal standard which was applied to the specimen prior to freezing and analysed simultaneously.Supported by grants from the Deutsche Forschungsgemeinschaft     

The distribution of potassium,sodium and chloride across the apical membrane of renal tubular cells: effect of acute metabolic alkalosis

Studies were undertaken to define the effect of acute metabolic alkalosis (hypertonic sodium bicarbonate i.v.) on the chemical gradients for potassium, sodium and chloride across the apical membrane of individual renal tubule cells. Electron microprobe analysis was used on freeze-dried cryosections of the rat renal cortex to measure electrolyte concentrations in proximal tubule cells and in the various cell types of the superficial distal tubule. Analyses were also performed in fluid samples obtained by micropuncture from proximal and early and late distal collection sites. Compared with the appropriate controls (hypertonic sodium chloride i.v.), administration of sodium bicarbonate resulted only in small and mostly insignificant increases in cell potassium concentrations and induced only minor alterations in the cell/tubule fluid potassium concentration gradient for all cell types analysed. This observation suggests that under this condition factors other than an increase in cell potassium concentration are important in modulating potassium transfer across the apical membrane of potassium secreting cells. Nevertheless, since in alkalosis phosphorus and cell dry weight were decreased, and hence cell volume increased, in all but the intercalated cells, actually the potassium content of most tubular cells was higher under this condition. In comparison with animals infused with isotonic saline at low rates (hydropenic controls), infusion of either hypertonic sodium chloride or sodium bicarbonate led to a sharp increase in distal tubule fluid sodium concentrations and in the sodium concentrations of distal convoluted tubule, connecting tubule and principal cells, indicating that under both conditions the primary event causing enhanced transepithelial sodium absorption is stimulation of the sodium entry step. The ensuing rise in cell sodium concentration shold lead secondarily to stimulation of active basolateral sodium extrusion. Intercalated cell sodium concentration was higher only in alkalosis which supports the notion that this cell type is not involved in transepithelial sodium transport.     

Effect of potassium adaptation on the distribution of potassium,sodium and chloride across the apical membrane of renal tubular cells

To assess the effect of K adaptation on the electrolyte concentrations of renal tubular cells and on the concentration gradients across the luminal membrane, electron microprobe analysis was employed on freeze-dried cryosections of the renal cortex and on freeze-dried samples of tubular fluid in control and high-K rats. The measurements were performed in individual cells of the proximal and superficial distal tubule and on samples of tubular fluid obtained by free flow micropuncture from proximal and early and late distal collection sites. The ingestion of a potassium-rich diet for at least 10 days together with an acute potassium load of 0.4 mmol/kg/h led to a small increase in potassium concentration of about 7 mmol/kg wet weight (w.w.) in all cell types analysed. In distal convoluted tubule, connecting tubule and principal cells sodium concentration was markedly decreased by 4, 4, and 6 mmol/kg w.w., respectively, while no significant changes in sodium concentration were found in proximal tubule and intercalated cells. No consistent changes in cell chloride could be observed under K adaptation. Analysis of the tubular fluid samples showed that the K concentration gradient across the apical cell membrane of all distal tubular cell types investigated was diminished in the high-K rats. The concentration gradient for sodium entry, however, was clearly enhanced in the distal convoluted tubule, connecting tubule and principal cells. These data suggest that an increment in cell potassium concentration is not a major functional determinant for the increased distal K secretion observed in high-K rats and that the enhanced distal sodium absorption under this condition may be due to a stimulation of the Na exit step across the basolateral cell membrane in excess of the luminal entry step in most distal tubular cell types.     

Proximal tubular cell sodium concentration in early diabetic nephropathy assessed by electron microprobe analysis

Electron microprobe X-ray analysis techniques were employed in order to assess the changes that occur in proximal tubular cell sodium concentration during the hyperfiltration phase of early diabetes mellitus induced by streptozotocin in Sprague Dawley rats. Intracellular rubidium accumulation following intravenous infusion of rubidium chloride was used as a marker of basolateral Na/K-ATPase activity. The diabetic animals studied had a significantly higher glomerular filtration rate compared with controls [1.44±0.07 vs. 1.00±0.07 ml min^–1 (100 g body weight)^–1; mean±SEM, P<0.001]. Intracellular Na concentration was significantly higher in diabetic animals (19.5±0.6 vs. 17.8±0.4 mmol/kg wet weight; P<0.01). Concurrent measurement of Rb demonstrated significantly higher intracellular accumulation in the proximal tubules of diabetic animals compared with control (7.9±0.5 vs. 5.5±0.5 mmol/kg wet weight; P<0.001). These results indicate that proximal tubular Na/K-ATPase activity is enhanced in the hyperfiltration phase of diabetes mellitus. Since, however, intracellular Na concentration is increased under these conditions, it may be inferred that apical Na entry into proximal tubular cells is stimulated beyond the rate of basal exit during the initial development of hyperfiltration. The reasons for these alterations in cellular Na transport are unclear but similar changes have been implicated in the pathogenesis of cell growth.