Roger Rick

Osmoregulation of renal papillary cells

Element concentrations were determined in various extra- and intracellular compartments of the rat renal papilla in antidiuresis and after furosemide-induced diuresis using electron microprobe analysis to elucidate further how the cells adapt osmotically to different osmolatities. In antidiuresis and diuresis the sum of intracellular cations (sodium and potassium), accompanying anions and urea was insufficient in both cases to provide cell osmolatities similar to those in extracellular compartments. This finding provides further evidence that the papillary cells achieve osmoadaptation to widely differing extracellular electrolyte concentrations mainly by varying the cellular concentrations of osmotically-active substances other than urea and electrolytes.

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.

Intracellular ion concentrations in the isolated frog skin epithelium: Evidence for different types of mitochondria-rich cells

SummaryIntracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Under control conditions, principal cells and mitochondria-rich cells (MR cells) had a similar intracellular ion composition, only the Cl concentration in MR cells was significantly lower. Inhibition of transepithelial Na transport by low concentrations of ouabain (2 × 10−6m, innerbath) resulted in a Na concentration increase of principal cells from 10.9 to 54.3 mmol/kg wet wt. The increase was completely abolished by simultaneous application of amiloride (10−4m, outer bath). Amiloride alone resulted in a significant decrease of the Na concentration to 6.1 mmol/kg. w. w. Among MR cells, two different groups of cells could be distinguished; cells that showed a Na increase after ouabain which was even larger than that in principal cells and cells that did not respond to ouabain. In about half of all ouabain-sensitive MR cells the Na increase could be prevented by amiloride. According to these results, a subpopulation of MR cells displays the transport characteristics expected for a transepithelial Na transport compartment, an apical amiloride-sensitive Na influx and abasal ouabain-inhibitable Na efflux. Given the small number of cells, however, it is unlikely that this subtype of MR cells contributes significantly to the overall rate of transepithelial Na transport.

Cl transport across the basolateral membrane in frog skin epithelium

Cellular Cl concentrations were determined by electron microprobe analysis to obtain further insight into the Cl transport across the basolateral membrane of the frog skin epithelium. Cl-free media on the serosal side led in all epithelial layers within 1 h to a decrease in cellular Cl concentration from about 40 to 15 mmol/kg wet wt, whereas the application of Cl-free solutions or amiloride to the apical side had no effect. Na-free media, furosemide or bumetanide on the serosal side had little effect on cellular Cl but abolished the Cl-reuptake into Cl-depleted cells. It is concluded that cellular Cl concentration is maintained above electrochemical equilibrium by a co-transport system, which is relatively silent under control conditions.

Differential effects of aldosterone and ADH on intracellular electrolytes in the toad urinary bladder epithelium

SummaryQuantitative electron microprobe analysis was employed to compare the effects of aldosterone and ADH on the intracellular electrolyte concentrations in the toad urinary bladder epithelium. The measurements were performed on thin freeze-dried cryosections utilizing energy dispersive x-ray microanalysis. After aldosterone, a statistically significant increase in the intracellular Na concentration was detectable in 8 out of 9 experiments. The mean Na concentration of granular cells increased from 8.9±1.3 to 13.2±2.2 mmol/kg wet wt. A significantly larger Na increase was observed after an equivalent stimulation of transepithelial Na transport by ADH. On average, the Na concentration in granular cells increased from 12.0±2.3 to 31.4±9.3 mmol/kg wet wt (5 experiments). We conclude from these results that aldosterone, in addition to its stimulatory effect on the apical Na influx, also exerts a stimulatory effect on the Na pump. Based on a significant reduction in the Cl concentration of granular cells, we discuss the possibility that the stimulation of the pump is mediated by an aldosterone-induced alkalinization.Similar though less pronounced concentration changes were observed in basal cells, suggesting that this cell type also participates in transepithelial Na transport. Measurements in mitochondria-rich cells provided no consistent results.

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.

Short-term bromide uptake in skins of Rana pipiens

Intracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Based on the 1 min Br uptake from the apical bath, two types of mitochondria-rich (MR) cells could be distinguished: active cells which rapidly exchanged their anions with the apical bath and inactive cells which did not. Br uptake and frequency of active MR cells were closely correlated with the skin conductance, gt. Replacing Cl in the apical bath with an impermeant anion significantly lowered gt and the Br uptake and Na concentration of active cells. Even larger reductions were observed after apical amiloride (0.1 mm). The inhibition of the Br uptake was reversible by voltage clamping (100 mV, inside positive). Cl removal and amiloride also led to some shrinkage of active cells. The results suggest that the active cell is responsible for a large part of gt. Inactive MR cells had much lower Br and Na concentrations which were not significantly affected by Cl removal, amiloride, or voltage clamping. Principal cells, which represent the main cell type of the epithelium, showed only a minimal Br uptake from the apical side which was not correlated with gt. Moreover, Cl removal had no effect on the Na, Br, and Cl concentrations of principal cells.

Na transport compartment in rabbit urinary bladder

Electron microprobe analysis was used to determine cellular electrolyte concentrations in rabbit urinary bladder. Under control conditions the mean cellular electrolyte concentrations were for Na 11.6±2.0, for K 124.1±15.3, and for Cl 26.0±5.1 mmol/kg wet weight. The dry weight content was 19.0±2.0 g/100 g. Inhibition of the Na/K-pump with ouabain resulted in drastic changes of the cellular element concentrations. Similar changes also occurred when in addition to ouabain the apical side was kept Na-free. In all epithelial layers the Na and Cl concentrations increased by 90 and 30 mmol/kg wet weight, whereas the K concentration and the dry weight content decreased by 90 mmol/kg wet weight and 6 g/100 g wet weight, respectively. With Na-free choline-Ringers solution on the basal side ouabain led to a decrease in the K concentration by about 60 mmol/kg wet weight while the Na and Cl concentrations remained unchanged. These data indicate that the basolateral membrane is permeable to Na, choline, Cl, and K. Nystatin produced drastic changes in the cellular electrolyte concentrations when Na- or Rb-sulfate Ringers solutions were present on the apical side. With Na-sulfate Ringers solution the Na concentration increased by about 25, the Cl concentration by 30 mmol/kg wet weight and the dry weight content decreased by 4.5 g/100 g, respectively. With Rb-Ringers solution about 20 mmol/kg wet weight of the cellular K was exchanged against Rb. The concentration changes were identical in all epithelial layers supporting the idea that the rabbit urinary bladder represents a functional syncytium with regard to the transepithelial Na 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.

Chapter 4 Electron Microprobe Analysis of Cell Sodium in Epithelia

Publisher Summary Originally designed for nonbiological materials (Castaing, 195 l), during recent years electron microprobe analysis has become widely used for biological specimens. Compared to other techniques for chemical analysis of intracellular ion concentrations, this method excels by allowing the simultaneous analysis of a large number of elements and by affording a very high spatial resolving power. Application of the technique to biological soft tissues is, however, not without problems. For the analysis of readily diffusible ions such as Na + and K + , perhaps the most important consideration is whether the specimen preparation preserves their original distribution. Conventional preparation techniques used for electron microscopy are certainly not appropriate. It is now generally accepted that the only method that satisfactorily maintains the original distribution of ions is fast-freezing, followed by cryosectioning and analysis in the frozen-hydrated state or, after additional freeze-drying, analysis in the frozen-dried state. The studies in the chapter are performed on thin, freeze-dried cryosections in a scanning transmission electron microscope using an energy dispersive X-ray detection system.