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The effects of and the interaction between local perivascular variation in potassium and bicarbonate concentration on the diameter of pial arteries were studied in cats by the microapplication technique. At 11 mEq/liter of bicarbonate, a direct correlation existed between vascular diameter and K +

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For elucidation of the functional organization of frog skin epithelium with regard to transepithelial Na transport, electrolyte concentrations in individual epithelial cells were determined by electron microprobe analysis. The measurements were performed on 1-micron thick freeze-dried cryosections by an energy-dispersive X-ray detecting system. Quantification of the electrolyte concentrations was achieved by comparing the X-ray intensities obtained in the cells with those of an internal albumin standard. The granular, spiny, and germinal cells, which constitute the various layers of the epithelium, showed an identical behavior of their Na and K concentrations under all experimental conditions. In the control, both sides of the skin bathed in frog Ringer's solution, the mean cellular concentrations (in mmole/kg wet wt) were 9 for Na and 118 for K. Almost no change in the cellular Na occurred when the inside bathing solution was replaced by a Na-free isotonic Ringer's solution, whereas replacing the outside solution by distilled water resulted in a decrease of Na to almost zero in all layers. Inhibition of the transepithelial Na transport by ouabain (10(-4) M) produced in increase in Na to 109 and a decrease in K to 16. The effect of ouabain on the cellular Na and K concentrations was completely cancelled when the Na influx from the outside was prevented, either by removing Na or adding amiloride (10(-4) M). When, after the action of ouabain, Na was removed from the outside bathing solution, the Na and K concentration in all layers returned to control values. The latter effect could be abolished by amiloride. The other cell types of the epithelium showed under some experimental conditions a different behavior. In the cornified cells and the light cells, which occurred occasionally in the stratum granulosum, the electrolyte concentrations approximated those of the outer bathing medium under all experimental conditions. In the mitochondria-rich cells, the Na influx after ouabain could not be prevented by adding amiloride. In the gland cells, only a small change in the Na and K concentrations could be detected after ouabain. The results of the present study are consistent with a two-barrier concept of transepithelial Na transport. The Na transport compartment comprises all living epithelial layers. Therefore, with the exception of some epithelial cell types, the from skin epithelium can be regarded as a functional syncytium for Na.

In antidiuresis, the cells of the renal medulla are exposed to high extracellular concentrations of NaCl and urea. Since urea equilibrates with the intracellular compartment and is known to perturb intracellular macromolecules, high urea concentrations may well disturb the structure and function of cell proteins. Two types of organic substances are believed to counteract the adverse effects of high intracellular urea concentrations: specific organic osmolytes of the trimethylamine family [betaine and glycerophosphorylcholine (GPC)], which accumulate in renal medullary cells during prolonged periods of antidiuresis and cytoprotective heat shock proteins (HSPs), the tissue content of two of which (HSPs 27 and 72) is much higher in the inner medulla than in the iso-osmotic renal cortex. To evaluate the contribution of trimethylamines and HSPs to cytoprotection in the presence of high urea concentrations, the effect of HSP induction and osmolyte accumulation prior to exposure to high urea concentrations was examined in Madin-Darby canine kidney (MDCK) cells. Accumulation of organic osmolytes and synthesis of HSP27 and HSP72 was initiated by hypertonic stress (increasing the osmolality of the medium from 290 to 600 mosmol/kg H2O by NaCl addition). Control, non-conditioned cells remained in the isotonic medium for the same period. Upon subsequent exposure to an additional 600 mM urea in the medium for 24 h, 90% of the osmotically conditioned cells but only 15% of non-conditioned cells survived. The HSP72 and trimethylamine contents of the NaCl-conditioned MDCK cells, but not HSP27 content, correlated positively with cell survival. To separate the effects of organic osmolytes and HSP72, chronically NaCl-adapted MDCK cells were returned to isotonic medium for 1 or 2 days, so depleting them of trimethylamine osmolytes. HSP72, with its longer half life, remained elevated. Subsequent exposure of these cells to 600 mM urea in the medium resulted in about 80% survival. These results suggest that in MDCK cells and probably in the renal medulla, HSP72 and perhaps additional protective factors contribute substantially to the resistance against high urea concentrations.

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The distribution of heat shock proteins (HSP) HSP60, HSP73, HSP72 and HSP25 in the isoosmotic cortex and the hyperosmotic medulla of the rat kidney was investigated using Western blot analysis and immunohistochemistry. HSP73 was homogeneously distributed throughout the whole kidney. The level of HSP60 was high in the renal cortex and low in the medulla. HSP25 and HSP72 were present in large amounts in the medulla. Only low levels of HSP25 and almost undetectable amounts of HSP72 were found in the cortex. HSP25 exists in one nonphosphorylated and several phosphorylated isoforms. Western blot analysis preceded by isoelectric focussing showed that HSP25 predominates in its nonphosphorylated form in the outer medulla but in its phosphorylated form in cortex and inner medulla. Although this intrarenal distribution pattern was not changed during prolonged anaesthesia (thiobutabarbital sodium), a shift from the nonphosphorylated to the phosphorylated isoforms of HSP25 occurred in the medulla. The characteristic intrarenal distribution of the constitutively expressed HSPs (HSP73, HSP60, HSP25) may reflect different states of metabolic activity in the isoosmotic (cortex) and hyperosmotic (medulla) zones of the kidney. The high content of inducible HSP72 in the medulla most likely is a consequence of the osmotic stress imposed upon the cells by the high urea and salt concentrations in the hyperosmotic medullary environment.