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.

SUMMARY The use of propane/isopentane mixtures as cooling media is proposed since they remain fluid at liquid nitrogen temperature. The attainable cooling rates are virtually identical to those obtained in liquid propane.

During recent years our group has employed the technique of electron microprobe analysis to determine the electrolyte concentrations in various epithelial tissues. The specimen preparation is characterized by shock-freezing of small tissue pieces in liquid propane/isopentane mixtures at 77 K, cryosectioning of 1 pm thick serial sections at 170 K and subsequent freezedrying at 190 K andPa. The analysis of the frozen dried cryosections is performed in a scanning electron microscope which is equipped with an energy dispersive X-ray detector. The measuring conditions selected are 17-20 kV acceleration voltage and 0.1-05 nA probe current. For quantification, the cellular X-ray spectra are compared with those of an internal albumin standard layer. The evaluation of the characteristic X-ray intensities is performed using a computer program. Some critical points of this technique will be discussed.

The element concentrations in various intra- and extracellular compartments of the tip of the rat renal papilla were determined during antidiuresis using electron microprobe analysis. Urinary concentrations (means +/- SEM) were: urea, 1509 +/- 116; potassium, 268 +/- 32; sodium, 62 +/- 19 mmoles X 1(-1); and osmolality, 2548 +/- 141 mOsm X kg-1. Electrolyte concentrations in the interstitial space were: sodium, 437 +/- 19; chloride, 438 +/- 20; and potassium, 35 +/- 2 mmoles X kg-1 wet wt. The vasa recta plasma exhibited almost identical element concentrations. The values in the papillary collecting duct cells were: sodium, 28 +/- 1; chloride, 76 +/- 3; potassium, 135 +/- 3; and phosphorus, 316 +/- 7 mmoles X kg-1 wet wt. Similar concentrations were observed in the papillary epithelial cells. In interstitial cells potassium and phosphorus concentrations were virtually identical to those of the collecting duct cells, whereas sodium and chloride concentrations were higher by about 30 mmoles X kg-1 wet wt. The element composition of the various papillary cells is, thus, not substantially different from that of proximal tubular cells. This finding demonstrates that cellular accumulation of electrolytes is not the regulatory mechanism by which papillary cells adapt osmotically to their high environmental osmolality and sodium chloride concentration.

The concentrations of intracellular elements were determined by electron microprobe analysis in the nucleus and cytoplasm of freeze-dried cryosections of superficial proximal and distal tubules of the rat kidney. For the nucleus of the proximal tubular cell, the concentrations of sodium and chloride were 20 and 23 mmoles/kg of wet wt, and those of potassium and phosphorus were 144 and 150 mmoles/kg wet wt. For the nucleus of the distal tubular cell, the concentrations of sodium and chloride were significanlty lower (11 and 13 mmoles/kg wet wt), that of potassium was unchanged (143 mmoles), and that of phosphorus was significantly higher (175 mmoles). Towards the basolateral infoldings of the proximal and distal tubules and the brush border of the proximal tubules, the concentrations of sodium and chloride were higher and those of potassium and phosphorus were lower than those obtained in the nucleus, indicating the presence of extracellular compartments in these regions. Measurements performed in the centrally located cytoplasm of proximal and distal tubular cells, close to the nucleus, showed sodium and potassium concentrations to the indistinguishable from those in the nucleus, whereas chloride and phosphorus concentrations were considerably higher. These data demonstrate differences in the intracellular concentrations of phosphorus, sodium, and chloride between proximal and distal tubular cells. In neither the proximal nor the distal tubular cells, however, could a concentration difference for sodium and potassium between nucleus and cytoplasm be determined.