Electron microprobe analysis of frog skin epithelium: Evidence for a syncytial sodium transport compartment

Abstract

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.

Cellular composition and ultrastructure of the gill epithelium of larval and adult lampreys

“…Furthermore, studies on the cellular distributions of Na + /K + -ATPase in the toad and turtle urinary bladders and frog skin have shown that the vast majority of Na + pumps are localised in the granular cells and that the intercalated MR cell possesses only a few of these pumps. This distribution pattern is consistent with the model that the granular cell compartment is responsible for Na + uptake (Rick et al, 1978;Durham and Nagel, 1986;Nagel and Dörge, 1996) and suggests that the few Na + pumps present in the intercalated MR cells are sufficient to maintain the balance of the Na + and K + gradients across the basolateral membrane but do not significantly contribute to Na + uptake.…”

“…This mechanism is dependent on the activities of both cytosolic carbonic anhydrase in the H + secreting cell and Na + /K + -ATPase in the basolateral membrane of the cell whose apical membrane contains the ENac (Harvey and Ehrenfeld, 1986;Harvey et al, 1988;Nagel and Dörge, 1996;Ehrenfeld and Klein, 1997). Furthermore, the toad and turtle urinary bladders, in which the epithelium has a cellular composition comparable with that of amphibian skin and consists also of granular cells and mitochondria-rich (MR) cells (Wade et al, 1975;Wade, 1976;Rick et al, 1978;Durham and Nagel, 1986;Brown and Breton, 1996), employ essentially the same mechanisms for reabsorbing Na + from the urine and for secreting H + into that urine (Stetson and Steinmetz, 1985;Durham and Nagel, 1986;Lang, 1988). In addition, the immunocytochemical demonstration that H + -ATPase and ENac are present in the gill epithelium of teleosts (Sullivan et al, 1995;Wilson et al, 2000a;Marshall, 2002) is consistent with the hypothesis that this mechanism for taking up Na + from a hypotonic environment is universal amongst vertebrates and invertebrates such as crustaceans, annelids and molluscs (Kirschner, 1983).…”

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

“…The polarity is maintained by the tight junctions between cells of the outer S. granulosum, which also restricts diffusion between the outer solution and the intercellular spaces (Martlnez-Palomo, Erlij, and Bracho, 1971). These structural observations, together with the demonstration of the syncytial behavior of the epithelium (Rick et al, 1978) and the large capacitance of the basolateral membrane of the intact epithelium (Smith, 1971 ;Garcia-Dfaz and Essig, 1985;Schoen and Erlij, 1985) indicates that, functionally, all innermost cell membranes behave as basolateral membrane. This implies that the majority of isolated cells will exhibit macroscopic properties that correspond to the basolateral membrane of the intact epithelium and there would be a much higher probability of recording single channels of basolateral origin/ One can also make the argument that, since the apical membrane of R. pipiens skin does not exhibit any significant conductive permeability to ions other than Na (Helman and Fisher, 1977;Nagel, 1977;Garcia-Diaz et al, 1985;Stoddard, Jakobsson, and Helman, 1985;D6rge, Beck, Wienecke, and Rick, 1989) the K and CI selective channels found in this study are necessarily of basolateral origin.…”

“…Cells in this layer are joined by tight junctions (Farquhar and Palade, 1964) maintaining the epithelial polarity. Cells from all epithelial layers are interconnected by intercellular junctions, forming a syncytium (Farquhar and Palade, 1964;Rick, D6rge, Arnim, and Thurau, 1978). The inner membrane of the S. granulosum and all membranes of the innermost layers contain Na-K ATPase (Farquhar and Palade, 1966;Mills, Ernst, and DiBona, 1977) and, presumably, the K channels that confer high K selectivity to the basolateral membrane.…”

Life with Tracers

“…Some studies have given clear evidence for the coupling in frog skins (20) while others have indicated very limited coupling (33). Current work by our group (R. Nielsen and H. H. Ussing, in preparation) indicates that both views may be correct, depending on the experimental circumstances.…”