Microelectrode studies of the active Na transport pathway of frog skin.

Abstract

A B S T R A C T When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V0, was concurrent with the observations that (a) a high resistance outer barrier Ro accounting for approximately 75% or more of the transcellular resistance of control skins had been penetrated and that (b) 10 -5 M amiloride and reduced [Na] outside caused the values of V o to increase towards mean values near -130 mV while the values of %Ro increased to >90%. It was of interest to observe that the values of E1 observed in studies of the current-voltage relationships were the same as the values of E~ defined as the values of voltage at the inner barrier when the Vo of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of El, --130 mV, represent the E Na of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of Vo to low negative values in approximately 10-15 min. For all values of transepithelial voltage <E~ the V 0 was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.

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

“…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.…”

Micro‐electrode studies on the effects of exogenous cyclic adenosine monophosphate on active sodium transport in frog skin.

“…We have accepted, as before, that E. is not significantly different from zero (Helman & Fisher, 1977; and the outer barrier may be modelled by a simple Thevenin slope conductance or resistance, Ro. The Thevenin electromotive force for transepithelial sodium transport in frog skin, 540 (ENa), as originally defined by Ussing & Zerahn (1951), could be estimated directly from the values of E' as previously described (Helman & Fisher, 1977). Briefly, with a micro-electrode in position, values for El were determined by hyperpolarizing the tissues to values of VT which caused VO to equal zero.…”

“…Studies were done with isolated abdominal skins of the African frog Rana angolersis mounted horizontally in a specially designed Lucite chamber for micro-electrode studies (Helman & Fisher, 1977). Both the mucosal and serosal surfaces of the skins were continuously perfused, from reservoirs, with a C1--HC03-Ringer solution containing 100 mM-NaCl, 2-4 mM-KHCO3, 2-0 mmCaCl2 and 11 1-mM glucose (pH = 8-1-8-2) which was aerated with 100% 02 immediately before use.…”

“…The technique used was essentially the same as originally described by Helman & Fisher (1977). In order to monitor the individual changes occurring at the outer and inner barriers of the transporting cells, skins were impaled vertically from above with the micro-electrode in the outer solution.…”

“…These and other similar studies were done mainly by recording the responses of the short-circuit current (ISC) to applied cyclic AMP and could not tell us much about the mechanism whereby cyclic AMP elicited its effects. We chose to investigate the effects of cyclic AMP on active transepithelial sodium transport in frog skin with intracellular micro-electrodes and analysed our data according to an electrical model ofthe transport pathway as proposed by Helman & Fisher (1977). This allowed us to assess in more detail the mechanism by which cyclic AMP might modify the individual electrical characteristics of apical and basolateral membranes of the sodium-transporting cells of frog skin.…”