Cell K activity in frog skin in the presence and absence of cell current

Abstract

Cell K activity, acK, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30-60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO3 Ringer on the apical surface. Under control conditions acK was nearly constant among skins (mean +/- SD = 92 +/- 8 mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 microA/cm2). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase in acK after inhibition of cell current was observed, on the average the change was not significant (98 +/- 11 mM after amiloride, 101 +/- 12 mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from -42 to -77 mV, hyperpolarized after inhibition of cell current, initially to -109 +/- 5 mV, then depolarizing to a stable value (-88 +/- 5 mV) after 15-25 min. At this time K was above equilibrium (EK = 98 +/- 2 mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport. The measurement of acK permitted the calculation of the passive K current and pump current under control conditions, assuming a "constant current source" with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.

Voltage dependence of cellular current and conductances in frog skin

“…It is not possible, of course, completely to rule out errors, particularly because the complex protocols of the present study prevented the application of usual criteria to avoid microelectrode offset potentials. However, there is no basis for suspecting major bias; promptly after blockage of cell current with amiloride Va averaged -103 -+ 11 mV, in good agreement with the mean value of -109 -+ 5 mV in an earlier study in which it was possible to apply the most rigorous criteria for the exclusion of artifacts [4] . 4 At the reversal potential, the apical amiloride-sensitive current is zero.…”

“…However, there is no basis for suspecting major bias; promptly after blockage of cell current with amiloride Va averaged -103 -+ 11 mV, in good agreement with the mean value of -109 -+ 5 mV in an earlier study in which it was possible to apply the most rigorous criteria for the exclusion of artifacts [4] . 4 At the reversal potential, the apical amiloride-sensitive current is zero. If the apical membrane were perfectly selective for sodium, the absence of amiloride-sensitive current despite a finite amiloride-sensitive conductance would demonstrate that sodium is at thermodynamic equilibrium across the apical membrane.…”

“…The mucosal fluid was either NaCI Ringer or Ringer in which 110 mM NaCl was replaced by the Na salt of nitrate or gluconate. The latter solutions were employed if the tissues had excessive conductance which disappeared after replacement of mucosal chloride or addition of amiloride [4,14]. (We made no adjustments for the lowering of calcium activity in gluconate because we have noted no change in short-circuit current and membrane conductance even on lowering calcium to trace levels.)…”

“…They were advanced across the apical border into individual cells of the epithelium with the aid of a stepping motor micromanipulator (MF500, Biomedizinische Instrumente, Germering, FRG). Criteria for acceptable intracellular recordings have been described previously [4,11].…”

“…One technique often applied to this problem is the sequential determination of current/voltage (I/V) relationships, first under control conditions and then following abolition of transcellular transport by amiloride [1,18,21,23]. However, as previously reported [4,15], exposure to amiloride importantly affects paracellular conductance (particularly in the presence of apical CI solutions), complicating the interpretation of I/V relationships. We have characterized further the problems encountered using amiloride and found that, with the use of substitutes for apical C1 and brief intermittent exposure to amiloride at each setting of Vt, it was possible to minimize changes of paracellular conductance.…”