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Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage ( I/ V) and conductance/voltage ( g/ V) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuring I and g before and after brief (<10 sec) exposure to amiloride at each setting of V. I/V relationships were characterized by these means in frog skins ( Rana pipiens, Northern variety, and Rana temporaria). Cellular current, I, decreased with hyperpolarization (larger serosa positive clamps) of V. Derived I/ V relationships between V=0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance, g, remained finite, it was possible to demonstrate reversal of I. Values of the reversal potential V' averaged 156±14 ( sd, n=18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances, g and g. The apical conductance decreased monotonically with increasing positivity of V (and V). In contrast, in the range in which the basolateral conductance could be evaluated adequately ( V<125 mV), g increased with more positive values of V (and V). That is, there was an inverse relation between g and cellular current at the quasi-steady state, 10-30 sec after the transepithelial voltage step. [ABSTRACT FROM AUTHOR]

In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltage Vt clamped (except for brief perturbations delta Vt) at zero. The skins were impaled from the outer surface with 1.5 M KCl-filled microelectrodes (Rel greater than 30 M omega). The transepithelial (short-circuit) current It and conductance gt = -delta It/delta Vt, the outer membrane voltage Vo (apical reference) and voltage-divider ratio (Fo = delta Vo/delta Vt), and the microelectrode resistance Rel were recorded continuously. Intermittent brief apical exposure to 20 microM amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values of gt and Fo before and after amiloride or as the ratio of changes (-delta Ic/delta Vi) induced by amiloride. On serosal substitution of Na by K, within about 10 min, Ic declined and gt increased markedly, mainly as a consequence of increase in gp. The basolateral membrane voltage Vi (= -Vo) was depolarized from 75 +/- 4 to 2 +/- 1 mV [mean +/- SEM (n = 6)], and was partially repolarized following amiloride to 5 +/- 2 mV.(ABSTRACT TRUNCATED AT 250 WORDS) [ABSTRACT FROM AUTHOR]

The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringer's the intracellular potential difference Vsc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25 mM or exposure to 2 mM BaCl2 (K+ channel blocker). With Ba2+ the depolarization of the Vsc by 25 mM K+ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistance Ro remained unchanged, the relative basolateral membrane resistance Ri, which includes the lateral intercellular space, increased at the most by less than twofold after Ba2+. These effects in conjunction with the depolarization of the Vsc by 62 mV after increasing the stromal-side K+ from 2.5 to 100 mM in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a = Ro/Ri) from 13 to 32 are all indicative of an appreciable basolateral membrane K+ conductance. This ratio decreased significantly after exposure to either 25 mM K+ or Ba2+. The decline of Ro/Ri with 25 mM K+ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25 mM K+, but rather perhaps a larger decrease of Ro than Ri. Also an increase of lateral space resistance may offset the effect of decreasing Ri with 25 mM K+. In contrast, Ro/Ri did transiently increase during voltage clamping of the apical membrane potential difference Vo and exposure to 25 mM K+ on the stromal side.(ABSTRACT TRUNCATED AT 250 WORDS) [ABSTRACT FROM AUTHOR]

Previous studies in anuran epithelia have shown that, after clamping the transepithelial voltage in symmetrical sequences for 4-6 min there is near-constancy of the rate of active Na transport and the associated oxidative metabolism, with a near-linear potential dependence of both. Here we have investigated in frog skin the cellular electrophysiolgical events associated with voltage clamping (Vt = inside-outside potential). Increase and decrease of Vt produced converse effects, related directly to the magnitude of Vt. Hyperpolarization resulted in prompt decrease in inward transepithelial current It and increase in fractional outer membrane resistance fRo (as evaluated from small transient voltage perturbations) and in outer membrane potential Vo. Overshoot of Vo was followed by relaxation to a quasi-steady state in minutes. Changes in fRo were progressive, with half times of some 1-5 sec. Changes in transepithelial slope conductance gt were more variable, usually preventing precise evaluation of the outer and inner cell membrane conductances go and gi. Nevertheless, it was shown that go is related inversely to Vt and Vo. Presuming insensitivity of gi to Vt, the dependence of Go on Vo in the steady state much exceeds that predicted by the constant field equation. Apparent inconsistencies with earlier results of others may be attributable to differences in protocol and the complex dependence of go on Vo and/or cellular-current. In contrast to previous findings in tight epithelia at open circuit, differences in Vt were associated with substantial differences in fRo and inner membrane potential Vi. Hyperpolarization of Vt over ranges commonly employed in studies of active transport ad metabolism appears to increase significantly the electrochemical work per Na ion transported. [ABSTRACT FROM AUTHOR]

Microelectrodes were used to investigate the effect of 0.5 mm mucosal lanthanum (La) on the intracellular potential and the resistance of outer and inner isolated frog skin ( Rana esculenta) cell membranes. Under short-circuit conditions, the transapical membrane potential V (mean value=−65.4±3.2 mV, inside negative) hyperpolarized to −108.7±2.3 mV in control skins, after addition of the sodium blocker amiloride. Current-voltage curves for the outer and inner membranes were constructed from the amiloride-inhibitable current versus the outer membrane potential V or the inner membrane potential V. The outer, and to a lesser degree the inner, membrane showed a characteristic nonlinearity with two slope resistances. Addition of La to the outer medium increased the short-circuit current to 190% of the control value. V concomitantly changed to −28±3.5 mV and outer and inner membrane resistances fell, considerably attenuating the nonlinearity seen in control skins. La is suggested to raise the conductance by its effect on the surface potential. A secondary long-term inhibitory effect of La on short-circuit current has been observed. It is ascribed to the penetration of La into the sodium channels. [ABSTRACT FROM AUTHOR]

Intracellular Na+, K+, and Cl- activities (aiNa, aiK, aiCl) and transapical membrane potentials (V0) were measured with liquid ion-exchanger and open-tip microelectrodes in isolated short-circuited frog skins (R. pipiens) incubated at 23 degrees C in normal amphibian Ringer's solution. Under control conditions aiNa = 14 +/- 3 mM, aiK = 132 +/- 10 mM and aiCl = 18 +/- 3 mM (SD). The value of aiCl is 4.4 times the value corresponding to electrochemical equilibrium for this ion. Thus, Cl- is actively accumulated by epithelial cells of the frog skin. Shortly after addition of amiloride (2--5 microM) to the apical bathing medium, aiK, aiNa, and aiCl were essentially unchanged although V0 had hyperpolarized by about 30--40 mV. During long-term exposure to amiloride aiK and aiCl did not change significantly, V0 depolarized by about 16 mV from the maximal value and aiNa decreased to 8 +/- 3 mM. Immediately after exposure to amiloride the transmembrane driving force for Na+ increased from 124 to 154 mV. During further exposure to amiloride, despite changes in both V0 and aiNa, this driving force remained virtually constant. Since Isc during this period was close to zero, it is suggested that the observed driving force for Na+ under these condition approximates the maximal driving force generated by the Na+--K+ ATP-ase pump in the basolateral cell membrane. [ABSTRACT FROM AUTHOR]

Skins from Rana temporaria, investigated with microelectrode techniques in the absence of Na uptake across the outer border (Na-free epithelial solution or amiloride), were found to be permeable to K+ at the apical membrane in 10-20% of the experiments. Full development of the K+-permeable state requires the absence of Na+ uptake for certain periods of time, which suggests that the K+-permeability of the apical membrane is higher at lower intracellular [Na]. The addition of Ba++ reduces the K+-permeability of the apical membrane. These skins may provide a model for the study of transcellular K+ movements. [ABSTRACT FROM AUTHOR]

The unidirectional Na influx j12, and Na efflux, j12, at the epithelial surface of the frog skin were determined under various experimental conditions. The j21 was taken as the difference between j12 and the simultaneously measured shortcircuit current (SCC). Errors in j12 determination originating from various transport rates within the skin were kept to a minimum using a normalization procedure. Under control conditions, j12 (1.20 muEquiv/cm-2. hr) was found to be only slightly larger than the SCC (1.10 MUEquiv/cm-2. hr). After inhibition of the transepithelial Na transport by amiloride, ouabain, low temperature and low Na concentration, the reduction of j12 and SCC was almost identical, indicating that the entrance of Na into the epithelium is rate limiting for the transepithelial transport. Compared to the control, j21 remained unchanged after amiloride and ouabain, but was insignificantly reduced at low temperature and significantly reduced at low Na concentration. These data are consistent with the assumption that the Na efflux follows mainly an extracellular pathway.

The effect of Li upon the intracellular potential of frog skin (Rana esculenta) was investigated. In the range between 1 and 25 mM Li in the epithelial bathing solution, a semilogarithmic linear relationship between [Li] and intracellular potential under short circuit conditions was obtained. The intracellular potential at all [Li] is quantitatively sufficient to explain the previously reported accumulation of Li in the intracellular space of the frog skin epithelium (Leblanc, G. 1972. Pfluegers Arch. 337:1) on the basis of a passibe entrance step at the outer border. A reduction of the intracellular potential by Li is also observed in the presence of 6 mM Na in the epithelial bathing solution. Consequences regarding the mechanism of uptake of Na across the outer border of the frog skin are discussed.

The effect of ADH upon the intracellular potential and the resistance of inner and outer borders of the transport pathway was investigated on isolated skins of Rana temporaria. Within 40 min after ADH (100--300 mU/ml), the intracellular potential under short-circuit conditions decreased to about 40% of the control value (--79 +/- 4 mV), concomitant with an increase in the short-circuit current to about 160% of the control value. Amiloride, applied when steady values under ADH had been reached, caused an immediate rise of the intracellular potential to values typical for control conditions. This confirms (i) the intracellular location of the microelectrode and the absence of impalement artifacts, and (ii) the ineffectiveness of ADH upon the electromotive forces of the inner border. ADH had no effect upon the intracellular potential after blockage of the Na entry by Amiloride. The equilibrium potential of the outer border was estimated to be about +20mV under the influence of ADH. As this value is considerably less positive than might be expected for the chemical potential of Na, a significant contribution of ions other than Na to the outer border conductance and equilibrium potential is implicated. The resistance of the outer border was more significantly decreased than that of the active transcellular pathway after ADH due to an increase in the inner border resistance, which exceeded that of the outer border after ADH. The effect of ADH upon the outer membrane characteristics would be underestimated by a factor of two, if the alterations of the electrical potential difference were not taken into consideration.

Skins from the frog Rana pipiens pipiens were studied under short-circuited conditions during the course of removing and replacing potassium in the inner bathing media in 14 experiments. The intracellular potential (Vsc), fractional resistance (FR), short-circuit current (Isc) and total tissue conductance (gr) were constantly monitored during impalements of the epithelial cells. The mean value (+/- SE) for Vsc was --79 (+/- 3) mV under baseline conditions. Removal of potassium from the inner bathing solution transiently stimulated the short-circuit current and hyperpolarized the basolateral membrane; with sufficiently long incubations, the basolateral membrane was eventually depolarized. Restoration of potassium to the inner solution within 43 min after initiating the perfusion with K+-free solution depolarized the basolateral membrane. However, restoration of potassium after at least 1 1/2 hr of incubation hyperpolarized the membrane. Ouabain consistently depolarized the basolateral membrane, even after extended periods of potassium depletion as long as 320 min. In the presence of ouabain, restoration of potassium depolarized the basolateral membrane. The data provide further evidence for the concept that the Na--K exchange pump of frog skin is rheogenic. Furthermore, the results suggest that the pump continues to be active even during prolonged periods of potassium depletion, reaccumulating potassium which has leaked out of the epithelial cells.