Although a variety of hormones and other agents modulate renal Na+ transport acting by way of the epithelial Na+ channel (ENaC), the mode(s), pathways, and their interrelationships in regulation of the channel remain largely unknown. It is likely that several hormones may be present concurrently in vivo, and it is, therefore, important to understand potential interactions among the various regulatory factors as they interact with the Na+transport pathway to effect modulation of Na+ reabsorption in distal tubules and other native tissues. This study represents specifically a determination of the interaction between two hormones, namely, aldosterone and insulin, which stimulate Na+ transport by entirely different mechanisms. We have used a noninvasive pulse protocol of blocker-induced noise analysis to determine changes in single-channel current ( i Na), channel open probability ( P o), and functional channel density ( N T) of amiloride-sensitive ENaCs at various time points following treatment with insulin for 3 h of unstimulated control and aldosterone-pretreated A6 epithelia. Independent of threefold differences of baseline values of transport caused by aldosterone, 20 nM insulin increased by threefold and within 10–30 min the density of the pool of apical membrane ENaCs ( N T) involved in transport. The very early (10 min) increases of channel density were accompanied by relatively small decreases of i Na(10–20%) and decreases of P o (28%) in the aldosterone-pretreated tissues but not the control unstimulated tissues. The early changes of i Na, P o, and N T were transient, returning very slowly over 3 h toward their respective control values at the time of addition of insulin. We conclude that aldosterone and insulin act independently to stimulate apical Na+ entry into the cells of A6 epithelia by increase of channel density.

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

Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.

Isolated renal cortical collecting tubules from rabbits were studied at random (control) and from rabbits maintained on diets containing either high or low Na and K content or injected with DOCA for up to 31 days. In general, the values of transepithelial voltage (VT) and electrical resistance (RT) varied considerably between tubules. When rabbits were fed a high K, low Na diet or were injected with DOCA the values of VT increased. Of interest was the observation that tubules obtained from rabbits maintained on DOCA for 11-18 days or longer possessed markedly elevated values of VT (mean VT, -54 mV) and a marked increase in their ability to reabsorb Na and secrete K at rates well above the levels observed even at 3-6 days of treatment when it was expected that maximal transport rates would have been achieved. From the estimates of the conductances of Na, K, and Cl, the data could be interpreted to mean that DOCA caused a decrease in the conductance of Cl, perhaps via the shunt pathway, while elevating the conductances of Na and K, thereby enhancing active Na and especially K transport by this nephron segment.

To study and define the early time-dependent response (≤6 h) of blocker-sensitive epithelial Na+channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current ( i Na) channel open probability ( P o), and channel density ( N T) under transient conditions of transport as measured by macroscopic short-circuit currents ( I sc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 μA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. N T varied linearly over a 70-fold range of transport (0.5–35 μA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of P o occurred during control (10–20% over 2 h) and aldosterone experimental periods (10–30% over 6 h). When the P o of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, P o was observed to vary inversely with I sc, falling from ∼0.5 to ∼0.15 at the highest rates of Na+ transport or ∼25% per 3-fold increase of transport. Because decreases of P o from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane N T.