Amiloride-sensitive sodium channels in rabbit cortical collecting tubule primary cultures.

Patch-clamp methodology was applied to principal cell apical membranes of rabbit cortical collecting tubule (CCT) primary cultures grown on collagen supports in the presence of aldosterone (1.5 microM). The most frequently observed channel had a unit conductance of 3-5 pS, nonlinear current-voltage (I-V) relationship, Na permeability (PNa)-to-K permeability (PK) ratio greater than 19:1, and inward current at all applied potentials (Vapp) less than +80 mV (n = 41). Less frequently, an 8- to 10-pS channel with a linear I-V curve, PNa/PK less than 5:1, and inward current at Vapp less than +40 mV was also observed (n = 7). Luminal amiloride (0.75 microM) decreased the open probability (Po) for both of these channels. Mean open time for the high-selectivity Na+ channel was 2.1 +/- 0.5 s and for the low-selectivity Na+ channel was 50 +/- 12 ms. In primary cultures grown without aldosterone the high-selectivity Na+ channel was rarely observed (1 of 32 patches). Lastly, a 26- to 35-pS channel, nonselective for Na+ over K+, was not activated by cytoplasmic Ca2+ or voltage nor inhibited by amiloride (n = 17). We conclude that under specific growth conditions, namely permeable transporting supports and chronic mineralocorticoid hormone exposure, principal cell apical membranes of rabbit CCT primary cultures contain 1) both high-selectivity and low-selectivity, amiloride-inhibitable Na+ channels and 2) amiloride-insensitive, nonselective cation channels.

Potassium permeable channels in primary cultures of rabbit cortical collecting tubule.

Rabbit cortical collecting tubule (RCCT) primary cultures, were grown on permeable, collagen supports with 1.5 microM aldosterone. Single K+ permeable channels in principal cell apical membranes were examined. At applied patch pipette potential (Vapp) from -60 to +60 mV (cell interior with respect to pipette interior), outward currents (cell to pipette) with a unitary conductance of 8 to 10 pS were seen in cell-attached (N = 31) and excised inside-out (N = 15) patches. At resting membrane potential (Vapp = 0 mV), mean open probability (Po = 0.85 +/- 0.16) decreased by 50% with 0.75 mM luminal BaCl2 exposure. In cell-attached patches, a second type of outward current was seen only at extreme depolarization, Vapp greater than +80 mV (N = 9). Usually in the closed state (Po less than 0.0005) at no applied potential, Po for this 150 pS channel increased dramatically with depolarization and/or raising cytoplasmic Ca2+. With a calculated K+ equilibrium potential of -84 mV, excised patch reversal potentials were less than -50 mV for both the above channel types, indicating high selectivity for K+ over Na+. In cultures grown without aldosterone low conductance K+ channels were rarely observed, while mineralocorticoid status did not appear to affect high conductance K+ channel frequency. Finally, a 30 pS cation channel was found to be nonselective for K+ over Na+, and insensitive to voltage, intracellular Ca2+ or luminal Ba2+. We conclude that: 1) Principal cell apical membranes from aldosterone-stimulated, RCCT primary cultures contain (a) low conductance, Ba(2+)-inhibitable and (b) high conductance, Ca2+/voltage-dependent K+ channels; and c) nonselective cation channels. 2) The low conductance K+ channel may play an important physiologic role in native RCCT mineralocorticoid-controlled K+ secretion, while the latter two channels' functions are unknown, although similar channels have been suggested to play a role in cell volume regulation.

Regulation of the amiloride-blockable sodium channel from epithelial tissue.

The first step in net active transepithelial transport of sodium in tight epithelia is mediated by the amiloride-blockable sodium channel in the apical membrane. This sodium channel is the primary site for discretionary control of total body sodium and, therefore, investigating its regulatory mechanisms is important to our understanding of the physiology of fluid and electrolyte balance. Because essentially all of the regulatory sites on the channel are on the intracellular surface, patch clamp methods have proven extremely useful in the electrophysiological characterization of the sodium channel by isolating it from other channel proteins in the epithelial membrane and by allowing access to the intracellular surface of the protein. We have examined three different regulatory mechanisms. (1) Inhibition of channel activity by activation of protein kinase C; (2) activation of the channel by agents which activate G-proteins; and (3) modulation of channel kinetics and channel number by mineralocorticoids. Activation of protein kinase C by phorbol esters or synthetic diacylglycerols reduces the open probability of sodium channels. Protein kinase C can be activated in a physiological context by enhancing apical sodium entry. Actions which reduce sodium entry (low luminal sodium concentrations or the apical application of amiloride) increase channel open probability. The link between sodium entry and activation of protein kinase C appears to be mediated by intracellular calcium activity linked to sodium via a sodium/calcium exchange system. Thus, the intracellular sodium concentration is coupled to sodium entry in a negative feedback loop which promotes constant total entry of sodium. Activation of G-proteins by pertussis toxin greatly increases the open probability of sodium channels. Since channels can also be activated by pertussis toxin or GTP gamma S in excised patches, the G-protein appears to be closely linked in the apical membrane to the sodium channel protein itself. The mechanism for activation of this apical G-protein, when most hormonal and transmitter receptors are physically located on the basolateral membrane, is unclear. Mineralocorticoids such as aldosterone have at least two distinct effects. First, as expected, increasing levels of aldosterone increase the density of functional channels detectable in the apical membrane. Second, contrary to expectations, application of aldosterone increases the open probability of sodium channels. Thus aldosterone promotes the functional appearance of new sodium channels and promotes increased sodium entry through both new and pre-existant channels.

Expression of amiloride-blockable sodium channels in Xenopus oocytes.

This report describes the expression of a sodium-selective, amiloride-blockable conductance in Xenopus oocytes that have been injected with RNA prepared from a distal nephron cell line (A6). After injecting the RNA into mature oocytes (stage V or VI) and incubating the oocytes for 2-4 days, the oocytes were examined for amiloride-blockable current. The RNA induced a substantial amiloride-blockable current. Uninjected or water-injected oocytes had no measurable amiloride-blockable current. RNA prepared from aldosterone-treated A6 cells was much more effective in inducing amiloride-blockable sodium current than RNA prepared from aldosterone-depleted A6 cells. Oocytes injected with RNA prepared from mineralocorticoid-depleted cells appeared very similar to water-injected oocytes. The amiloride-blockable current in oocytes has a reversal potential of approximately +50 - +60 mV, which varies 61 mV/decade change in external sodium concentration, suggesting that the current is highly selective for sodium over other ions. In addition, the concentration of amiloride that produces half block of the current is 48 +/- 8 nM. Thus the current expressed in oocytes appears very similar to sodium-selective currents observed from the apical membranes of various tight epithelial tissues.

Alpha 2-receptors mediate catecholamine-stimulated acid secretion in Amphiuma jejunum.

The receptors mediating adrenergic stimulation of acid secretion by Amphiuma jejunum were characterized in this study using alpha- and beta-adrenergic agonists and antagonists. Isolated segments of jejunum were mounted in Ussing chambers and bathed in Cl- -free (SO4(2-] medium. Shortcircuit current (Isc) and acid secretion (JH) were recorded, the latter by measuring the rate of alkalinization of the serosal medium. The beta-adrenergic receptor antagonist, propranolol (10(-4) M), had no effect on the Isc and JH stimulated by norepinephrine (NE). The alpha 2-adrenergic agonists, clonidine and UK-14,304, mimicked the effect of NE, with effective concentrations providing 50% maximal delta Isc of 2.0 X 10(-7) and 9.0 X 10(-8) M, respectively. NE added subsequently produced no greater stimulation. In contrast, the alpha 1-adrenergic agonists, phenylephrine and methoxamine, produced little stimulation of JH and Isc; NE added subsequently stimulated the Isc. The alpha 1-adrenergic antagonist prazosin had no effect on the NE-induced Isc or JH, whereas the alpha 2-adrenergic antagonist yohimbine inhibited the NE-stimulated Isc with a half-maximal effective concentration of 3.5 X 10(-7) M. Yohimbine (10(-4) M) reduced the NE-stimulated Isc by 88%, whereas the spontaneous Isc was reduced by only 12%. These results demonstrate that alpha 2-adrenergic receptors on the basolateral membrane of Amphiuma enterocytes mediate NE-enhanced, but not spontaneous, intestinal acid secretion.

Norepinephrine induces Na+-H+ and Cl -HCO3 exchange in Amphiuma intestine: locus and response to amiloride.

Catecholamines stimulate Na+-dependent acid secretion by Amphiuma small intestine. Studies were undertaken to localize the response within the mucosa and characterize the effect on Na+ and Cl- transport. Stripped segments of jejunum were mounted in tissue chambers that permitted isolation of villus or intervillus epithelium. In Cl-free medium, norepinephrine (NE) stimulated the transepithelial voltage (Vms) in both villus and intervillus epithelium, whereas galactose and valine elevated Vms predominately in the villus. Paired segments of whole mucosa were maintained under short circuit while the rate of acid secretion (JH) was measured by titration of the unbuffered serosal medium and unidirectional fluxes of Na+ were measured by 22Na. NE significantly stimulated net Na+ absorption (JNanet), short circuit current (Isc), and JH. Amiloride reduced JH and Isc in NE-stimulated tissues and blocked the stimulation of JNanet by NE. The NE-induced current was nearly completely and reversibly inhibited by replacement of luminal medium HCO3- or CO2. NE significantly stimulated net Cl- absorption without changing Isc or JH. It is concluded that cells throughout the mucosa respond to catecholamines with enhanced Na+ and Cl- absorption, possibly through induction or stimulation of Na+-H+ and Cl- -HCO3- exchange.