Enhanced Intracellular Sodium Concentration in Kidney Cells Recruits a Latent Pool of Na-K-ATPase Whose Size Is Modulated by Corticosteroids*

Besides its role in the control of the rate of functioning of each Na-K-ATPase unit (as a substrate of the enzyme), the intracellular sodium concentration also regulates the number of active Na-K-ATPase units, as previously described in cultured cells. To evaluate such a possibility in kidney epithelial cells, the intracellular concentration of sodium in rat cortical collecting tubules (CCT) maintained in vitro was altered by the use of the sodium ionophore nystatin. When CCT were preincubated for 2-3 h at 37 degrees C in the presence of nystatin, the enzymatic activity of Na-K-ATPase was markedly stimulated as compared to tubules preincubated without nystatin or in the presence of the ionophore but in the absence of extracellular sodium. Although nystatin increased both Na-K-ATPase activity and [3H]ouabain specific binding in CCT, its action was independent of de novo synthesis of the pump since neither actinomycin D nor cycloheximide abolished it. It is suggested that increasing the sodium concentration in CCT cells induces the recruitment of a latent pool of Na-K-ATPase units. The size of this latent pool of enzyme is under the control of corticosteroids as it is markedly decreased in CCT from adrenalectomized rats.

Besides its role in the control of the rate of functioning of each Na-K-ATPase unit (as a substrate of the enzyme), the intracellular sodium concentration also regulates the number of active Na-K-ATPase units, as previously described in cultured cells. To evaluate such a possibility in kidney epithelial cells, the intracellular concentration of sodium in rat cortical collecting tubules (CCT) maintained in vitro was altered by the use of the sodium ionophore nystatin. When CCT were preincubated for 2-3 h at 37 "C in the presence of nystatin, the enzymatic activity of Na-K-ATPase was markedly stimulated as compared to tubules preincubated without nystatin or in the presence of the ionophore but in the absence of extracellular sodium. Although nystatin increased both Na-K-ATPase activity and [3H]ouabain specific binding in CCT, its action was independent of de nova synthesis of the pump since neither actinomycin D nor cycloheximide abolished it. It is suggested that increasing the sodium concentration in CCT cells induces the recruitment of a latent pool of Na-K-ATPase units. The size of this latent pool of enzyme is under the control of corticosteroids as it is markedly decreased in CCT from adrenalectomized rats.
In the collecting tubule of the mammalian nephron, the capacity for sodium reabsorption varies within a wide range in response to several hormones and/or factors (1). In these cells, sodium reabsorption from the luminal medium to the peritubular fluid is accomplished through a passive luminal entry via amiloride-sensitive sodium channels (2) and active basolateral pumping catalyzed by Na-K-ATPase (Na+/K+transporting ATPase, EC 3.6.1.37). Under normal conditions, the activit,y of Na-K-ATPase balances the sodium influx through the luminal membrane to maintain the constancy of the intracellular sodium concentration. When luminal sodium conductance increases, the intracellular sodium concentration rises and stimulates, by a substrate effect, the activity of each catalytic unit of Na-K-ATPase.
If this increase of sodium entry is sustained, the sodium efflux through the pump can balance the sodium influx through the luminal membrane only if Na-K- pumping rate, one possibility is to enhance the V,., of Na-K-ATPase.
The purpose of our study was to investigate the mechanisms through which the V,,, of the enzyme may increase and, in particular, whether an increase in the intracellular sodium concentration may stimulate Na-K-ATPase. For this purpose, changes in the intracellular sodium concentration were induced in microdissected rat cortical collecting tubules (CCT)' by the use of the polyene antibiotic nystatin, a sodium ionophore (3). Since stimulation of the V,,,,x of an enzyme may result from increasing either the number of active catalytic units or the activity of each catalytic unit, or both, we determined the number of active pump units present in collecting tubules incubated in the absence or presence of nystatin by measuring the specific binding capacity of [3H]ouabain.
The use of an in vitro system allowed us to further evaluate the molecular events underlying the action of an increased intracellular concentration on Na-K-ATPase. Finally, the possible relationship between adrenal steroids, which are known to induce Na-K-ATPase synthesis in rat collecting tubule (4,5), and increased intracellular sodium concentration was investigated.

AND DISCUSSION
Stimulation of Na-K-ATPase Activity in CCT from Normal Rats by Increased Intracellular Sodium Concentration--Results depicted in Fig. 1 indicate that incubation of CCT from normal rats for 3 h in the presence of nystatin (0.1 unit/pi) induced a marked stimulation of Na-K-ATPase activity (control, 880 + 48 pmol. mm-'. h-l; nystatin, 1443 f 63 pmol. mm-'. h-l; mean + SE., n = 19, p < 0.001). When added during Na-K-ATPase assay, nystatin had no effect on enzyme activity (data not shown). Na-K-ATPase activity was also stimulated after preincubation of CCT in the presence of amphotericin B (1 fig/ml), another sodium ionophore (Fig. 2). ouabain, a procedure also aimed at increasing the intracellular sodium concentration, stimulated Na-K-ATPase activity (determined after washing off ouabain) (Fig. 2). Furthermore, the stimulatory action of nystatin was totally abolished when choline was substituted for sodium in the incubation solution (Fig. 3). These results demonstrate that stimulation of Na-K-ATPase maximal activity is not due to a direct effect of nystatin.
Rather, it is likely related to alterations in the intracellular sodium (or potassium) concentration.
Alternately, an increase in Na-K-ATPase activity in response to nystatin could conceivably result from a rise in the intracellular calcium ion concentration through a secondary inhibition of Na+/Ca*+ exchange. However, in one experiment in which we attempted to increase the intracellular Ca*+ concentration by preincubating CCT for 3 h in the presence of the calcium ionophore ionomycin (1.7 X 10m6 M) and Ca2+ (1 mM), Na-K-ATPase activity did not increase, whereas it did in response to ouabain and nystatin in the same experiment ( The stimulation of Na-K-ATPase activity in response to an increased intracellular sodium level was observed after a lag period of 15 min and reached its maximum within 1 h (Fig. 4). This latency is much shorter than that previously observed in cultured cells (ll-16) reported for rabbit CCT (8). That nystatin altered the binding capacity and not the affinity of collecting tubules for ouabain was assessed by the finding that the apparent K, of Na-K-ATPase for ouabain was similar with and without nystatin pretreatment (Fig. 5B). At this point, it should be stressed that in these experiments, ouabain binding was measured in intact nonpermeabilized cells so that only ouabain-binding sites accessible in the plasma membrane were detected. Thus, stimulation of Na-K-ATPase in response to nystatin involves the setting in place of new catalytic sites.
In vitro up-regulation of Na-K-ATPase was reported in several cell types (reviewed in Ref. 17) in response to an increased intracellular sodium concentration brought about by growing cells in low potassium medium or in the presence of either sublethal doses of ouabain or veratridine (in those cells which have voltage-sensitive sodium channels). Such stimulation of Na-K-ATPase peaked after about 24 h. It corresponded to an induction of Na-K-ATPase synthesis, as assessed by increased incorporation of [?S]methionine into immunoprecipitable or-subunits of Na-K-ATPase in myocytes (16), enhanced rates of synthesis of (Y-and P-mRNAs in Madin-Darby canine kidney cells (12), and/or an inhibition of the degradation rate of pre-existing pump units (15,16). We therefore evaluated whether stimulation of Na-K-ATPase activity in response to nystatin was brought about by increased synthesis of Na-K-ATPase.
Results in Fig. 6 indicate that neither actinomycin D nor cycloheximide affected the effect of nystatin when added concomitantly to it. Although these drugs were shown to efficiently inhibit Na-K-ATPase synthesis in rat CCT when used under similar conditions (4), one might argue that their action could be altered in the presence of nystatin. To exclude this possibility, we evaluated the effect of cycloheximide on [35S]methionine incorporation in single CCT incubated in the presence or absence of nystatin. Data in Fig. 7 indicate that under these two conditions, cycloheximide inhibited by 95% [35S]methionine incorporation into proteins of CCT. They also indicate that nystatin alone had no detectable effect on [35S]methionine incorporation.
In parallel experiments, we verified that cycloheximide did not alter nystatin-induced stimulation of Na-K-ATPase (Fig. 7). Thus, up-regulation of Na-K-ATPase observed in rat CCT in response to nystatin cannot be accounted for by increased synthesis of Na-K-ATPase, neither can it be attributed to a decreased rate of Na-K-ATPase degradation, as its fast kinetics (see Fig. 4) is incompatible with the halflife (-15 h) of the enzyme (18). Results from these experiments instead suggest that an increased intracellular sodium concentration triggers the rapid redistribution of pre-existing inactive enzyme molecules to the plasma membrane of CCT cells. Presently, we have no information concerning the intracellular localization and the molecular form of this latent pool of Na-K-ATPase.
This hypothesis does not preclude that other mechanisms, including stimulation of Na-K-ATPase synthesis, may occur later in CCT cells, especially since these cells share several properties with the Madin-Darby canine kidney cell line, in which stimulation of the transcription of genes encoding for Na-K-ATPase subunits was reported in response to an enhanced intracellular sodium level (12). steroids-In contrast to what was observed in normal rats, preincubation of CCT in the presence of nystatin did not alter Na-K-ATPase activity in tubules isolated from adrenalectomized rats (adrenalectomized, 199 + 47 pmol. mm-'. h-l; adrenalectomized + nystatin, 213 f 43 pmol. mm-'. h-'; mean + S.E., n = 6, not significantly different), suggesting that there is no latent pool of Na-K-ATPase in CCT of adrenalectomized animals (Fig. 8, inset). This is consistent with the previous finding that preincubation with ouabain for 6 h did not increase Na-K-ATPase activity in suspensions of outer medullary collecting tubules from adrenalectomized rats (19). To study the kinetics of decay of the latent pool of pumps following adrenalectomy, Na-K-ATPase activity of CCT in- cubated for 2 h in the absence or presence of nystatin was determined at various times (from 12 h to 1 week) after adrenalectomy.
Results in Fig. 8 indicate that Na-K-ATPase activity measured after incubation without nystatin (activity of the pool of active Na-K-ATPase) remained constant during the first 24 h following adrenalectomy and then decreased to the values observed 1 week after adrenalectomy.
In contrast, Na-K-ATPase activity measured after stimulation by nystatin, which corresponds to both active and latent pools of Na-K-ATPase, decreased as soon as 12 h following adrenalectomy and equaled the unstimulated activity at 1 day after adrenalectomy and thereafter.
These results suggest that the latent