Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium.

Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.

bility to the basolateral Na+ gradient (9-13).  reported that incubating apical vesicles derived from toad bladder mucosal cells with Ca" ions decreases their amiloride-sensitive Na+ permeability. The Ca2+ effects were restricted to the cytoplasmic phase of the membrane and half-maximal inhibition was induced by 0.5 FM free Ca2+. The ability of Ca2+ ions to inhibit Na+ transport in isolated membrane vesicles suggests that this inhibition is mediated by a direct noncovalent Ca2+-apical membrane interaction. In a recent work (15), we provided evidence for a different type of Ca2+-dependent down-regulation of channels. It was found that the amiloride-sensitive "Na+ uptake by apical vesicles strongly depends on the conditions at which the cells were incubated prior to their homogenization. Accordingly, maximal channel activity in vesicles was obtained if the epithelial cells were preincubated in a Ca2+-free EGTA' solution for at least 30 min at 25 "C. Including Ca2+ (1 mM) in the cell incubation medium almost completely abolished the amiloride-sensitive uptake into vesicles, in spite of the fact that the membranes were isolated and assayed in Ca2+-free solutions.
In the current paper we further characterize this Ca2+dependent temperature-sensitive regulation of Na+ channels. The Ca2+-induced inhibition was found to be a highly cooperative process (Hill coefficient of nearly 4) with a Ki that varied, in different vesicle preparations, between 6.4 x lo-' and 8.15 X M Ca2+. Reagents known to influence several Ca2+-dependent cellular events such as calmodulin inhibitors, phorbol ester, and cytoskeleton disrupting agents had no effect on this process. CAMP, which mediates the antidiuretic hormone-induced activation of Na+ channels and the methylation inhibitor 3-deazaadenosine were also ineffective. Vanadyl (V02+) a potent inhibitor of alkaline phosphatase could mimic the Ca" effects, but vanadate (VO:) had no effect.

Vesicle Preparation-Toads (Bufo marinus, Mexican origin, obtained from Lemberger, Oshkosh, WI) were doubly pithed and de-
blooded by transventricular perfusion with 300-600 ml of Ringer's solution containing (in mM): NaC1,llO.O; CaC12,l.O; MgC12,0.5; and K-phosphate buffer, 3.5 (pH = 7.5). The urinary bladders were excised and rinsed several times in an ice-cold Ca2+-free medium composed of (in mM): KC1, 90; sucrose, 45; MgCl,, 5; EGTA, 10; and a pH buffer, 10. The buffer used varied according to the experimental design and was Tris. HCI (pH = 7.81, TES (pH = 7.3), or PIPES (pH = 6.8). The epithelium was scraped off the underlying connective tissue with a glass slide, and the cells were dispersed in the above medium by rapidly drawing them in and out of a Pasteur pipette at least 10 times. The epithelial cells derived from a single animal were divided among 6-8 test tubes and pelleted by centrifugation at 1000 The abbreviations used are: EGTA, Iethylenebis(oxyethy1enenitrilo)] tetraacetic acid; PIPES, 1,4-piperazinediethanesulfonic acid;TES, 2-([2-hydroxy-l,l-bis(hydroxymethyl)ethyl]amino~ethanesulfonic acid; 8Br-cAMP, 8-bromo-CAMP. X g for 5 min at 2 "C. The cell pellets were washed twice by resuspension and centrifugation and then resuspended in either one of the above media or similar media containing various concentrations of CaClz in addition to EGTA (see below). Different reagents were added according to the experimental design, and the suspensions were incubated for 30 min at 25 "C. At the end of this period they were cooled back to 0 "C, diluted 5-fold in homogenizing solution composed of (in mM): KCl,90;sucrose,45;MgC12,5;EGTA,10; and Tris . HCI, 10 (pH = 7.8), pelleted, suspended in 3 ml of this (Ca2+-free) medium, and immediately sheared by a single 10-s burst of a Polytron homogenizer (Ystral GmbH, Dottingen, Federal Republic of Germany) at highest speed. In one set of experiments the cells were suspended and homogenized in medium containing 90 mM KzS04 instead of KCl. Intact cells, nuclei, and debris were removed by centrifuging the homogenate5 at 1000 X g for 5 min. The supernatants were then centrifuged for another hour at 27,000 X g to pellet the membrane fraction. The microsomal pellets were suspended in minimal volumes (200-400 pl) of the homogenizing medium, maintained at 0 "C, and used the same day.
Transport Assay-"Na+ uptake into toad bladder vesicles was measured in the presence of a membrane potential induced by a KC1 gradient + valinomycin, as described previously (15)(16)(17). Microsomal preparations were first eluted through short Dowex (50W-X8,50-100 mesh, Tris form) columns with 0.9 ml of 175 mM sucrose to exchange the extravesicular K+ by Tris. The pH of the eluted suspension was increased to 8.2 to maximalize the tracer uptake (17), and valinomycin (3 pM) was added. Two 300-pl aliquots of the eluted vesicles were thereupon added to reaction mixtures containing 60 pl of 175 mM sucrose, 5 pl of carrier-free 22NaC1 (final concentration 4 pCi/ml, 0.2-0.4 p~ Na+) and 6 1. 11 of either amiloride (final concentration 1.5 pM) or water diluent. The tracer uptake was measured by removing 150pl aliquots (4-15 pg of protein) at 1.5 and 3.5 min from the radioactive suspensions and eluting them through Dowex columns into counting vials with 1.5 ml of ice-cold 175 mM sucrose. Usually the uptake was linear for at least 3.5 min. The amount of radioactivity associated with the particles was insensitive to the time the vesicles were on the column (20-60 s) or to the time delay between the formation of membrane potential and the addition of "Na+ (0.5-2 min). This advantage is a consequence of the fact that in the absence of permeable cations in the external medium, the membrane potential dissipates very slowly, and internal Na+ does not leak after the removal of external radioactivity. The channel-mediated flux was calculated from the difference in internal radioactivity between the aliquots incubated with and without amiloride, and expressed as pmol of 22Na+.mg of protein".min".
In vesicles prepared from cells incubated in Caa+-free solution, the amiloride-sensitive flux accounted for 70.5 & 3.2% [mean of 31 preparations] of the total uptake. If valinomycin was omitted from the reaction mixture, the total uptake was much smaller and the flux component blocked by 1.5 FM amiloride was negligible, suggesting that amiloride-sensitive adsorption of =Na+ to the vesicles does not significantly contribute to the measured fluxes. In determining the full time course of 22Na+ uptake (Fig. l), the vesicles were initially eluted with 2.1 ml of sucrose, 900 pl of eluant was added to each of the reaction mixtures, and aliquots were sampled at times ranging from 2 to 60 min. The above transport assay was also used to measure the steady state %Rb' accumulation.  Harafuji and Ogawa (20) and correcting it for the different pH, temperature, and ionic strength of our experiments, using the equations suggested by them. %'a2+ Flux Measurements in Whole Cells-Scraped epithelial cells were suspended in EGTA-free, homogenizing medium to a final concentration of 4.5 mg of protein/ml. The cells were either incubated with 45CaC12 (10 pCi/ml, -1 p~ Ca") for different periods of time (influx assay) or incubated with the tracer for 10 min, followed by addition of 10 mM EGTA and incubation for additional timed periods (efflux assay). Sampling was done by filtering 100-pl aliquots through polycarbonate membrane filters (0.6 pm, Nucleopore Corp., Pleasanton, CA) and washing the cells with 4 ml of homogenizing medium 7401 containing 10 mM EGTA. A high vacuum pump which gave a filtration rate of about 4 ml/s was used in order to minimalize loss of internal radioactivity during the washing. The filters were immersed in scintillation fluid (xylene based) and counted in a @ scintillation counter. The background radioactivity estimated from the sampling of a cell-free solution was less than 1% of the maximal *Ca2+ uptake.

Ca2+/EGTA
Statistics-Data are expressed as mean +. SE and the number of measurements is given in brackets. Hill coefficients and inhibition constants were obtained by a linear regression analysis of Hill plots.

RESULTS
Inhibition of Nu+ Transport by Ca2+-As was recently reported (15), incubating scraped toad bladder epithelial cells for 30 min at 25 "C in a Ca2+-free solution increases by more than 4-fold the amiloride-sensitive "Na+ flux measured in membrane vesicles derived from them. This activation was prevented if Ca" ions (>1 pM) were present in the cell incubating media (even though removed before the homogenization) or if cells were maintained a t 0 "C. On the basis of these data it was concluded that the toad bladder Na+ channels are down-regulated by a temperature-sensitive Caz+dependent reaction which takes place in whole cells and induces a stable change in the membrane lipids and/or proteins preserved by the isolated vesicles.
The ability of Ca" ions to prevent the activation of channels was further examined in the experiments summarized in Table I. Vesicles prepared from cells incubated under the control conditions (i.e. for 30 min at 25 "C in a Ca2+-free TABLE I Ca2+-induced inhibition of "Na+ uptake Toad bladder cell aliquots were incubated at 25 "C at pH = 7.8 under 4 different conditions: A, for 30 min in a Ca2+-free homogenizing solution containing 10 mM EGTA; B, for 30 min in a homogenizing solution containing 10 mM EGTA + 10 m~ CaClz (10" M free CaZ+ at pH = 7.8); C, for 30 min in a solution containing 1 mM EGTA + 1 mM BaC12; D, as for B plus an additional 30-min incubation in a Ca%-free 10 mM EGTA medium; E, as for A. The incubations were terminated by cooling the suspensions to 0 "C and diluting them 5fold with the Ca2+-free homogenizing medium. Cells were pelleted, suspended in the same medium, and homogenized. To aliquot E, Ca2+containing solution was added after the homogenization to a final concentration of 10 mM EGTA + 10 mM CaClz (lou5 M free Ca"), and this was incubated for an additional 15 min at 25 "C. Vesicles were isolated and =Na+ uptake measured as described under "Materials and Methods." The mean amiloride-blockable and amilorideinsensitive fluxes, expressed as a fraction of the control values (condition A), are presented. The mean amiloride-sensitive and amilorideinsensitive fluxes under condition A were 11.4 +. 1.5 1311 and 3.4 k 0.3 [31] pmol of =Na+. me of Dr0tein-l. min". remectivelv.  EGTA solution) had mean amiloride blockable and amiloride insensitive initial rates of 11.4 +. 1.5 [31] and 3.4 +-0.3 [31] pmol of 22Na+. mg of protein-'. min-l, respectively? Including M free Ca2+ in the cell incubating medium lowered the amiloride-sensitive uptake to 2.24 f 0.12 pmol of "Na+.mg of protein-l-min-l (19.7 k 2.5% of the control value) and hardly affected the amiloride-insensitive flux (3.11 -+ 0.16 pmol of 22Na+. mg of protein-'. min-' or 91.6 k 4.9% of the control value). This inhibition was not observed by incubating cells with Ba2+ (1 mM) which is known to mimic some of the Ca2+ effects in adrenal medulla cells (21,22). The decrease in Na+ uptake induced by Ca2+ was irreversible; incubating Ca" treated cells for an additional period of 30 min in a Ca2+-free EGTA solution did not increase the Na+ flux in vesicles. In another set of experiments Ca2+ was applied to homogenates of cells preactivated in EGTA. Addition of Ca" ions to a broken cell suspension followed by their removal before the transport assay had no effect on the Na+ permeability in vesicles, i.e. the above inhibition can be induced in whole cells only. It therefore either involves a structural component which is destroyed by the homogenization or requires a cytoplasmic factor which is largely diluted during the shearing of the cells.
Possible Sources of the Ca2' Effect-The observed Ca2+induced decrease in amiloride-sensitive Na+ uptake may in principle result from one of three events. 1) A decrease in the apical Na+ permeability, i.e. closure or internalization of Na+ channels. 2) An increase in the apical permeability to other ion, in particular C1-(23) and therefore depolarization of the driving potential. 3) A decrease in the yield or dimensions of the channel containing particles. Elimination of the two last, less interesting, possibilities was made by substituting $0;for C1-, looking at the details of the full time course of "Na+ uptake, and comparing the uptake of =Rb+ into control and Ca2+-inhibited vesicles. First, the tracer fluxes were measured in vesicles prepared to contain 90 mM K2S04 instead of KC1.3 It was found that pretreatment of cells with M Ca2+ lowered the amiloride-sensitive uptake into these vesicles to 13 & 2% [3] of the control value, i.e. the Ca2+-induced inhibition of transport can be demonstrated in the complete absence of C1-and therefore cannot stem from an increased permeability of this ion. The second protocol used, to exclude the possibility that Ca" acts on the permeability of other ions, was to measure the full time course, i.e. both the initial accumulation and the following efflux of "Na+ ( Fig. 1). As shown previously, both experimentally and by computer simulations, a decrease in the initial uptake induced by an increase in the permeability to other ions, is accompanied by a faster efflux due to the enhanced dissipation of membrane potential (16,24). The data of Fig. 1 clearly show that the inhibition of 22Na+ uptake is not accompanied by an earlier and faster loss of internal radioactivity, i.e. the time course of "Na+ fluxes measured in vesicles derived from Ca2+-treated cells is as expected from Ca2+ affecting the Na+ permeability and is different from the kinetic behavior predicted by an increased leakiness of the particles! Note that the assay mixture contained 1.5 pM amiloride only, i.e. the "amiloride-blockable" component in this study is the "high affhity phase" of (15, 16).
Since the activity coefficient of K+ in SO:solutions is about 0.55, the K+ activity of 90 mM KzS04 is only slightly higher than of 90 mM KC1.
Unlike before (16, 24), lowering the Na+ permeability did not considerably slow down "Na+ efflux. The reason for this difference is that in the present study the potential was induced by a KC1 gradient and not by a NaCl gradient. Thus, the decrease of Na+ permeability had no direct effect on the potential dissipation rate. "Na+ uptake into the isolated vesicles was measured as described under "Materials and Methods," and the amiloride-sensitive uptake is plotted versus time.

TABLE I1
=Rb+ and "Na+ accumulation in vesicles derived from control and Ca2+-treated ceUs =Rb+ and =Na+ uptake were measured in vesicles derived from cells incubated with either EGTA or M Caz+ as described under "Materials and Methods." Results are expressed as a percentage of the total radioactivity taken up during a 2-min incubation. This is a steady state accumulation in the case of ffiRb+ and initial uptake in the case of "Na+. =Rb+ uptake was measured in the presence and absence of 3 ELM valinomycin and KC1, NaC1, or Tris.HC1, added to the assay mixture to a final concentration of 25 mM. The data in the table come from a single experiment, but similar results were obtained in 3 additional preparations. Amil, amiloride.

External additions
Tracer uptake Possible effects of Ca2' on the yield or dimensions of the channel containing vesicles can be assessed by comparing the intravesicular spaces in membrane preparations derived from control and Ca2+-treated cells. The common methods for measuring an intravesicular space (e.g. comparing the space accessible to t3H]water and a nonpermeable solute) will estimate the overall intravesicular volume of our crude membrane preparation and may therefore overlook Ca2+-induced changes in a subpopulation of the particles. An alternative measurement which could be more sensitive to a change of the apical volume is evaluating the steady state distribution of "Rb+ across the vesicle membrane. In the presence of valinomycin =Rb+ will equilibrate according to the membrane potential (A$) and its steady state distribution (cpmh/cpmout) will be equal to (Vout/Vin)eFA+'RT (when Vi, and Vout are the intraand extravesicular volumes). The 86Rb+ accumulation will therefore preferentially measure the internal volume of "tight" (maximal A$) vesicles. Moreover, by comparing the steady itate 86Rb+ uptake in the presence and absence of external Na", it is possible to estimate the volume of a vesicle subpopulation which is impermeable to C1-and permeable to Na+, i.e. the apical particles. Table I1 summarizes measure-ments of =Rb+ and "Na+ uptake in vesicles derived under matched conditions from control and Ca2+-treated cells. Unlike "Na+ fluxes which were relatively slow, =Rb+ readily equilibrated across the membrane of valinomycin-treated vesicles, and the internal activity measured after a 2-min incubation represents its equilibrium distribution. As seen from Table 11, =Rb' was taken up to a large extent only in the presence of valinomycin. This accumulation was completely prevented if K+ (25 mM) was present in the external solution, but an equivalent concentration of Tris' had no effect. Na+, added to the assay mixture to a final concentration of 25 mM, lowered the =Rb+ uptake by nearly 40%. This effect, however, was independent of the presence of amiloride, suggesting that external Na+ can depolarize the K+ diffusion potential also by entering through amiloride-insensitive electrogenic pathways. The significant finding from Table I1 is that under no circumstances could a difference between control and Ca2+treated membranes be observed. On the other hand, "Na+ uptake by the same vesicles differed by almost 5-fold (last row in Table 11). Thus, it seems that the observed ea2+induced inhibition of "Na+ flux reflects a decrease of the Na+ permeability rather than a decrease of the "capacity" of apical vesicles to accumulate Na+.
Ca" Dose-Response Relationships-Valuable information on the nature of the above Ca" effect and its possible involvement in the physiological control of Na+ transport can be obtained from measurements of the Ca2+ dose-response relationships. Such measurements are meaningful only if the cell membrane is sufficiently permeable to Ca" ions, i.e. setting the external Ca" activity at a given value will buffer the cytoplasm at the same value. Normally the toad bladder plasma membrane is Ca2+-impermeable, and a substantial influx of Ca2+ into intact cells can only be induced with an ionophore or a favorable basolateral Na+ gradient (11,13,25). However, it is possible that the scraping and dispersion of epithelial cells induces a limited mechanical damage of the plasma membrane and permeabilizes them to Ca" ions (15,26). The Ca2+ permeability of whole cells was evaluated by measuring 45CaZ+ fluxes under conditions similar to those used to pretreat them with Ca2+/EGTA mixtures (Fig. 2). Indeed, saturable 45Ca2+ influx and efflux in a time scale of a few minutes could be observed. Thus, it is feasible that buffering the external Ca2+ activity at a given value will soon clamp the cytoplasmic free Ca" to the same value.

L 1 0
Tlme ( m i d

FIG. 2. 46Caz+ fluxes in whole cells. *Ca2+ influx (closed triangles)
and efflux (open triangles) into and out of epithelial cells were measured as described under "Materials and Methods." In the influx measurements %a2' was added at t = 0. In the efflux measurement the tracer was applied at t = -10 min, and efflux was initiated at t = 0 by adding 10 mM EGTA. Each experimental point is the mean of two measurements. assaying the amiloride-blockable fluxes in vesicles derived from cell aliquots incubated with different Ca2+/EGTA mixtures. In one series of 9 experiments carried out during November 1984January 1985, the amiloride-sensitive "Na+ uptake was almost completely blocked by M free Ca". The Ca2+ inhibition curve was measured in 6 of these preparations by incubating cell aliquots at pH = 7.8. This pH was chosen in order to match the effective buffering range of EGTA (apparent Ca2+/EGTA dissociation constant of 9.5 X lo7 M" at pH = 7.8) to the inhibitory concentrations of free Ca". The experimental data, depicted in Fig. 3A, could be fitted to an inhibition curve with a Ki value of 6.4 X lo-' M Ca2+ and a Hill coefficient of 3.9 & 0.4. In another series of 8 experiments carried out during September-November 1985, using the same protocols, M Ca2+ had no effect, but the uptake could still be inhibited with M Ca2+. The Ca2+ inhibition curve was then remeasured in another five preparations in which the cells were incubated at pH = 6.8 (apparent Caz+/ EGTA dissociation constant of 1.0 X lo6 " l ) . The results of these measurements, shown in Fig. 3B, could be fitted to an inhibition curve with a Ki value of 8.15 X M Ca2+ and a Hill coefficient of 3.7 & 0.6. Moreover, incubating cells with low concentrations of Ca" (1-4 pM) seemed to produce a slight activation of transport. It should be emphasized that the shift in the Ca" inhibition curve observed was not the result of a different experimental protocol or different batch of EGTA (27). We attribute this shift to physiological changes in the intracellular activities of factors which modulate the Ca2+-induced reaction. This behavior is in fact reminiscent of the changes observed in the affinity of protein kinase C to Ca" ions in the presence of different lipids (28,29). However, including the phorbol ester 4-phorbol lL-myristate 13-acetate

FIG.
3. Caa' dose-response relationship. Cells scraped from a single bladder were divided to several aliquots, pelleted and suspended in 2 ml of media containing different Ca2+/EGTA mixtures buffered to pH of either 7.8 (A) or 6.8 (B). The suspensions were incubated at 25 "C for 30 min. At the end of this period they were cooled to 0 "C, diluted with 10 ml of Ca2+-free homogenizing medium (10 mM EGTA, pH = 7.8) pelleted, and suspended in 2 ml of this medium. The cells were broken and membrane vesicles isolated and assayed for =Na+ uptake as described under "Materials and Methods." The amilorideblockable tracer uptake, expressed as a percentage of the value obtained in vesicles isolated from cells maintained in Ca2+-free solutions, is plotted against the free Ca2+ concentration of the incubating medium. The different symbols refer to different experiments. The insets are Hill plots of the experimental data. The best fits were: A, slope (Hill coefficient): 3.9 k 0.4, x intercept: -7.19 (Ki = 6.4 X lo-' M Ca"), and correlation coefficient: 0.95; B, slope: 3.7 0.6, 2 intercept: -5.09 (Ki = 8.15 X M Ca"), and correlation coefficient:

ea2+-induced Down-regulation
of Na+ Channels (100 ng/ml) in the cell incubating medium failed to shift the inhibition curve shown in Fig. 3B to lower values. Such a shift is expected if Ca2+ inhibits Na+ transport by activating protein kinase C. Consequently, some other physiological change must be involved. Possible Mechanisms of the Ca2+-induced Inhibition of Channels-In an attempt to shed light on the molecular events that mediate the permeability changes, we studied effects of several reagents on the activation of channels in EGTA solutions and their inhibition by M Caz+ (Table  111). The preparations used in this set of experiments had a Ca2+ inhibition constant in the micromolar range, i.e. at M Ca2+ most of the channels were closed, but the divalent ion was not present in a large excess. Preincubating cells with the lipid-soluble calmodulin inhibitor trifluoperasine had no significant effect on the base-line channel-mediated uptake or its inhibition by Ca". Thus, Ca2+-calmodulin interaction does not seem to play a role in the regulation of Naf channels. Vanadate (VO:), known to inhibit many phosphate transfer reactions (30), did not influence the Na+ permeability changes. On the other hand, vanadyl (V02+) strongly inhibited Na+ uptake into vesicles derived from cells incubated in EGTA and increased the inhibition observed with M Ca2+. These effects were observed only with freshly made vanadyl solutions presumably due to its atmospheric oxidation to vanadate. No major effect on transport in vesicles was observed upon incubating scraped cells and whole bladders with the cytoskeleton-disrupting agents colchicin and cyto-

TABLE I11 Effects of various reagents on the amiloride-sensitive flux in vesicles
Scraped cells were suspended at 0 "C either in a Caz+-free EGTA solution or in solution buffered to 10" M Ca2+ (pH = 7.3). Half of each suspension received one of the above listed reagents at the indicated concentration and the other half an equal volume of diluent. After an initial period of 5 min at 0 'C (to enable the penetration of the tested drug), the cells were incubated at 25 "C for 30 min. The incubation was terminated by cooling the cells to 0 "C and diluting them into a Ca2+-free EGTA solution. Cells were pelleted, suspended in the Caz+-free medium, and homogenized. Membrane vesicles were prepared and assayed for amiloride-sensitive uptake as described under "Materials and Methods." The amiloride-blockable fluxes, expressed as a fraction of the control values (no added reagents, Caz+free solution) in the same experiment, are presented.
Amiloride-sensitive "Na+ uptake * This relatively impermeable reagent was introduced into the cells by a different procedure. Paired hemibladders were immersed in 10 ml of Ringer's solution containing 10 mM glucose and incubated under aeration at 25 'C for 4 h in the presence or absence of colchicine (lou4 M). At the end of this period, the bladders were rinsed well in an ice-cold Ca2+-free homogenizing medium, and the epithelium was scraped and treated as above. Colchicine was readded to the scraped cell incubation medium.

TABLE IV Effects of 8Br-CAMP on the amiloride-sensitive "Nu" uptake
Scraped cells were divided into 6 aliquots and incubated under various conditions in the presence of either 8Br-CAMP (1 mM) + 3isobutyl-1-methylxanthine (IBMX) (0.1 mM) (treated) or water di-Iuent (control). At the end of the incubation period the aliquots were cooled to 0 "C, pelleted, resuspended in the standard Ca2+-free medium, and immediately homogenized. Vesicles were isolated and "Na+ fluxes measured as described under "Materials and Methods." The amiloride-blockable fluxes presented are expressed as fractions of the fluxes measured in vesicles isolated from cells incubated for 30 min at 25 "C in Ca2+-free solutions.
Amiloride-blockable 22Na+ uptake chalasin B or the methylation inhibitor 3-deazaadenosine. It was recently suggested that Na+ channels in cultured toad kidney cells (A6) are activated by methylation of either lipids or proteins (31). The above data indicate that this process is not the same process studied by us.
It is well established that augmentation of the apical amiloride-blockable Na' conductance in toad bladder can be induced by an increase of the cytoplasmic CAMP, and this is the mechanism by which antidiuretic hormones mediate their natriferic action (1, 32). Recently, it was shown that vasopressin induces a small decrease of the cytoplasmic Ca2+ activity in toad bladder cells, and the possibility was raised that cellular Ca2+ plays a role at least in the hydroosmotic response to the hormone (33). To examine the possible involvement of CAMP in the processes examined in the present study, we measured the effects of 8Br-CAMP and 3-isobutyl-I-methylxanthine (a CAMP phosphodiesterase inhibitor), added to the cell incubation medium, on the channel activity recovered in vesicles. Effects of the permeable cyclic nucleotide were measured in cells incubated under three different sets of conditions: 1) for 30 min at 25 "C in a Ca2+-free solution, a condition which induces maximal activation of channels; 2) for 30 min at 25 "C in a solution buffered to 7 X 10"' M Ca", a concentration which in this set of experiments blocked about half of the uptake; 3) for 10 min at 25 "C in Ca2'-free solution, an incubation period which is sufficient to activate only part of the channels (cf. Fig. 3 in Ref. 15), but is long enough to enable 8Br-CAMP to induce maximal transport changes (34). It was found that under none of the above conditions could 8Br-CAMP increase the apical permeability measured in vesicles (Table IV). Thus, the stable transport changes induced by Ca" at 25 "C are not affected by the cellular level of CAMP.

DISCUSSION
The present paper examines the effects of Ca" ions interacting with whole, permeabilized, toad bladder cells on the channel-mediated 22Na+ fluxes assayed in isolated membrane vesicles. As before (15) we observed that the amiloride-sensitive Na+ flux in vesicles is large if cells are preincubated at 25 "C in a Ca2+-free EGTA solution and is greatly reduced if the cells are exposed to 10" M Ca". By monitoring the full time course of tracer uptake and measuring the steady state %Rb+ distribution, we were able to exclude the possibilities that inhibition of transport in vesicles results from depolari-zation of the membrane or a decrease in the vesicular volume. The data of Table I1 do not totally exclude the possibility that Ca2+ ions act by preventing the vesiculation of a membrane fraction which is so minor as not to be detected by 86Rb+ uptake, but happens to contain all the Na+ channels. However, such an effect induced by a transient exposure of whole cells to Ca2+ seems unlikely. The likely remaining possibility is that Ca" induces a reaction which lowers the Na+ permeability of the apical membrane. This putative closure of channels should not be confused with the inhibition observed by interaction of the isolated membrane with Ca2+ ions (14,16). We too observed a direct inhibition of channels by including Ca2+ ions in the isolated vesicles, and this process is different from the currently discussed effect in many aspects?
The inhibition of transport could not be reversed by transferring cells from the Ca2+ incubation medium to a Ca2+-free EGTA solution (Table I). One explanation for this irreversibility is that the prolonged incubation of permeabilized cells in the homogenizing buffer depletes them of a factor required for the activation of channels upon the removal of Ca2+. Similarly we observed that Ca" ions applied to cells preincubated for 30 min in EGTA induced only a partial inhibition of "Na+ flux (cf. Table IIID in Ref. 15). An alternative explanation is that the recovery from an increase in the cytoplasmic Ca2+ activity involves relatively slow processes and cannot be detected within h. Prolonged effects induced by a transient increase of cell Ca2+ were observed in other systems too (35). The apparent irreversibility of the inhibition also raises the possibility that the permeability change reflects unspecific damage to the apical surface by a Ca2+-activated lipase or protease, released to the external medium from lysed cells. This potential artifact is excluded by the observation that the Ca2+-dependent down-regulation of channels cannot be induced in broken cell suspensions. The fact that the inhibition of transport depends on the cell integrity indicates that it either involves structural components which are destroyed by the shearing of cells (e.g. membrane-cytoskeleton or plasma-membrane-internal vesicles interactions) or requires a soluble component which is diluted too much during the homogenization.
The Ca" dose-response relationships were measured by incubating aliquots of scraped, permeabilized cells with different Ca2+/EGTA mixtures. This procedure yielded inhibition curves characterized by an Hill coefficient of 3.5-4.0 and two different values of Ki. The variability in the measured Ki is too large to be accounted for by EGTA impurities (27) or experimental errors. It was also independent of the source of chemicals used or the cell incubating pH. The most likely explanation for the different Ki values obtained in different experiments is spontaneous variations in the concentration of cellular Ca2+ binding proteins or in their affinity to this ion. One example for such behavior is the dramatic effect of the lipid composition on the activation of protein kinase C by Ca2+ (28). The possibility that Ca2+ blocks channels by inducing protein phosphorylation is supported by the inhibitory effect of V02+. On the other hand, the fact that VO: and 4phorbol 12-myristate 13-acetate had no effect on the Ca2+ inhibition curve argues against this view. Regardless of the measured value of the inhibition constant, it is clear from the data that closure of Na+ channels is induced by Ca2+ concentrations which are within the physiological range of intracellular Ca2+ and the process is highly cooperative. The strong dependence of Na+ transport on cell Ca2+ means that large H. Garty and C. Asher, manuscript in preparation.
permeability changes can be induced by small variations in cell Ca". This point is of special importance since recent estimations of the intracellular Ca2+ in toad bladder cells failed to indicate large changes in its activity in response to changes in the external osmolarity, Na+ activity, or the presence of vasopressin (33,36,37).
In an attempt to identify the Ca2+-activated reaction which down-regulates Na+ channels we examined the influence of various reagents, added to the cell incubating medium, on the transport in vesicles. Most of the reagents tested should readily permeate the cell membrane and reach their potential intracellular target site within minutes. Two others, V02+ and colchicine, are less permeable but may enter the scraped, permeabilized cells. In addition, the bladders were exposed to colchicine for 4 h, a period that was sufficient to evoke the inhibitory effect of this drug on the hydroosmotic response (38). None of the tested reagents besides vanadyl appeared to have an effect on the base-line channel-mediated flux or on the ability of Ca2+ ions to inhibit it. Thus, the current data do not support the possibilities that the transport changes measured in this study require a Ca2+-calmodulin interaction, involve methyl transfer reaction, or are mediated by Ca2+dependent membrane-cytoskeleton interactions. In addition, 8Br-CAMP failed to enhance the 22Na+ uptake in vesicles or reduce its inhibition by Ca2+ ions, in spite of the well established augmentation in Na+ transport induced by this cyclic nucleotide in intact bladders. This result suggests that the mechanism we have identified is different from the one involved in the natriferic response to antidiuretic hormones. Alternatively, it is possible that CAMP mediates its action on Na+ permeability by lowering Ca?: and, thus, is ineffective once the cell Ca" had been buffered to a given value, by suspending permeabilized cells in the Ca2+/EGTA mixture.
Complete inhibition of the channel-mediated flux in vesicles was also induced by incubating cells with 1 mM V02+, and vanadyl also abolished transport in vesicles derived from cells that were incubated with submaximal concentration of Ca2+. From the fact that an equivalent amount of vanadate had no effect on "Na+ uptake, one may conclude that the active ion is indeed V02+ and not one of its oxidation products (39). The observed effects of vanadyl may be accounted for by at least 3 mechanisms. One possibility is that V02+ acts as a "Ca" analog" and activates the same process triggered by Ca2+ ions. A second possibility is that V02+ causes a transient increase of the intracellular level of free Ca2+, either by blocking Ca2+-ATPase or by displacing bound Ca2+. The possibility that V02+ displaces Ca2+ bound to EGTA can, however, be excluded since full inhibition is observed upon incubation of cells with 10 mM EGTA, 1 mM V02+, and no added Ca2+. Finally it is possible that vanadyl may act by inhibiting a phosphatase (40). According to this interpretation Na+ channels are activated by protein dephosphorylation and can be down-regulated either by inhibiting this process with V02+ or activating phosphorylation with Ca2+.
In summary, the above data establish the finding that Ca2+ can down-regulate Na+ channels by activating a process which takes place in whole cells only but induces a stable modification of a membrane component preserved by the isolated vesicles. This process is presumably involved in the physiological control of Na+ transport, but the,molecular events which mediate it are as yet unknown.