Evidence for a phorbol ester-insensitive phosphorylation step in capacitative calcium entry in rat thymic lymphocytes.

Experiments were undertaken to investigate the regulation of capacitative Ca2+ entry by phorbol ester-sensitive protein kinase C and serine/threonine protein phosphatase activity. The thapsigargin-activated Ca2+ entry pathway was probed in control cells and cells treated with phosphatase type 1/2A inhibitors, okadaic acid and calyculin A, or with the phorbol ester, phorbol 12-myristate 13-acetate. The permeability state of this pathway was monitored in the presence or absence of these agents using fluorometric measurements of intracellular Ca2+ concentration, unidirectional Mn2+ entry, and membrane potential and unidirectional measurements of Ca2+ uptake using 45Ca2+. The results of these studies demonstrate that modification of the phosphorylation state of target protein(s) on serine/threonine amino acid residues by inhibition of phosphatase type 1/2A inhibits the capacitative Ca2+ entry pathway in rat thymic lymphocytes. Importantly, the capacitative Ca2+ entry pathway in rat thymic lymphocytes is not modulated by activation of phorbol ester-sensitive protein kinase C.

While it is generally accepted that the Ca 2ϩ permeability of the plasma membrane in nonexcitable cells is regulated in large part by the degree of filling of the intracellular storing compartments (capacitative Ca 2ϩ entry), the mechanisms underlying this phenomenon remain obscure. A wide range of mechanisms have been proposed or implicated in the communication step and have been discussed in detail in recent reviews on this subject (1,2).
Several of the proposed mechanisms describe events whereby protein phosphorylation plays an important modulatory role in the control of capacitative Ca 2ϩ entry, either at the level of the communication step or at the Ca 2ϩ entry pathway. However, many of the reports implicating phosphorylation/ dephosphorylation events in the regulation of capacitative Ca 2ϩ entry appear to be contradictory. For example, a number of studies have demonstrated an enhancement of the Ca 2ϩ entry pathway using phosphatase inhibitors that promote serine/threonine phosphorylation (3)(4)(5). Consistent with this notion, it has been reported that modest activation of phorbol ester-sensitive PKC 1 enhances capacitative Ca 2ϩ entry in oo-cytes and a pancreatic cell line (6,7). In contrast, phorbol ester activation of PKC has been shown to inhibit capacitative Ca 2ϩ entry in the lymphocytic cell line Jurkat, human neutrophils, and peripheral T-lymphocytes, Drosophila photoreceptors, and rat basophilic leukemia cells (8 -12). In accordance with an inhibition of capacitative Ca 2ϩ entry by hyperphosphorylation of serine/threonine residues, serine/threonine phosphatase inhibitors have been reported to inhibit this pathway (13). Clearly, the role of phosphorylation in the modulation of capacitative Ca 2ϩ entry is not consistent, and it may represent differences between cell types.
The existence of capacitative Ca 2ϩ entry in rat thymic lymphocytes has been well documented. Release of endosomal Ca 2ϩ via inhibition of endosomal Ca 2ϩ -ATPase activity, elevations in inositol 1,4,5-trisphosphate, or brief incubation in Ca 2ϩ -free medium activates an indistinguishable plasma membrane Ca 2ϩ influx pathway (14 -18). Recent work from our laboratory is indicative of a role of high energy phosphate donors in the activation and sustained activation of the capacitative Ca 2ϩ entry pathway in this cell type (19). Such a finding may be indicative of an important modulatory role of protein phosphorylation in the control of capacitative Ca 2ϩ entry in rat thymic lymphocytes. The purpose of the present experiments was to investigate the regulation of capacitative Ca 2ϩ entry by phorbol ester-sensitive PKC and serine/threonine protein phosphatase activity. To address these questions we have utilized non-invasive fluorometric and isotopic techniques.

EXPERIMENTAL PROCEDURES
Reagents and Solutions-The AM derivatives of indo-1 and BCECF were purchased from Teflabs (Austin, TX). The AM derivatives of Bapta and EGTA, and bis-oxonol were purchased from Molecular Probes (Eugene, OR). Ionomycin, thapsigargin, and HEPES were obtained from Calbiochem. Gramicidin and EGTA were purchased from Sigma. Charybdotoxin was purchased from Peninsula Laboratories (Belmont, CA). NMG, NiCl 2 hexahydrate, and Me 2 SO were purchased from Aldrich. NaCl, KCl, CaCl 2 , MgCl 2 , MnCl 2 , D-glucose, NaOH, and KOH were purchased from Fisher. Nor-okadaone, calyculin A, and the Na ϩ salt of okadaic acid were purchased from LC Laboratories (Woburn, MA). PMA was purchased from multiple vendors. Stock solutions of indo-1-AM, Bapta-AM, thapsigargin, PMA, and gramicidin were made up in Me 2 SO. Ionomycin, nor-okadaone and calyculin A were dissolved in ethanol.
The basic Na ϩ medium employed in the fluorescence experiments contained 140 mM NaCl, 3 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM D-glucose, and 20 mM HEPES-free acid, titrated to pH 7.3 at 37°C with NaOH. Ca 2ϩ -free solutions were made by omitting Ca 2ϩ and adding 200 M EGTA. NMG ϩ medium was made by equimolar replacement of sodium. When Ca 2ϩ and Mn 2ϩ were added to cell suspensions, these divalents were added as chloride salts. All solutions and stocks were stored at Ϫ20°C.
Cell Isolation and Bapta Loading-Thymic lymphocytes were iso-lated from 120 -200-g male Wistar rats (Charles River Breeding Laboratories) as described previously (20). The cells were counted using a model ZM Coulter Counter (Coulter Electronics, Hialeha, FL) and maintained at room temperature in basic Na ϩ medium. Where required, the rate of change of [Ca 2ϩ ] i was slowed by loading cells with the Ca 2ϩ chelator Bapta as previously reported (15,16,18,21). This procedure also facilitated the detection of E m and prolonged the linear phase of Ca 2ϩ uptake in 45 Ca 2ϩ experiments. Cells were loaded by incubation with 20 M of the AM derivative of Bapta at 37°C in basic Na ϩ medium. To control for differences in the efficacy of Bapta loading, all comparisons between experimental conditions were made in cells taken from the same batch of Bapta-loaded cells.
Fluorescence Determinations-All experiments were performed at 37°C using a Photon Technology International fluorescence spectrophotometer (Delta Scan) equipped with a magnetic stirrer and temperature control. The cells were counted immediately after the last manipulation, prior to addition to the cuvette, to ensure that the appropriate number were added.
Determination of Free Cytosolic Calcium Concentration-[Ca 2ϩ ] i was determined by measuring the fluorescence of indo-1 as previously reported by our laboratory (15,16). The excitation and emission wavelengths used were 331 nm (3-nm slit width) and 405 nm (10-nm slit width), respectively. Cell suspensions (25 ϫ 10 6 cells/ml) were loaded with indo-1 by incubation with a 4 M concentration of the AM precursor for 30 min at 37°C in basic Na ϩ medium. When necessary, cells were simultaneously loaded with Bapta. The cells were then sedimented, resuspended in basic Na ϩ medium, and kept at room temperature until required. Cells were added to the cuvette at a final concentration of 2 ϫ 10 6 cells/ml. Fluorescence was calibrated using ionomycin and Mn 2ϩ as described previously (15,16,22) using a dissociation constant of 250 nM (23).
Determination of Mn 2ϩ Influx-Mn 2ϩ uptake was monitored as the rate of quenching of indo-1 fluorescence at the isosbestic point for the Ca 2ϩ -indo-1 complex (excitation 331 nm (3-nm slit width), emission 443-447 nm (10-nm slit width)) determined daily under our experimental conditions. When measured at the isosbestic wavelength, the rate of fluorescence decrease is insensitive to changes in the [Ca 2ϩ ] i and is proportional to the rate of Mn 2ϩ accumulation in the cytosol (15,16).
Determination of E m -Determinations of E m were made by measuring the fluorescence of the negatively charged dye bis-oxonol (24) at excitation and emission wavelengths of 540 nm (3-nm slit width) and 580 nm (10-nm slit width), respectively, as described previously (18,21,25). Cells were added to the cuvette at a final concentration of 2 ϫ 10 6 cells/ml. bis-Oxonol was then added to a final concentration of 0.15 M, and fluorescence was monitored. External calibration was made by adding 40 nM gramicidin to cells suspended in media containing varying ratios of Na ϩ and NMG ϩ . A calibration curve was constructed as described previously (18 -21) assuming comparable gramicidin-induced Na ϩ and K ϩ conductances and/or that intra-and extracellular concentrations of alkali cations are identical at equilibrium.
Determination of Cytosolic pH-Intracellular pH was measured fluorimetrically using BCECF as previously reported (19,26). The excitation and emission wavelengths used were 495 nm (3-nm slit width) and 525 nm (10-nm slit width), respectively. Loading with BCECF was achieved by incubating 25 ϫ 10 6 cells/ml with 2 g/ml of the AM precursor for 30 min at 37°C. To monitor fluorescence, aliquots containing the required cell number were sedimented, resuspended in the appropriate medium devoid of Ca 2ϩ , and added to the cuvette at a final concentration of 2 ϫ 10 6 cells/ml. Calibration curves of pH against fluorescence were generated by lysing the cells with Triton X-100 (0.05% (v/v) final) at the end of the experiment and titrating the medium by the addition of small amounts of NaOH or HEPES while measuring the solution pH with a semimicro combination pH electrode (Accu-pHast, Fisher Scientific). A correction factor was independently determined for each batch of BCECF-loaded cells to offset the red shift undergone by the dye inside the cells (20,25). 45 Ca 2ϩ Influx Determinations-Cells were Bapta-loaded as detailed above, sedimented, and kept at room temperature until required. All experiments were conducted at 37°C. Unidirectional Ca 2ϩ uptake was measured essentially as described previously (15,16) with the following exceptions.
For all uptake measurements, 250 ϫ 10 6 cells treated as described above were sedimented, resuspended in 650 l of Ca 2ϩ -free Na ϩ medium, and incubated at 37°C for a further 5 min in the presence of 100 nM thapsigargin. 600 l of the suspension was transferred to a 1.5-ml microcentrifuge tube, while the remaining 50 l was saved for cell counting. Uptake was initiated by the addition of 400 l of appropriate solution containing concentrations of cold and hot Ca 2ϩ to result in a final [Ca 2ϩ ] of 4 mM and an isotope concentration of 10 Ci/ml. In experiments using the phorbol ester, PMA, cells were exposed to this agent at the initiation of Ca 2ϩ uptake (time zero).
For comparison purposes, basal Ca 2ϩ uptake rates were also determined in cells incubated for identical time periods in solution containing 1 mM Ca 2ϩ and devoid of thapsigargin.
Treatment with Phosphatase Inhibitors-Cells treated with 500 nM okadaic acid, 500 nM nor-okadaone, or 100 -200 nM calyculin A were incubated for 30 min at 37°C in normal Na ϩ solution unless stated otherwise. Control cells were treated with vehicle in parallel experiments devoid of either phosphatase inhibitor.
Data are presented as representative records and/or the mean Ϯ S.E. of the number of experiments indicated. Statististical analysis was performed using Student's paired t test or one way analysis of variance as appropriate.

RESULTS
We have monitored the effects of phorbol ester stimulation of PKC on capacitative Ca 2ϩ entry activated by the addition of the endosomal Ca 2ϩ -ATPase inhibitor thapsigargin. The increase in [Ca 2ϩ ] i measured fluorometrically with indo-1 following the addition of 33 nM thapsigargin to cells suspended in NMG ϩ medium containing 1 mM Ca 2ϩ is shown in Fig. 1A. Thapsigargin treatment results in a marked increase in [Ca 2ϩ ] i that declined somewhat to a sustained plateau level. At this stable value, the phorbol ester, PMA (100 nM) was added. Fig. 1A shows that the addition of PMA results in a modest reduction in [Ca 2ϩ ] i , consistent with either a minor decrease in Ca 2ϩ entry or a PMA-mediated increase in Ca 2ϩ efflux in accordance with the documented stimulation of the plasma membrane Ca 2ϩ pump by phorbol esters (27,28).
To address whether the small changes in [Ca 2ϩ ] i occur as a result of changes in Ca 2ϩ influx or by altered efflux mechanisms, we have monitored unidirectional Mn 2ϩ influx as measured by indo-1 quench. When measured at the isosbestic wavelength, the rate of fluorescence decrease is insensitive to changes in the [Ca 2ϩ ] i and is proportional to the rate of Mn 2ϩ accumulation in the cytosol (15,16). Representative experi-  (Fig. 1B). The subsequent addition of 1 M ionomycin, which can transport Mn 2ϩ , abolished the remaining fluorescence. The addition of either 100 nM PMA or vehicle alone had no effect on the rate or magnitude of the fall in indo-1 fluorescence (Fig. 1B). To ensure that cells were exposed to PMA for a sufficient time to stimulate PKC, similar experiments were conducted in which cells were treated with PMA for 100 s prior to the addition of Mn 2ϩ . As shown in Fig. 1C, exposure to PMA for 100 s, under conditions where [Ca 2ϩ ] i is elevated due to capacitative Ca 2ϩ entry, failed to alter the rate or magnitude of Mn 2ϩ influx. Such results are not consistent with inhibition of the capacitative Ca 2ϩ entry pathway by PMA.
Given the lack of effect of phorbol esters, it was necessary to test the efficacy of this dose of PMA to stimulate PKC. To address this issue, identical experiments to those described above were conducted to monitor the activity of the Na ϩ /H ϩ antiporter known to be present in this cell type (20). Previous studies have demonstrated the ability of phorbol ester-stimulated PKC activity to enhance basal activity of the Na ϩ /H ϩ antiporter, resulting in modest alkalinization of cells (29 -31), which can be inhibited either by PKC depletion (30,31) or by pharmacological inhibitors of PKC (29,31). The pH changes associated with the addition of PMA were measured using the pH-sensitive fluorescent dye BCECF. As is evident in Fig. 1D, the addition of PMA following activation of capacitative Ca 2ϩ entry with thapsigargin results in an increase in intracellular pH. In the absence of external Na ϩ (equimolar substitution of Na ϩ by NMG ϩ ) no change in intracellular pH was detected following PMA addition (data not shown), consistent with the alkalinization observed in Na ϩ medium occurring as a consequence of increased Na ϩ /H ϩ antiporter activity as previously reported (29 -31). These data confirm the efficacy of PMA to stimulate PKC activity under our experimental conditions.
To directly address the question of whether the modest fall in [Ca 2ϩ ] i seen in Fig. 1A was due to altered influx via the capacitative Ca 2ϩ entry pathway, we have measured unidirectional 45 Ca 2ϩ uptake in thapsigargin-treated Bapta-loaded cells in the absence and presence of 100 nM PMA. Cells were incubated for 5 min at 37°C in Ca 2ϩ -free NMG ϩ solution in the presence of 100 nM thapsigargin to ensure maximal depletion of intra-cellular Ca 2ϩ stores. Control cells were incubated under identical conditions in the absence of thapsigargin. Uptake was initiated by the addition of 4 mM Ca 2ϩ containing 10 Ci of 45 Ca 2ϩ , in the absence or presence of PMA. Fig. 2A shows the time course of 45 Ca 2ϩ uptake under these experimental conditions (n ϭ 4 experiments). Unidirectional uptake rates were calculated from the linear time course data presented in Fig.  2A and are shown in Fig. 2B. Unidirectional uptake rates show no effect of PMA in either thapsigargin-stimulated or control cells (Fig. 2B). To address the concern that the Ca 2ϩ chelator, Bapta, itself alters PKC activity or interferes with the actions of PMA, we have performed similar experiments in which cells were loaded with EGTA in place of Bapta and have found no significant effect of PMA on unidirectional Ca 2ϩ entry (data not shown). Taken in concert, these experiments are consistent with the hypothesis that activation of phorbol ester-sensitive PKC does not modulate thapsigargin-mediated Ca 2ϩ influx. Furthermore, phorbol ester treatment does not alter basal plasma membrane Ca 2ϩ permeability in rat thymic lymphocytes. However, these data do not preclude the involvement of phorbol ester-insensitive serine/threonine phosphorylation/dephosphorylation events in capacitative Ca 2ϩ entry.
To further investigate the role of serine/threonine protein phosphorylation in the regulation of the capacitative Ca 2ϩ entry pathway, we have investigated the effects of okadaic acid, a potent type 1/2A phosphatase inhibitor (32), on this pathway. Fluorescence experiments were conducted to monitor the changes in [Ca 2ϩ ] i in the presence and absence of okadaic acid (Fig. 3A). Following activation of the capacitative Ca 2ϩ entry pathway, reintroduction of 4 mM Ca 2ϩ to Bapta-loaded control cells resulted in an increase in [Ca 2ϩ ] i from 172 Ϯ 6 nM to 1023 Ϯ 71 nM (ϮS.E., n ϭ 12 experiments). In contrast, Ca 2ϩ uptake via capacitative Ca 2ϩ entry in cells treated with 500 nM okadaic acid for 30 min was markedly inhibited, showing an increase in [Ca 2ϩ ] i to only 348 Ϯ 21 nM from a resting value of 165 Ϯ 6 nM (n ϭ 12, p Յ 0.05) (Fig. 3A). To ensure that this inhibitory effect was due to the ability of okadaic acid to inhibit protein phosphatases and not some ancillary effect, identical experiments were performed using the related analogue norokadaone, which exhibits little or no inhibitory effect upon 1/2A phosphatase activity (33). Fig. 3A shows the results of such an experiment. These data illustrate the lack of effect of norokadaone upon the Ca 2ϩ entry pathway. Experiments were undertaken to address the possibility that prior incubation with okadaic acid interferes with the ability of thapsigargin to deplete intracellular Ca 2ϩ stores and hence, the activation of capacitative Ca 2ϩ entry. Indo-1-loaded cells were treated with okadaic acid for 30 min in normal Na ϩ medium prior to exposure to thapsigargin in Ca 2ϩ -free medium. Treatment with okadaic acid failed to alter the magnitude or kinetics of the transient increase in [Ca 2ϩ ] i attributable to the release of the intracellular Ca 2ϩ stores by thapsigargin (data not shown). These findings are consistent with the conclusion that okadaic acid does not interfere with the ability of thapsigargin to deplete intracellular Ca 2ϩ stores and, hence, its ability to activate capacitative Ca 2ϩ entry.
To exclude the possibility that the inhibition by okadaic acid of the increase in [Ca 2ϩ ] i presented in Fig. 3A is due to stimulation of Ca 2ϩ extrusion mechanisms, measurements of unidirectional Ca 2ϩ uptake were performed. Bapta-loaded cells were incubated in basic Na ϩ medium for 30 min at 37°C in the presence or absence of 500 nM okadaic acid. Cells were further incubated in the presence or absence of okadaic acid for 5 min at 37°C in Ca 2ϩ -free Na ϩ medium in the presence of 100 nM thapsigargin to ensure maximal depletion of intracellular Ca 2ϩ stores. To investigate the effect of okadaic acid on basal Ca 2ϩ influx, cells that were not treated with thapsigargin were incubated under identical conditions in the presence and absence of okadaic acid. In all cases, uptake was initiated by the addition of 4 mM Ca 2ϩ containing 10 Ci of 45 Ca 2ϩ . Fig. 3B shows the time course of 45 Ca 2ϩ influx upon readdition of 4 mM Ca 2ϩ under these experimental conditions (n ϭ 4 experiments). Uptake rates were calculated from the time course data and are presented in Fig. 3C. The unidirectional uptake rate accompanying activation of capacitative Ca 2ϩ entry by thapsigargin was markedly reduced from a value of 830 Ϯ 80 pmol/min/15 ϫ 10 6 cells (ϮS.E., n ϭ 4) to a value of 160 Ϯ 10 pmol/min/15 ϫ 10 6 cells (ϮS.E., n ϭ 4, p Յ 0.05) in cells pretreated with okadaic acid. Control cells that were not exposed to thapsigargin, and hence displayed no stimulated capacitative Ca 2ϩ entry, showed no difference in basal Ca 2ϩ uptake rates between okadaic acid-treated and -untreated cells (Fig. 3B). The inhibitory effect of okadaic acid on capacitative Ca 2ϩ entry is consistent with a requirement for sustained protein phosphatase activity for the continued activation of the Ca 2ϩ influx pathway.
To further address this hypothesis we have investigated the effect of calyculin A, a phosphatase inhibitor structurally distinct from okadaic acid (32), on capacitative Ca 2ϩ entry. A 30-min prior exposure to 100 nM calyculin A resulted in almost total inhibition of the changes in [Ca 2ϩ ] i measured fluorometrically following reintroduction of 4 mM Ca 2ϩ to thapsigargintreated Bapta-loaded cells (Fig. 4A). Reintroduction of Ca 2ϩ to cells following incubation with calyculin A resulted in an increase in [Ca 2ϩ ] i to only 183 Ϯ 12 nM from a resting value of 153 Ϯ 12 nM, compared with the increase in [Ca 2ϩ ] i seen in control cells, from a value of 166 Ϯ 9 to 1011 Ϯ 182 nM (n ϭ 3, p Յ 0.05) (Fig. 3A). In agreement with the observations using okadaic acid, pretreatment of indo-1-loaded cells with calyculin A for 30 min failed to alter the magnitude or kinetics of the transient increase in [Ca 2ϩ ] i attributable to the release of the intracellular Ca 2ϩ stores by thapsigargin in Ca 2ϩ -free medium (data not shown). These data indicate that calyculin A does not interfere with the ability of thapsigargin to deplete intracellular Ca 2ϩ stores and, hence, its ability to activate capacitative Ca 2ϩ entry. In addition, 30-min preexposure to 200 nM calyculin A in normal Na ϩ medium totally abolished thapsigarginmediated 45 Ca 2ϩ influx (n ϭ 3 experiments) (Fig. 4B). Uptake rates were calculated from the time course data and are presented in Fig. 4C. Control cells that were not exposed to thapsigargin, and hence displayed no stimulated capacitative Ca 2ϩ entry, showed no difference in basal Ca 2ϩ uptake rates between calyculin A-treated and -untreated groups (Fig. 4C).
To address the possibility that inhibition of capacitative Ca 2ϩ entry by phosphatase inhibitors occurs only in Baptaloaded conditions, we have conducted experiments in which cells were loaded with indo-1 alone, under conditions in which the intracellular Ca 2ϩ pool was depleted with thapsigargin prior to the addition of calyculin A. Indo-1-loaded cells suspended in Ca 2ϩ -free Na ϩ medium were treated with thapsigargin in the presence of 150 nM charybdotoxin. Charybdotoxin was added to eliminate the Ca 2ϩ -dependent hyperpolarization brought about by the activation of Ca 2ϩ -activated K ϩ channels, known to be present in these cells (18,34). Following Ca 2ϩ store depletion, cells were exposed to calyculin A for 100 or 500 s prior to the readdition of 1 mM Ca 2ϩ . Representative results from such experiments are shown in Fig. 5 and demonstrate a time-dependent inhibition by calyculin A of the increases in [Ca 2ϩ ] i via capacitative Ca 2ϩ entry. In addition to demonstrating a clear time dependence for the inhibitory effect of calyculin A, these data allow us to rule out any effect of Bapta loading in the interpretation of the present results.
In order to ensure that the inhibitory effect of phosphatase inhibitors was not directly attributable to a collapse of resting E m , experiments were performed using bis-oxonol to measure E m in cells in the presence and absence of phosphatase inhibitors. Resting E m in Bapta-loaded cells suspended in Ca 2ϩ -free Na ϩ medium was not significantly different in control and inhibitor-treated cells (Ϫ59.3 Ϯ 5.5 mV in okadaic acid treated cells and Ϫ58.0 Ϯ 1.5 mV in paired control cells (n ϭ 3); Ϫ59.5 Ϯ 2.6 mV in calyculin A-treated cells and Ϫ54.8 Ϯ 2.1 mV in paired control cells (n ϭ 4)). On the strength of these results it is not possible to attribute the inhibitory effect of either of these agents to differences in E m .
Experiments were performed to investigate the effect of phosphatase inhibition on the depolarization previously attributed by our laboratory to electrogenic Ca 2ϩ influx via capacitative Ca 2ϩ entry (18,19,21). The capacitative Ca 2ϩ entry pathway was activated in control cells and in cells treated with calyculin A (100 nM) for 10 min, and E m was monitored fluorometrically using bis-oxonol. Fig. 6 shows representative E m changes associated with the addition of 4 mM Ca 2ϩ to control cells suspended in Ca 2ϩ -free medium containing 150 nM charybdotoxin to inhibit Ca 2ϩ -dependent K ϩ channel activity. Ca 2ϩ addition to control cells resulted in a depolarization of 30.6 Ϯ 5.5 mV (n ϭ 7). Consistent with the inhibitory effect of calyculin A on changes in [Ca 2ϩ ] i and unidirectional Ca 2ϩ uptake, the addition of 4 mM Ca 2ϩ to calyculin-treated cells resulted in a significantly smaller depolarization of 10.0 Ϯ 4.2 mV (n ϭ 4, p Յ 0.05) (Fig. 6). Okadaic acid treatment also significantly attenuated the depolarization observed upon readdition of Ca 2ϩ to the external medium although to a lesser degree than that seen following calyculin A treatment, producing a maximum depolarization of 19.7 Ϯ 1.5 mV (n ϭ 3, p Յ 0.05).

DISCUSSION
The present results are consistent with the regulation of capacitative Ca 2ϩ entry by a dephosphorylation event in rat thymic lymphocytes. This conclusion is based upon the finding that the structurally distinct type 1/2A serine/threonine phosphatase inhibitors, okadaic acid and calyculin A, dramatically inhibit the Ca 2ϩ influx induced by thapsigargin-mediated release of Ca 2ϩ from intracellular stores. In addition, the E m depolarization previously attributed to an increase in plasma membrane Ca 2ϩ conductance following activation of capacitative Ca 2ϩ entry (18,19,21) is markedly attentuated when cells are treated with okadaic acid or calyculin A. While it is not possible to completely rule out a direct inhibitory influence of these agents on capacitative Ca 2ϩ entry independent of their

FIG. 5. Time-dependent inhibition of thapsigargin-mediated rises in [Ca 2؉ ] i by calyculin A.
Indo-1-loaded rat thymic lymphocytes were suspended in Ca 2ϩ -free Na ϩ medium containing 150 nM charybdotoxin and 200 M EGTA, and fluorescence of indo-1 was monitored. Cells were exposed to 33 nM thapsigargin for 5 min prior to the addition of 100 nM calyculin for 100 (A) or 500 (B) seconds before the addition of Ca 2ϩ . Control cells were exposed to thapsigargin for 5 min prior to the addition of vehicle alone for 500 s before the addition of Ca 2ϩ (C). Where indicated, 1 mM Ca 2ϩ was added.
inhibitory effect on type 1/2A phosphatase activities, two pieces of evidence argue against such a postulate. First, nor-okadaone, a compound structurally related to okadaic acid but possessing little or no ability to inhibit type 1/2A phosphatase activity (33) has no effect on Ca 2ϩ influx mediated via depletion of Ca 2ϩ i stores. Second, the degree of inhibition produced by calyculin A demonstrates a clear time dependence, inconsistent with a simple blocking effect of this agent on the Ca 2ϩ influx pathway.
Interestingly, activation of phorbol ester-sensitive PKC activity by exposure to PMA had no effect upon thapsigarginmediated Ca 2ϩ influx measured fluorimetrically using Mn 2ϩ as a Ca 2ϩ surrogate or directly as unidirectional Ca 2ϩ influx determined isotopically. This lack of effect was found despite the ability of PMA to induce a Na ϩ -dependent alkalinization previously shown to be a result of PKC-stimulated Na ϩ /H ϩ antiport activity (29). Taken in concert, these data are consistent with the regulation of capacitative Ca 2ϩ entry by phorbol esterinsensitive serine/threonine kinase activity. The marked and relatively rapid onset of inhibition of capacitative Ca 2ϩ entry following the addition of calyculin A highlights a potentially critical point of modulation of this Ca 2ϩ influx pathway. Interestingly, okadaic acid and calyculin A were ineffective in activating capacitative Ca 2ϩ entry on their own. Such a result points to a important modulatory role of capacitative Ca 2ϩ entry by type 1/2A protein phosphatase activity independent of the activation mechanisms. However, modulation of type 1/2A phosphatase activity by events associated with depletion of Ca 2ϩ stores must not be ruled out.
While these data are consistent with an important modulatory role of a phorbol ester-insensitive serine/threonine kinase activity in the modulation of capacitative Ca 2ϩ entry in thymic lymphocytes, alternative interpretations must be entertained.
Experiments were performed to ensure that the inhibitory effect of phosphatase inhibitors was not directly attributable to a collapse of resting E m . This is especially important in the light of the fact that capacitative Ca 2ϩ entry occurs via an inwardly rectifying pathway in the range 0 to Ϫ53 mV in this cell type. 2 As a result of this rectification, depolarization will have a marked inhibitory effect on Ca 2ϩ influx. Measurements of E m rule out this possibility, given that no difference in resting E m in control or phosphatase inhibitor-treated cells was detected. The E m values measured in the present experiments are similar to previously reported normal E m values in rat thymic lymphocytes (18). Furthermore, such normal resting E m values ensure that the observed inhibition cannot be explained on the basis of diminished cell viability following exposure to either okadaic acid or calyculin A.
The possibility exists that the inhibition of the capacitative Ca 2ϩ entry pathway by phosphatase inhibitors occurs as a result of a direct inhibitory effect of these agents on the ability of thapsigargin to release Ca 2ϩ from the intracellular Ca 2ϩ stores. Such a phenomenon has previously been reported in rabbit platelets (35). To address this issue, we have conducted experiments in which thapsigargin was added to cells in Ca 2ϩfree medium in the presence and absence of okadaic acid or calyculin A. In the absence of extracellular Ca 2ϩ the magnitude of the transient rise in [Ca 2ϩ ] i is proportional to the amount of Ca 2ϩ released from the stores. We have found that a 30-min treatment with either calyculin A or okadaic acid failed to alter the magnitude or kinetics of the [Ca 2ϩ ] i changes following the addition of thapsigargin. As a result, the inhibition seen after the addition of phosphatase inhibitors cannot be explained in terms of interference with the ability of thapsigargin to deplete intracellular Ca 2ϩ stores.
Recent work from other laboratories has implicated protein phosphorylation in the control of capacitative Ca 2ϩ entry (for review see Ref. 2). Such regulation may occur either at the level of the communication step between the depleted Ca 2ϩ stores and the plasma membrane or at the Ca 2ϩ entry pathway itself. There is, however, a great deal of discrepancy between the results obtained. For example, two studies have demonstrated an enhancement of capacitative Ca 2ϩ entry following phorbol ester activation of PKC (6,7). In these studies, phorbol esters stimulated capacitative entry in the pancreatic cell line, RINmF5 (7), while low doses of PMA enhanced Ca 2ϩ influx in Xenopus oocytes (6). However, in the lymphocytic cell line Jurkat (8), human neutrophils (9) and peripheral T-lymphocytes (11), Drosophila photoreceptors (10), and rat basophilic leukemia cells (12), phorbol ester activation of PKC has been reported to inhibit the capacitative Ca 2ϩ entry pathway. These findings are in contrast to the present study, in which no effect of phorbol ester was found (see Figs. 1 and 2).
Previous work using type 1/2A phosphatase inhibitors has also yielded conflicting results. In accordance with an observed inhibition of the capacitative Ca 2ϩ entry pathway by phorbol ester stimulation of PKC, an inhibition of this pathway by phosphatase inhibitors has been reported in human neutrophils (13). Such a finding is in agreement with the present study in which we demonstrate a dramatic inhibition of the capacitative Ca 2ϩ entry pathway using identical phosphatase inhibitors. In contrast, Ca 2ϩ influx mediated by the putative "Ca 2ϩ influx factor" has been shown to be enhanced following treatment with type 1/2A phosphatase inhibitors that promote serine-threonine phosphorylation (3)(4)(5).
The present study indicates the existence of a PP1/2A-mediated dephosphorylation event in the maintenance of the capacitative Ca 2ϩ entry pathway. In contrast to other cell types, this inhibition resulting from enhanced phosphorylation of serine/ threonine residues is not subject to exacerbation by phorbol ester-sensitive serine/threonine kinases. These data could be explained by the existence of a phorbol ester-insensitive PKC isotype that exerts an inhibitory effect upon the Ca 2ϩ influx pathway. Interestingly, the dominant isotypes of PKC in rat thymic lymphocyte are the "conventional" isotype ␣, and the phorbol-insensitive, "atypical," isotype , as determined by immunoblot analysis. 3 Given the presence of the isotype, it is tempting to speculate on a role for this PMA insensitive kinase in the serine/threonine phosphorylation-mediated inhibition of capacitative Ca 2ϩ entry as evidenced by its susceptibility to PP1/2A inhibitors. As a corollary, such a marked and rapid FIG. 6. Effect of calyculin A on the E m changes attributable to capacitative Ca 2؉ entry following reintroduction of Ca 2؉ . Baptaloaded cells were incubated for 30 min in normal Na ϩ medium in the absence (Control) or presence (Calyculin) of 100 nM calyculin A. Cells were then suspended in Ca 2ϩ -free Na ϩ medium containing 150 nM charybdotoxin and 200 M EGTA, and exposed to 33 nM thapsigargin for 5 min while bis-oxonol fluorescence was monitored. Where indicated, 4 mM Ca 2ϩ was added. Experiments were performed with cell aliquots taken from the same Bapta-loading procedure. sensitivity of the pathway to phosphatase inhibitors may implicate the control of phosphatase activity as being an important site in the overall regulation of capacitative Ca 2ϩ entry.
The data presented in this study suggest that modification of phosphorylation of target protein(s) on serine/threonine amino acid residues exerts an important modulatory effect upon the capacitative Ca 2ϩ entry pathway but that this phosphorylation event does not occur as a result of phorbol ester-sensitive PKC activity. Whether these results point to the existence of a phorbol ester-insensitive PKC isotype that provides a potent inhibitory influence on this pathway remains to be resolved, and further studies are required to address this issue.