1 a ,25-Dihydroxy-vitamin-D 3 -induced Store-operated Ca 2 1 Influx in Skeletal Muscle Cells MODULATION BY PHOSPHOLIPASE C, PROTEIN KINASE C, AND TYROSINE KINASES*

In skeletal muscle cells the steroid hormone 1 a ,25-dihydroxy-vitamin-D 3 (1,25(OH) 2 D 3 ) nongenomically promotes Ca 2 1 release from intracellular stores and cation influx through both L -type and store-operated Ca 2 1 (SOC) channels. In the present work we evaluated the regulation and kinetics of the 1,25(OH) 2 D 3 -stimulated SOC influx in chick muscle cells. Stimulation with 10 2 9 M 1,25(OH) 2 D 3 in Ca 2 1 -free medium resulted in a rapid (40–60 s) but transient [Ca 2 1 ] i rise, which correlated with sterol-dependent inositol 1,4,5-trisphosphate pro-duction. The SOC influx stimulated by the hormone was insensitive to both L -type channel antagonists and polyphosphoinositide-specific phospholipase C (PPI-PLC) inhibitors but was fully inhibitable by La 3 1 and Ni 2 1 . PPI-PLC blockade prior to 1,25(OH) 2 D 3 stimulation suppressed both the [Ca 2 1 ] i transient and the SOC influx. 1,25(OH) 2 D 3 -induced SOC entry was markedly increased after 3 min of treatment (30% above basal) and then rapidly reached a steady-state level. The sterol-stimulated SOC influx was prevented by protein kinase C and tyrosine kinase inhibitors but unaffected by blockade of the protein kinase A pathway. None of these inhibitors altered the thapsigargin-induced SOC entry, suggesting the operation of a signaling mechanism different

In skeletal muscle cells the steroid hormone 1␣,25dihydroxy-vitamin-D 3 (1,25(OH) 2 D 3 ) nongenomically promotes Ca 2؉ release from intracellular stores and cation influx through both L-type and store-operated Ca 2؉ (SOC) channels. In the present work we evaluated the regulation and kinetics of the 1,25(OH) 2 D 3 -stimulated SOC influx in chick muscle cells. Stimulation with 10 ؊9 M 1,25(OH) 2

D 3 in Ca 2؉ -free medium resulted in a rapid (40 -60 s) but transient [Ca 2؉ ] i rise, which correlated with sterol-dependent inositol 1,4,5-trisphosphate production. The SOC influx stimulated by the hormone was insensitive to both L-type channel antagonists and polyphosphoinositide-specific phospholipase C (PPI-PLC) inhibitors but was fully inhibitable by La 3؉ and Ni 2؉ . PPI-PLC blockade prior to 1,25(OH) 2 D 3 stimulation suppressed both the [Ca 2؉ ] i transient and the SOC influx. 1,25(OH) 2 D 3 -induced SOC entry was markedly increased after 3 min of treatment (30% above basal) and
then rapidly reached a steady-state level. The sterolstimulated SOC influx was prevented by protein kinase C and tyrosine kinase inhibitors but unaffected by blockade of the protein kinase A pathway. None of these inhibitors altered the thapsigargin-induced SOC entry, suggesting the operation of a signaling mechanism different from that for sterol-dependent SOC influx. The present results indicate that 1,25(OH) 2 D 3 -induced activation of PPI-PLC is upstream to Ca 2؉ influx through SOC channels and point for a role of both protein kinase C and tyrosine kinases but not protein kinase A in the regulation of the sterol-dependent SOCE pathway.
The steroid hormone 1␣,25-dihydroxy-vitamin-D 3 (1,25(OH) 2 -D 3 ) 1 modulates calcium homeostasis in skeletal muscle cells by two modes of action: a classical genomic one (long term responses) involving high affinity binding to an intracellular vitamin D receptor followed by association of the steroid-vitamin D receptor complex with vitamin D-responsive elements to elicit control of gene expression (1) and a nongenomic (rapid responses) mechanism that implies direct membrane effects of the hormone (2)(3)(4). Fast actions of 1,25(OH) 2 D 3 involve participation of diverse transmembrane signaling systems. In chick skeletal muscle cells the steroid rapidly modulates Ca 2ϩ influx by a complex mechanism that implies G protein-mediated activation of both PPI-PLC, thus generating diacylglycerol and IP 3 (5,6), and adenylyl cyclase with the concomitant acute increase in cyclic AMP levels (7). Altogether, these sterol-triggered signaling cascades drive, in an as yet not fully elucidated scheme, activation of PKA and PKC (5,(7)(8)(9), regulation of phospholipase A 2 and phospholipase D activities (10 -12), release of Ca 2ϩ from intracellular stores (13) ,and activation of voltage-dependent Ca 2ϩ channels (VDCC) pharmacologically identified as from the L-type on the basis of their sensitivity to dihydropyridines (14). These observations have led to the proposal that 1,25(OH) 2 D 3 activation of second messenger systems in muscle cells involves the existence of a putative cell surface receptor for the hormone (3), as proposed for this and other steroids in various cell types (15)(16)(17)(18). However, the existence of such a membrane receptor still remains as an open question. Recently, in a preliminary communication we examined the regulatory action of 1,25(OH) 2 D 3 on cytosolic Ca 2ϩ levels in skeletal muscle cells (13). The hormone triggers a complex [Ca 2ϩ ] i response that involves an initial rapid sterol-induced Ca 2ϩ mobilization from thapsigargin-sensitive internal stores followed by cation influx from the extracellular milieu. This hormone-induced Ca 2ϩ influx was shown to be contributed not only by the well established L-type VDCC-mediated Ca 2ϩ entry but also by a store-operated Ca 2ϩ influx (SOCE) pathway, thus introducing a novel aspect into the mechanism of 1,25(OH) 2 D 3induced Ca 2ϩ entry across the plasma membrane of muscle cells. SOCE has been observed in most cells that have been specifically examined, including both nonexcitable as well as excitable cell systems (19). Despite the plethora of messenger systems that have been related to SOCE regulation (20,21), mobilization and/or depletion of Ca 2ϩ from endogenous (both IP 3 -sensitive and -nonsensitive) stores seems to be an almost unavoidable step in activation of SOC channels.
In the present work we further evaluated SOC-mediated Ca 2ϩ influx in avian muscle cells by fluorimetry to analyze both the kinetics and regulation of the 1,25(OH) 2 D 3 -induced SOCE pathway in these cells. Sterol-induced activation of PPI-PLC seems to be a prerequisite for Ca 2ϩ influx through SOC channels to occur, apparently by mobilization and/or depletion of IP 3 -sensitive stores. Evidence points to a role for both PKC and tyrosine kinase (TK) activities but not the cyclic AMP/PKA cascade in the regulation of the sterol-dependent SOCE pathway. Interestingly, although a thapsigargin-sensitive intracellular Ca 2ϩ store apparently accounts for the 1,25(OH) 2 D 3 -induced, IP 3 -mediated Ca 2ϩ mobilization, different signaling mechanisms appear to be responsible for activation and/or regulation of sterol-and thapsigargin-dependent SOC influx.
Cell Culture-Undifferentiated, myogenic chick skeletal muscle cells (myoblasts) were isolated from the breast muscle of 13-day-old white leghorn chick embryos essentially as described before (14), seeded at appropriate density (120,000 cells/cm 2 ) in Petri dishes (80 mm) for IP 3 measurements or onto glass coverslips (24 ϫ 6 mm) for intracellular calcium measurements, and cultured at 37°C under a humidified atmosphere (air/5% CO 2 ). Cells were allowed to grow until confluence (4 -6 days after plating) before use. Under these conditions, myoblasts proliferate within the first 48 h and at the fourth day become differentiated into myotubes expressing both biochemical and morphological characteristics of adult skeletal muscle fibers. 2 Intracellular Calcium Measurements-Intracellular Ca 2ϩ changes were monitored by using the Ca 2ϩ -sensitive fluorescent dye Fura-2 (22). Cell dye loading was achieved by incubating the cells in buffer A containing (in mM): 138 NaCl, 5 KCl, 1 MgCl 2 , 5 glucose, 10 Hepes (pH 7.35), 1.5 CaCl 2 , plus 0.1% bovine serum albumin, 1 M Fura-2/pentaacetoxymethyl ester, and 0.012% pluronic F-127 in the dark during 45-60 min at room temperature. Unloaded dye was washed out, and cells were stored in buffer B (buffer A without bovine serum albumin, Fura-2/pentaacetoxymethyl ester, and pluronic F-127) in the dark at room temperature for at least 40 min prior to use. The coverslips were then placed into quartz cuvettes containing buffer B and introduced into the thermostatized (37°C) sample compartment of a SLM Aminco 8100 spectrofluorimeter (Spectronics Inc., Urbana, IL) under constant stirring. Fura-2 intracellular fluorescence intensity was monitored at an emission wavelength of 510 nm (8-nm band pass) by alternating the excitation wavelength between 340 and 390 nm with a dual excitation monochromator (4-nm band pass). Signals from short and long wavelength were divided (r ϭ 340/390), and calibration to calculate [Ca 2ϩ ] i was performed for each coverslip essentially as described previously by us (13). Maximal (R max ) and minimal (R min ) values were determined by adding 5 M ionomycin plus 3 mM Ca 2ϩ and 10 mM EGTA (pH 9.0), respectively. Under these conditions the dissociation constant (K d ) for the Ca 2ϩ -Fura-2 complex is assumed to be 225 nM, and [Ca 2ϩ ] i derives from (22) where R is the ratio of Fura-2 fluorescence at the selected wavelengths, R max and R min represent ratios from Ca 2ϩ -saturated and Ca 2ϩ -free intracellular dye, respectively, and ␤ is the ratio between the specific fluorescence of the Ca 2ϩ -free and Ca 2ϩ -bound forms of the dye at the longer wavelength (Sf 2 /Sb 2 ).
In some experiments, a Ca 2ϩ -free extracellular medium was used (free Ca 2ϩ concentration near 1 nM) by preparing a nominally Ca 2ϩ -free buffer B (see composition above) plus 1 mM EGTA. Free Ca 2ϩ levels were calculated by using the WinMaxc program (version 1.7; Ref. 23), with the association constants and K d for the EGTA-Ca 2ϩ complex corrected for pH, temperature, and ionic strength of the buffer (24,25). All buffers and saline solutions used were prepared with deionized water.
Measurement of Mn 2ϩ Influx-Fura-2 loaded cells were exposed to 1,25(OH) 2 D 3 or thapsigargin, and variations in [Ca 2ϩ ] i were monitored from the 340/390 fluorescence ratio. When [Ca 2ϩ ] i stabilized, dual wavelength excitation was changed to single wavelength excitation at 360 nm, and Mn 2ϩ influx was initiated (zero time for cation influx) by addition of 1 mM MnCl 2 to the medium. When the rate of Mn 2ϩ influx was measured, fluorescence quenching by Mn 2ϩ precluded calibration of the signals to calculate [Ca 2ϩ ] i . Maximal quenching (100%) at each condition was obtained by adding 0.05% Triton X-100. To compensate for differences in absolute fluorescence between experiments, the Mn 2ϩ influx was expressed as a percentage of quenching related to maximal cytosolic quenching.
Measurement of IP 3 Production-The cells were equilibrated in Krebs-Henseleit solution containing 10 mM glucose for 20 min at 37°C. Then 30 mM LiCl was added, and the incubation proceeded for an additional period of 10 min. The medium was aspirated, and vehicle (Ͻ0.1% ethanol) or 1,25(OH) 2 D 3 (10 Ϫ9 M) was added for the indicated times. Treatment was stopped by aspirating the medium and immediately freezing the cells in liquid air. Monolayers were then scraped off and transferred to plastic tubes, and ice-cold trichloroacetic acid (20% final concentration) was added. Samples were left on ice for 15 min and centrifuged at 1,000 ϫ g (10 min, 4°C). Trichloroacetic acid was extracted four times with 5 volumes of water-saturated diethylether. IP 3 was quantitated by a radioreceptor assay (26) using a commercially available kit.
Statistical Analysis-Statistical significance of data was evaluated using Student's t test (27), and probability values below 0.05 (p Ͻ 0.05) were considered significant. Quantitative data are expressed as the means Ϯ S.D. from the indicated set of experiments.

Effect of 1,25(OH) 2 D 3 on Intracellular Ca 2ϩ Levels in Skele-
tal Muscle Cells-Stimulation of chick muscle cells with 1,25(OH) 2 D 3 triggers a specific and rapid increase in [Ca 2ϩ ] i that stays elevated as long as the cells are exposed to the hormone ( Fig. 1 and Ref. 13). The plateau phase of this [Ca 2ϩ ] i response is due to Ca 2ϩ influx, partially contributed by a SOCE pathway. We next examined whether sterol-induced activation of PPI-PLC was needed for occurrence of such hormone-dependent SOC influx. When skeletal muscle cells were stimulated with 10 Ϫ9 M 1,25(OH) 2 D 3 in Ca 2ϩ -free extracellular medium, the hormone produced a rapid (40 -60 s) but transient [Ca 2ϩ ] i rise ( Fig. 2A) that was entirely due to mobilization from endogenous stores without contribution of cation influx, as there were no significant differences in average peak [Ca 2ϩ ] i FIG. 1. Intracellular Ca 2؉ response of chick skeletal muscle cells to 1,25(OH) 2 D 3 . Fura-2-loaded cells were exposed to 1,25(OH) 2 D 3 (10 Ϫ9 M, arrow) in Ca 2ϩ -containing (1.5 mM) medium, and intracellular Ca 2ϩ concentration was measured as described under "Experimental Procedures." A typical response to the hormone is observed (see also Ref. 13), with a fast rise in [Ca 2ϩ ] i due to store mobilization (see Fig. 2A) and a sustained phase corresponding to the influx pathway. Shown is a representative recording from at least 12 independent [Ca 2ϩ ] i recordings.
when Ca 2ϩ (1.5 mM; Fig. 1) or VDCC blockers (see Table I in Ref. 13) were present in the bath. Moreover, this sterol-induced Ca 2ϩ transient was totally blocked by pretreatment with the PPI-PLC inhibitors U73122 and neomycin ( Fig. 2B) but not by U73343 (not shown), an analogue of U73122 without effect on PPI-PLC (28), and was temporally correlated with the hormonedependent monophasic generation of IP 3 , which peaked at 60 s with a 2.3-fold induction over basal values (Fig. 3). To evaluate the hormone-dependent SOC influx we used the Ca 2ϩ readdition protocol, which has been shown to be a sensitive procedure to measure changes in Ca 2ϩ influx through the SOCE pathway (29,30). Fura-2 loaded cells were stimulated with 1,25(OH) 2 D 3 in the absence of extracellular Ca 2ϩ ; once the rapid and transient elevation in [Ca 2ϩ ] i occurred, Ca 2ϩ readdition (1.5 mM) was performed after [Ca 2ϩ ] i fell down to levels equal or close to basal. At this point, readdition of Ca 2ϩ resulted in a fast (30 -60 s) and sustained [Ca 2ϩ ] i rise (about 1.8 -2.2-fold over basal), thus evidencing Ca 2ϩ influx from the outside through a preactivated pathway ( Fig. 2A, right arrow). Ca 2ϩ readmission to cells not previously exposed to the sterol resulted in no detectable Ca 2ϩ influx (not shown). Functional isolation of a SOC entry pathway in excitable cells requires suppression of the large Ca 2ϩ influx that normally occurs through VDCCs. We achieved this by adding either nifedipine or verapamil at concentrations known to effectively block VDCC-mediated Ca 2ϩ influx in our cell system (5, 14) after the 1,25(OH) 2 D 3 -induced [Ca 2ϩ ] i transient but 2 min prior to Ca 2ϩ readmission. This did not affect the Ca 2ϩ influx that normally follows Ca 2ϩ readdition (Fig. 4); however, it was fully inhibited by La 3ϩ (10 M) and Ni 2ϩ (2 mM), which are known to block SOC influx in nonexcitable cell systems (31,32). Thus, the Ca 2ϩ entry observed by the Ca 2ϩ -free/Ca 2ϩ readdition protocol mainly reflects Ca 2ϩ entering the cell via SOC channels. Because Ca 2ϩ entry through a SOCE pathway is proportional to the degree of depletion of the intracellular Ca 2ϩ store (33), the magnitude of SOC entry measured by the Ca 2ϩ readdition protocol, which imposes store depletion without chance of refilling until the cation is added back to the bath, is exacerbated when compared with that of the total (VDCC plus SOC) Ca 2ϩ influx observed under physiological stimulation with the sterol (see plateau phase in Fig. 1). Incubation of muscle cells with U73122 or neomycin after the sterol-induced [Ca 2ϩ ] i transient had no effect on the SOCE pathway (Fig. 4). However, addition of these PPI-PLC inhibitors prior to 1,25(OH) 2 D 3 stimulation not only suppressed the sterol [Ca 2ϩ ] i response ( Fig. 2B) but also the SOC influx (not shown).

Effect of 1,25(OH) 2 D 3 on Mn 2ϩ
Influx through SOC Channels-The 1,25(OH) 2 D 3-dependent non VDCC-mediated Ca 2ϩ entry is permeable to Mn 2ϩ ions (13). Mn 2ϩ is a good Ca 2ϩ surrogate, because it is not pumped out of the cell and can be considered a selective tracer for Ca 2ϩ entry (34). Moreover, VDCCs share very low permeability for Mn 2ϩ (35,36). Thus, for a more close estimation of influx through the sterol-dependent SOCE pathway, the Ca 2ϩ readdition protocol was used, but Mn 2ϩ (1 mM) was added instead of Ca 2ϩ . The resultant Mn 2ϩinduced quenching of intracellular Fura-2 fluorescence was measured at the isosbestic (Ca 2ϩ -insensitive) wavelength of the dye, associated to SOC channel activity (37,38). In 1,25(OH) 2 D 3 -stimulated cells, the Fura-2 signal was quenched at a rate 2.5-3 times faster than in control cells (Fig. 5A). We previously observed that depletion of endogenous Ca 2ϩ stores by pretreating the cells with the sarcoplasmic/endoplasmic reticulum Ca 2ϩ -Mg 2ϩ -ATPase inhibitor thapsigargin effectively blocks the response to the sterol (13). In the present conditions, thapsigargin (1 M), similarly to the hormone, markedly increased Mn 2ϩ influx (3-5-fold over control, p Ͻ 0.001). As expected, the sterol-activated Mn 2ϩ entry was totally suppressed by both La 3ϩ (10 M) and Ni 2ϩ (2 mM) but unaffected by VDCC or PPI-PLC blockers (Fig. 5A). These results are similar to those observed with the Ca 2ϩ readdition protocol, thus indicating that the pathways of sterol-induced Mn 2ϩ entry and SOC influx are the same. To follow both the kinetics and time course of such cation entry activation, a set of experiments were performed in which Mn 2ϩ was simultaneously added with 1,25(OH) 2 D 3 (Fig. 5B). As shown, 1,25(OH) 2 D 3 -induced SOC influx was not significantly stimulated until at least 2-3 min of hormone treatment had elapsed, but once activated, it rapidly reached a steady-state level (45% above basal) with a half-time constant (t1 ⁄2 ) to peak of 0.5 min. These results show that a lag time exists between 1,25(OH) 2 D 3 coupling to its putative membrane receptor and SOC channel activation.
Effect of PKC, Adenylyl Cyclase, and Tyrosine Kinase Inhibitors on 1,25(OH) 2 D 3 -induced SOC Influx-To address the possibility that PKC, PKA, and TK could play a role in the modulation of 1,25(OH) 2 D 3 -induced SOC influx, we assessed the effect of specific inhibitors of these signaling cascades on the hormone-regulated process by using the Mn 2ϩ quench technique. All the inhibitors used were added into the incubation medium after occurrence of the 1,25(OH) 2 D 3 -induced [Ca 2ϩ ] i transient, and 2 min later Mn 2ϩ was added. It is pertinent to note that pretreatment of muscle cells with either of these inhibitors completely suppressed any [Ca 2ϩ ] i change due to sterol stimulation (see under "Discussion" for extensive considerations on this point). Bisindolylmaleimide I and calphostin C, cell-permeable, highly specific PKC inhibitors (39,40), were used to evaluate the involvement of this kinase on the 1,25(OH) 2 D 3 -dependent SOCE pathway. Fig. 6A shows that these two PKC inhibitors dose-dependently suppressed hormone-stimulated Mn 2ϩ influx in chick muscle cells, a complete inhibition being achieved at 50 and 100 nM for bisindolylmaleimide and calphostin, respectively. Notoriously, the thapsigargin-induced Mn 2ϩ influx was insensitive to these inhibitors (Fig. 6B). Moreover, addition of 1,25(OH) 2  specific adenylyl cyclase inhibitor, SQ22,536 (41), which has been shown to effectively block cyclase activation by the sterol in chick muscle cells (7). In consequence, SQ22,536 precludes PKA activation by suppressing 1,25(OH) 2 D 3 -stimulated increases in cyclic AMP levels. We observed here that SQ22,536 (0.5 mM) did not alter the SOCE pathway activated either by the sterol or thapsigargin (Fig. 6). Finally, the possible involvement of tyrosine kinase activity was examined by using the TK inhibitor genistein (42). As shown in Fig. 6, genistein (50 -100 M) completely prevented Mn 2ϩ influx in response to the sterol but not that induced by thapsigargin. The observed effects of the inhibitor are highly probable to be specific because the concentrations used here do not alter the activity of serine/ threonine kinases (43). DISCUSSION We recently described in a preliminary report (13) that the secosteroid hormone 1,25(OH) 2 D 3 triggers in avian skeletal muscle cells a rapid, nongenomic [Ca 2ϩ ] i response composed of an initial fast sterol-induced Ca 2ϩ release from endogenous thapsigargin-sensitive stores that is followed by cation influx from the extracellular milieu. This cation entry pathway accounts for the sustained, long lasting [Ca 2ϩ ] i phase, to which both the well established L-type VDCC-mediated Ca 2ϩ entry and a novel store-operated Ca 2ϩ entry (SOCE) pathway have been shown to contribute. In the present study we further evaluated the sterol-dependent SOC influx to obtain information about its regulation, kinetics, and localization into the hormone-signaling network. Stimulation of chick skeletal muscle cells with 1,25(OH) 2 D 3 results in rapid activation of PPI-PLC, with a concomitant generation of both IP 3 (Fig. 3) and diacylglycerol (5). The results obtained clearly support the concept that the rapid sterol-induced [Ca 2ϩ ] i transient is due to mobilization of the cation from IP 3 -sensitive stores and also show that such Ca 2ϩ release from internal stores does not last long enough to be responsible per se for the non-VDCC-mediated Ca 2ϩ entry phase seen after hormone stimulation of the cells in Ca 2ϩ -containing medium under conditions of VDCC blockade (13).
To date, neither direct activators or inhibitors of SOC influx nor sufficiently selective compounds with ability to affect the SOCE pathway have been developed. However, by means of simple experimental manipulations, it is possible to measure SOC influx by fluorimetric techniques. We assayed here the sterol-induced SOC influx by using a Ca 2ϩ readdition protocol, which relies on the fact that either mobilization or depletion of Ca 2ϩ from endogenous stores constitutes an absolutely essential step for activation of SOC channels. It was observed that 1,25(OH) 2 D 3 -dependent SOC influx in muscle cells is insensitive to the action of classical VDCC inhibitors, thus excluding the possibility of contribution from L-type channels in this cation entry phase. It has been reported that in certain cell types U73122 acts on one or more sites in addition to its interaction with PPI-PLC (44). Particularly, both U73122 and U73343 have been shown to inhibit SOC influx in hepatocytes (45). However, in our hands, these PPI-PLC inhibitors did not affect the SOCE pathway (Fig. 4). This evidence strongly supports the idea that activation of PPI-PLC is an unavoidable step in the mechanism by which the sterol induces SOC influx in these cells and that mobilization of Ca 2ϩ from IP 3 -sensitive stores occurs upstream from the activation of the SOCE-pathway. In line with this view, the kinetics of the sterol-induced SOC influx (Fig. 5B) indicates that SOC channels seem to be significantly activated after completion of the IP 3 -dependent Ca 2ϩ transient. On this basis, although not directly addressed here, it is tempting to speculate that activation of SOC channels within the 1,25(OH) 2 D 3 -induced signaling framework occurs before that of the VDCC-mediated Ca 2ϩ entry, as it was previously noted that dihydropyridine-sensitive Ca 2ϩ influx is fully operating after 3-5 min of exposure of skeletal muscle cells to the hormone (14). SOC entry, although quantitatively smaller than cation influx through the VDCC-mediated pathway, could be physiologically operating as a small depolarizing conductance which in turn triggers the much larger Ca 2ϩ influx through VDCCs. Evidence for the operation of such SOCEinduced VDCC influx has been obtained in mouse pancreatic ␤-cells (46) and GH 3 pituitary cells (47).
At present, the molecular regulatory mechanism responsible for signaling between Ca 2ϩ store depletion and SOC influx activation remains unsolved. Several candidates have been proposed as the coupling signal including Ca 2ϩ entry factors in Ca 2ϩ -free medium, and the Ca 2ϩ transient due to store mobilization was monitored from the 340/390 fluorescence ratio. When [Ca 2ϩ ] i levels returned to basal, excitation was changed to 360 nm (isosbestic wavelength for Fura-2), Mn 2ϩ (1 mM) was added to the medium (zero time), and Mn 2ϩ influx followed as the rate of fluorescence quenching (see text). When used, both the Ca 2ϩ channel blockers (nifedipine 2 M, ϫ; verapamil 5 M, f; La 3ϩ 10 M, Ⅺ; Ni 2ϩ 2 mM, ࡗ) and the PPI-PLC inhibitors (U73122 2 M, ‚; neomycin 0.5 mM, OE) were added 2 min prior to Mn 2ϩ addition. Control corresponds to cells exposed to vehicle (Ͻ0.1% ethanol, E); in this situation, as no changes in [Ca 2ϩ ] i occurred, Mn 2ϩ was added 3 min later. Mn 2ϩ influx is expressed as the percentage of maximal quenching (see "Experimental Procedures"). Data are the averages of three independent experiments. To simplify visual interpretation, error bars were not included in the graph. B, the kinetics of Mn 2ϩ entry was followed by the rate of Mn 2ϩinduced quenching of Fura-2 cytosolic fluorescence in cells incubated in a Ca 2ϩ -free bath to which 1,25(OH) 2 D 3 (10 Ϫ9 M) and Mn 2ϩ (1 mM) were simultaneously added. Results are expressed as the ratio between the rate of 1,25(OH) 2 D 3 -dependent Mn 2ϩ entry (sterol-induced) and the rate of basal cation entry (basal) and are the averages of two independent experiments. and G proteins (reviewed in Ref. 21), making the scenario of SOC influx regulation controversial and complicated. However, there is some consistent evidence indicating that both TK-and PKC-mediated protein phosphorylation is involved in modulation of SOCE (see Ref. 48 and references therein). In light of the fact that 1,25(OH) 2 D 3 promotes in muscle cells activation of PKC (5,9), PKA (7,8), and tyrosine kinases, 3 we also evaluated the possibility that these kinases could play a modulatory action in the 1,25(OH) 2 D 3 -induced SOC influx. Blockade of the adenylyl cyclase/cyclic AMP/PKA pathway had no effect on the sterol-induced SOC influx. Indeed, little is actually known about the involvement of PKA in store-operated Ca 2ϩ influx. However, our results suggest that sterol-dependent activation of both PKC and TK strongly contributes to stimulation and/or modulation of SOC influx. In the insulin-secreting cell line RINm5F (49), lymphocytes, and human fibroblasts (50,51), positive modulation of SOC influx by these kinases has also been observed. It was previously reported by us (9) that in skeletal muscle cells 1,25(OH) 2 D 3 -induced activation of PKC stimulates Ca 2ϩ influx through L-type channels. Moreover, a good correlation between the appearance of catalytic fragments of the PKC ␣ isoform and the increment in both Ca 2ϩ influx and membrane PKC activity in response to 1,25(OH) 2 D 3 stimulation was observed, 2 suggesting that this isoform could be mainly responsible for the hormone-induced activation of Ca 2ϩ channels.
None of the kinase-signaling cascade inhibitors used here was able to alter the thapsigargin-dependent SOC entry, suggesting that different mechanisms could be operating for regulation of sterol-and thapsigargin-dependent SOC influx. Because of this, it must be kept in mind that although a similar [Ca 2ϩ ] i response is seen after either sterol or thapsigargin stimulation of skeletal muscle cells (13), the sarcoplasmic/endoplasmic reticulum Ca 2ϩ -Mg 2ϩ -ATPase inhibitor, unlike the hormone, bypasses any possible membrane and/or receptorcoupled signaling step, making it possible that very different intracellular events could be triggered upon thapsigargin or hormone treatment, thus accounting for the aforementioned discrepancies. in Ca 2ϩ -free medium, and the hormone-dependent Ca 2ϩ transient was monitored until completion from the 340/390 fluorescence ratio; then either the PKC inhibitors bisindolylmaleimide (Bis, 20 and 50 nM) or calphostin (Cal, 50 and 100 nM), the adenylyl cyclase inhibitor SQ22,536 (SQ, 0.5 mM), the tyrosine kinase inhibitor genistein (Gen, 50 and 100 M) or vehicle (Ͻ0.1% ethanol, Control), were added 2 min prior to Mn 2ϩ addition to the incubation medium. Mn 2ϩ entry was followed as detailed under "Experimental Procedures." Results are expressed as the amplitude (percentage) of Mn 2ϩ influx, compared with controls (100%). The number of recordings for each group is given in parentheses in the graph bars. *, p Ͻ 0.001; ns, not statistically significant. B, the same as for A, but thapsigargin (1 M) was used instead of 1,25(OH) 2 D 3 to induce the Ca 2ϩ transient, and the concentrations used for bisindolylmaleimide, calphostin, SQ22,536, and genistein were 50 nM, 100 nM, 0.5 mM, and 100 M, respectively.