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Originally published In Press as doi:10.1074/jbc.M208077200 on October 3, 2002

J. Biol. Chem., Vol. 277, Issue 50, 48165-48171, December 13, 2002
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Epidermal Growth Factor-induced Depletion of the Intracellular Ca2+ Store Fails to Activate Capacitative Ca2+ Entry in a Human Salivary Cell Line*

Bin-Xian ZhangDagger §, Xiuye Ma, Chih-Ko Yeh||**, Meyer D. LifschitzDagger §, Michael X. ZhuDagger Dagger , and Michael S. Katz§||

From the Dagger  Medical Research Service and || Geriatric Research, Education and Clinical Center, South Texas Veterans Health Care System, Audie L. Murphy Division, San Antonio, Texas 78229, Departments of § Medicine,  Biochemistry, and ** Dental Diagnostic Science, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, and Dagger Dagger  Neurobiotechnology Center and Department of Neuroscience, Ohio State University, Columbus, Ohio 43210

Received for publication, August 7, 2002, and in revised form, September 26, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca2+ ([Ca2+]i). Generally, agonist-mediated Ca2+ mobilization involves both Ca2+ release from internal stores and Ca2+ influx activated by store depletion (i.e. capacitative or store-operated Ca2+ influx). However, the role of capacitative Ca2+ entry in EGF-mediated Ca2+ mobilization is still largely unknown. In this study, we compared [Ca2+]i signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca2+ entry. Unlike carbachol and Tg, EGF (5 nM) elicited a transient [Ca2+]i signal without a plateau phase in the presence of extracellular Ca2+ and also failed to accelerate Mn2+ entry. Repletion of extracellular Ca2+ to cells stimulated with EGF in the absence of Ca2+ elicited an increase in [Ca2+]i, indicating that EGF indeed stimulates Ca2+ influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca2+ release. Interestingly, the phospholipase C inhibitor U73122 completely inhibited Ca2+ release induced by both EGF and carbachol and also reduced Ca2+ influx responsive to carbachol but had no effect on Ca2+ influx induced by EGF. EGF-induced Ca2+ influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca2+ influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca2+ influx responsive to carbachol but did not increase EGF-activated Ca2+ influx. Both EGF and carbachol depleted internal Ca2+ stores. Our results demonstrate that EGF-induced Ca2+ release from internal stores does not activate capacitative Ca2+ influx. Rather, EGF stimulates Ca2+ influx via a mechanism distinct from capacitative Ca2+ influx induced by carbachol and Tg.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gq protein-coupled receptor mediated intracellular calcium ([Ca2+]i) mobilization in non-excitable cells is usually divided into two phases (i.e. an initial rapid rise followed by a sustained plateau). The initial rise in [Ca2+]i is mainly a result of Ca2+ release from the internal stores, whereas the sustained plateau phase is a result of subsequent Ca2+ influx across the plasma membrane (1, 2). Release of Ca2+ from the internal stores may be induced either by activation of inositol 1,4,5-trisphosphate (IP3)1 receptors or by inhibition of the Mg2+, Ca2+-ATPase on the endoplasmic reticulum (ER). Production of IP3 and activation of IP3 receptors occur after stimulation of either Gq protein-coupled receptor-phospholipase C (PLC) beta  or receptor tyrosine kinase-PLCgamma pathways. The nature of the coupling between depletion of the internal store Ca2+ contents and subsequent activation of Ca2+ influx, as well as the molecular identity of the Ca2+ entry channels, is still unknown.

A capacitative Ca2+ influx model (also referred to as the depletion-activated or store-operated Ca2+ influx model) has been proposed and widely accepted (1, 2). The basic concept of this model is that the fall of the Ca2+ contents in the internal stores provides a full and sufficient signal to activate Ca2+ entry across the plasma membrane (1-3). Thus theoretically capacitative Ca2+ influx in the cells is determined by the extent of Ca2+ depletion in the internal stores and is independent of the means employed to deplete the internal store Ca2+. Numerous studies have demonstrated in different cell types that depletion of internal store Ca2+ either by activation of the G protein coupled receptor signaling pathway or by inhibition of the ER Ca2+ pump stimulates capacitative Ca2+ influx (1-6).

The cellular actions of epidermal growth factor (EGF) are mediated by activation of EGF receptor tyrosine kinase cascades, which leads to a number of signaling events, including activation of PLCgamma and mobilization of [Ca2+]i (reviewed in Refs. 7, 8). However, whether capacitative Ca2+ influx pathways are involved in [Ca2+]i signaling by EGF has not yet been determined. In our previous study in a human salivary cell line (HSY), we have shown that EGF causes a transient [Ca2+]i signal that is distinct from the response to activation of the G protein-coupled muscarinic receptor (9). In this study, we demonstrate that EGF does not activate capacitative or store-operated Ca2+ influx; rather, EGF stimulates Ca2+ influx by alternative mechanisms distinct from capacitative influx.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant human EGF was purchased from Promega (Madison, WI). Fura-2 acetoxymethyl ester was from Molecular Probes (Eugene, OR). Thapsigargin (Tg) was purchased from Sigma/RBI (Natick, MA). Dulbecco's modified Eagle's medium, EGTA, trypsin-EDTA, and Opti-MEM were from Invitrogen. Phorbol 12-myristate 13-acetate (PMA), carbachol, U73122, oligomycin, and other chemicals were purchased from Sigma.

Cell Culture-- The HSY cell line was originally established by Yanagawa et al. (10) and was kindly provided by Dr. James Turner (NIDCR, National Institutes of Health, Bethesda, MD). Cells were plated at a density of about 2 × 104 cells/cm2 in 100-mm culture dishes and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and penicillin (100 units/ml)/streptomycin (100 µg/ml) at 37 °C in a humidified 5% CO2 atmosphere incubator. Cells were grown to near confluence at 72 h and were then harvested with 0.05% trypsin-0.02% EDTA for [Ca2+]i measurements.

Measurement of Intracellular Calcium-- [Ca2+]i was determined by spectrofluorometric measurements in cell suspensions. HSY cells were loaded with 1.2-1.5 µM Fura-2 acetoxymethyl ester in phosphate-buffered saline supplemented with 1 mM MgCl2 and 1 mM CaCl2 (PBS) at 37 °C for 30-45 min with gentle shaking (9). Loaded cells were washed with fresh buffer to remove the extracellular dye and resuspended in PBS at a density of 2.5-5 × 105 cells/ml. The [Ca2+]i signals were measured in suspensions of cells after stimulation with various agents at 37 °C. [Ca2+]i signals in the absence of extracellular Ca2+ were assayed using cells washed with and resuspended in Ca2+-free PBS (i.e. PBS containing 200 µM EGTA instead of 1 mM CaCl2). In cells initially suspended in Ca2+-free medium, Ca2+ influx was measured upon subsequent repletion of extracellular Ca2+ (2 mM). A basal Ca2+ entry occurred in nonstimulated cells. All measurements of Ca2+ influx in HSY cell suspensions exposed to stimulatory agents were accompanied by parallel determinations of basal Ca2+ influx (i.e. the Ca2+ influx observed when extracellular Ca2+ was replenished to nonstimulated cell suspensions maintained in Ca2+-free solution). The amplitude of basal Ca2+ influx in our studies averaged 0.3 units of 340/380 fluorescence ratio, and in each experiment reported basal Ca2+ influx has been subtracted.

Excitation ratio of 340/380 was measured with a PTI Delta Scan spectrofluorometer (Photon Technology International Inc., South Brunswick, NJ) using 340 and 380 nm wavelengths for excitation and 510 nm for emission (9). Changes of [Ca2+]i were measured and indexed by the alterations in the fluorescence ratio of 340/380.

Transfection of HSY Cells with pIRESneo or pIRESneo-hTRPC3-- The full-length human TRPC3 (hTRPC3) was subcloned into the pIRESneo (Clontech) mammalian expression vector at the EcoRI site (6). Transient transfection was performed in HSY cells grown to 40-50% confluence in 100-mm plates. The cells were washed with Opti-MEM once and incubated with pIRESneo (1.7 µg) or an equimolar amount of pIRESneo-hTRPC3 DNA with the LipofectAMINE plus system (Invitrogen) in Opti-MEM for 3 h. At the end of 3 h of transfection, Opti-MEM was replaced with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cells were continuously cultured for 24 h and then harvested with trypsin-EDTA for [Ca2+]i measurement. The transfection efficiency of HSY cells was 30% as determined by using the green-fluorescent-protein vector (Clontech).

Data Analysis-- Data from multiple experiments are presented as mean ± S.E. Statistical significance of single comparisons was determined using Student's t test. Multiple comparisons were performed using two-factor analysis of variance followed by Tukey-Cramer multiple comparison tests.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the presence of extracellular Ca2+, stimulation of HSY cells in suspension with the muscarinic receptor agonist carbachol induced a rapid rise in [Ca2+]i followed by a sustained plateau phase (Fig. 1). This pattern of Ca2+ mobilization resembled the typical [Ca2+]i response to Gq coupled receptor activation (1). Presumably, the initial rapid increase of [Ca2+]i mainly represented Ca2+ release from the internal stores, and the sustained plateau indicated capacitative Ca2+ influx activated by the depletion of internal store Ca2+. Thapsigargin (Tg), a specific inhibitor of the ER Ca2+ pump, caused a similar [Ca2+]i response, as observed with muscarinic receptor activation, although the rate of the initial rise was slower (Fig. 1). Compared with the biphasic [Ca2+]i response to carbachol and Tg, EGF, which activates receptor tyrosine kinase pathways and PLC gamma  (7), generated a very different [Ca2+]i signal in HSY cells (Fig. 1). The [Ca2+]i response to EGF (5 nM) peaked ~45-60 s after EGF stimulation and then gradually decayed toward the baseline (Fig. 1). These results suggested that the [Ca2+]i signal of EGF lacked the capacitative Ca2+ influx to sustain the elevated plateau phase.


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  		Fig. 1.   EGF-, carbachol-, and Tg-induced [Ca2+]i responses in HSY cells in the presence of extracellular Ca2+. Fura-2-loaded HSY cells in suspension were stimulated with EGF (5 nM), carbachol (100 µM), or Tg (10 nM) as indicated by the vertical arrows in the figure. Changes in [Ca2+]i were measured as described under "Experimental Procedures." The concentrations of agonists were chosen to produce maximal [Ca2+]i responses in HSY cells. The scale bars indicate the value of 340/380 fluorescence ratio and time in seconds, respectively. The traces shown in this figure are representative of three experiments.

The lack of capacitative or store-operated Ca2+ entry after EGF stimulation was further demonstrated by the experiments shown in Fig. 2. Carbachol and Tg are agents used most often to activate capacitative Ca2+ influx in various cells (1, 2) including HSY cells (9). We thus used carbachol and Tg to stimulate capacitative Ca2+ influx in HSY cells and compared their effects with that of EGF. The rate of Mn2+ entry, as measured by the quenching of cellular Fura-2 fluorescence after addition of Mn2+, has been used as a measure of capacitative Ca2+ influx (11). Compared with the basal Mn2+ quenching signal recorded in the absence of any agonists (Fig. 2a), stimulation of the cells with EGF (5 nM) before Mn2+ addition had no measurable effect on the rate of Mn2+ entry (Fig. 2b). In contrast, prior stimulation of the cells with carbachol (100 µM) or Tg (10 nM) dramatically increased the rate of Mn2+ entry (Fig. 2, c and d), indicating the activation of capacitative Ca2+ influx. These results demonstrated that, unlike carbachol and Tg, EGF did not activate capacitative Ca2+ entry in HSY cells.


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  		Fig. 2.   Effects of EGF, carbachol, or Tg on Mn2+ entry in HSY cells. Fura-2-loaded HSY cells in suspension were either unstimulated (a) or stimulated in PBS with 5 nM EGF (b), 100 µM carbachol (c), or 10 nM Tg (d). Addition of agonist in b-d is indicated by the first vertical arrow in each trace. The fluorescence was excited at 360 nm and measured at 510 nm. Quench of fluorescence was recorded as Mn2+ (200 µM) was added (indicated by the single vertical arrow in a and the second vertical arrow in b-d). The scale bars indicate arbitrary fluorescence intensity and time in seconds, respectively. The traces shown in this figure are representative of three experiments.

A unique feature of capacitative Ca2+ entry is that depletion of the internal Ca2+ stores provides the necessary and sufficient signal for activation (1-3). PLC-mediated generation of IP3 and subsequent IP3 receptor activation are required for Ca2+ release (i.e. depletion of the internal Ca2+ stores) by both carbachol and EGF (12). It is also known that inhibition of PLC and Ca2+ release results in blockade of capacitative Ca2+ influx (13). To determine whether EGF induces Ca2+ influx in HSY cells and, if so, whether the EGF-responsive Ca2+ influx is capacitative entry, we investigated the effects of EGF, carbachol, and Tg on Ca2+ release and influx in the absence and presence of the pharmacological PLC inhibitor U73122 (14). Ca2+ release from the internal stores was measured in the absence of extracellular Ca2+, whereas Ca2+ influx was subsequently observed after extracellular Ca2+ repletion. As shown in Fig. 3, stimulation of HSY cells with EGF (5 nM), carbachol (100 µM), and Tg (10 nM) induced Ca2+ release as well as Ca2+ influx (dark traces). Pretreatment with U73122 (5-10 µM, 2-10 min) completely blocked both EGF- and carbachol-mediated Ca2+ release from the internal stores (Fig. 3, a and b, light traces). The same treatment also dramatically inhibited the subsequent Ca2+ influx in carbachol-stimulated cells (Fig. 3b, light trace). However, pretreatment with U73122 failed to inhibit the Ca2+ influx in EGF-stimulated cells (Fig. 3a, light trace). Treatment with U73122 also reduced Tg-mediated Ca2+ release and the corresponding Ca2+ influx, indicating that Tg-mediated Ca2+ release from the internal stores was partially dependent on PLC in HSY cells (Fig. 3c, light trace). These results demonstrated that EGF-responsive Ca2+ influx was insensitive to PLC inhibition by U73122 and thus independent of Ca2+ release (i.e. depletion of the internal Ca2+ stores).


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  		Fig. 3.   Effect of the PLC inhibitor U73122 on EGF-, carbachol-, and Tg-induced Ca2+ release and Ca2+ entry in HSY cells. HSY cells were washed with and resuspended in Ca2+-free PBS. Suspensions of HSY cells were either untreated or treated with U73122 (5-10 µM) at room temperature for 2-10 min. Both untreated and treated cells were then stimulated with EGF (5 nM), carbachol (100 µM), or Tg (10 nM) as indicated in the figure to trigger the release of Ca2+ from the internal stores. Extracellular Ca2+ (2 mM) was replenished to trigger Ca2+ influx as indicated. Changes in [Ca2+]i were measured as described under "Experimental Procedures." The scale bars indicate the value of 340/380 fluorescence ratio and time in seconds, respectively. Traces from untreated (dark traces) and U73122 treated (light traces) cells are superimposed for comparison. The traces shown in this figure are representative of three experiments.

The relationship between EGF-responsive Ca2+ release and Ca2+ influx in HSY cells was further assessed by comparing the effects of increasing concentrations of EGF on Ca2+ release versus influx (Fig. 4). Ca2+ release and influx in response to increasing concentrations of carbachol and Tg were also measured to demonstrate the relationship between Ca2+ release (i.e. depletion of internal stores) and capacitative entry. With increasing concentrations of carbachol and Tg, the amplitudes of Ca2+ release and subsequent Ca2+ influx were enhanced accordingly (Fig. 4, B and C). No Ca2+ influx occurred after Ca2+ repletion if the cells were treated with carbachol and Tg at concentrations (10-7 and 10-10 M, respectively) insufficient to produce Ca2+ release from internal stores (Fig. 4, B and C). For both carbachol and Tg, the concentrations of agonist required for half-maximal Ca2+ release and influx were equivalent (carbachol, 8.3 × 10-6 M; Tg, 2.0 × 10-9 M) (Fig. 4, B and C). These data suggested that carbachol and Tg-responsive Ca2+ influx was coupled to Ca2+ release from the internal stores.


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  		Fig. 4.   Relationship of Ca2+ release induced by increasing concentrations of EGF, carbachol, or Tg and subsequent Ca2+ influx in HSY cells. Fura-2-loaded HSY cells suspended in Ca2+-free PBS were stimulated with increasing concentrations of EGF (A), carbachol (B), or Tg (C) to trigger the release of Ca2+ from the internal stores. Changes in [Ca2+]i were measured as described under "Experimental Procedures." The alteration in [Ca2+]i induced by EGF, carbachol, or Tg in the absence of extracellular Ca2+ was measured to monitor Ca2+ release from the internal stores (solid line), whereas the alteration in [Ca2+]i after subsequent repletion of extracellular Ca2+ (2 mM) was measured as an indicator of Ca2+ influx (dashed line). Both release and influx were measured as peak responses. In the influx experiments, the cells were stimulated in Ca2+-free medium for 3-5 min before repletion of extracellular Ca2+ as demonstrated by the time scale in Fig. 3. The values shown in A are means ± S.E. from five experiments; values in B and C are means ± S.E. from three experiments.

In contrast, EGF stimulated Ca2+ influx over a range of concentrations distinct from that required to induce Ca2+ release. At low concentrations (5 × 10-12-2.5 × 10-11 M), EGF triggered Ca2+ influx even in the absence of Ca2+ release from internal stores; activation of Ca2+ release required higher concentrations of EGF (>= 5 × 10-11 M) (Fig. 4A). The concentration of EGF required for half-maximal release (2.6 × 10-10 M) was 8-fold higher than that required for half-maximal influx (3.0 × 10-11 M) (Fig. 4A). Theoretically, the capacitative Ca2+ influx pathway should be activated after Ca2+ release at higher concentrations of EGF. Thus, we would expect substantial increases in Ca2+ influx to coincide with the progressive rise in Ca2+ release produced by EGF added at concentrations of 5 × 10-11-10-9 M. However, over this concentration range, the amplitude of Ca2+ influx remained essentially constant (Fig. 4A). The lack of additional Ca2+ influx at higher concentrations of EGF indicated that EGF-responsive Ca2+ release from internal stores might not activate capacitative Ca2+ entry. Moreover, the Ca2+ influx observed at low concentrations of EGF in the absence of Ca2+ release from the internal stores may not be capacitative Ca2+ influx.

It is still controversial whether activation of Ca2+ entry is graded (i.e. so that even a small decrease in free Ca2+ in the ER can produce a small increase in depletion-activated Ca2+ entry) or involves a threshold, in which case a fall in ER Ca2+ below a critical level triggers Ca2+ influx (4, 5). In HSY cells treated with carbachol and Tg, the relationship between Ca2+ release and Ca2+ influx demonstrated by the dose-response curves in Fig. 4 indicated that any small amount of Ca2+ release from internal stores was accompanied by a subsequent influx of Ca2+ upon repletion of extracellular Ca2+. Thus, our results did not support the existence of a minimal threshold of Ca2+ release from internal stores for activation of capacitative Ca2+ entry (Fig. 4, B and C).

Previous studies have demonstrated that capacitative Ca2+ entry is regulated by mitochondria (15, 16). A number of mitochondrial inhibitors, including oligomycin, have been shown to block capacitative Ca2+ influx in other systems (15, 16). We therefore tested the effect of oligomycin on Ca2+ release and influx responsive to EGF, carbachol, and Tg in HSY cells. Incubation of HSY cells with oligomycin (1-50 ng/ml) had little or no effect on EGF-, carbachol-, or Tg-mediated Ca2+ release in the absence of extracellular Ca2+ (Fig. 5A). After repletion of extracellular Ca2+, Ca2+ influx in EGF-stimulated cells was dramatically inhibited by 50 ng/ml oligomycin (>70% inhibition; Fig. 5B). However, similar treatment with oligomycin (50 ng/ml) only modestly inhibited carbachol and Tg-stimulated capacitative Ca2+ influx (~40%; Fig. 5B). Complete inhibition of EGF responsive Ca2+ influx was observed at 100 ng/ml oligomycin, whereas much higher concentrations of oligomycin (5-10 µg/ml) were required to eliminate carbachol or Tg stimulated Ca2+ influx in HSY cells (data not shown). Interestingly, at low concentrations (1 and 5 ng/ml) of oligomycin, Ca2+ influx was actually increased in EGF-stimulated HSY cells (Fig. 5B). These experiments suggested that in HSY cells, the effects of oligomycin on EGF-stimulated Ca2+ influx were significantly distinct from the concentration-dependent inhibitory action of this agent on carbachol- and Tg-responsive Ca2+ influx (i.e. capacitative entry).


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  		Fig. 5.   Effect of oligomycin on EGF-, carbachol-, and Tg-induced Ca2+ release and Ca2+ entry in HSY cells. HSY cells were washed with and resuspended in Ca2+-free PBS. Increasing concentrations of oligomycin or vehicle (Me2SO) were added into suspensions of HSY cells immediately before stimulation with EGF (5 nM) (open circle ------open circle ), carbachol (100 µM) (triangle ------triangle ), and Tg (10 nM) (------) to induce Ca2+ release. Extracellular Ca2+ (2 mM) was replenished to trigger Ca2+ influx after the return of [Ca2+]i toward the basal level (~200 s after agonist stimulation). Changes in [Ca2+]i were measured as described under "Experimental Procedures." Values of Ca2+ release in the absence of extracellular Ca2+ (A) and Ca2+ influx after extracellular Ca2+ repletion (B) are presented as means ± S.E. from seven experiments. Values in the presence of increasing concentrations of oligomycin are expressed as percentages of values in the absence of oligomycin (taken as 100%). Oligomycin exerted significantly different effects on EGF- versus carbachol- or Tg-induced Ca2+ influx, as determined by two-factor analysis of variance followed by Tukey-Cramer multiple comparison test (EGF versus carbachol, p = 0.007; EGF versus Tg, p = 0.015).

Protein kinase C (PKC) has been implicated in the regulation of capacitative Ca2+ entry (17, 18, 19). We compared the effects of PKC activation on Ca2+ influx responses to EGF, carbachol, and Tg. Ca2+ release in HSY cells suspended in Ca2+-free PBS was induced by EGF, carbachol, or Tg. During the course of decay of the [Ca2+]i signal to the basal level, HSY cells were incubated with 100 nM PMA for 2-3 min to activate PKC, and then Ca2+ influx was triggered by extracellular Ca2+ repletion. The results in Fig. 6 demonstrated that PMA treatment largely inhibited Ca2+ influx in both EGF- and carbachol-stimulated HSY cells (Fig. 6, a and b). These data indicated that the EGF-responsive non-capacitative Ca2+ influx, like the carbachol-responsive capacitative Ca2+ influx, was inhibited by PKC activation. Surprisingly, however, the Ca2+ influx in Tg-stimulated HSY cells was not affected by PMA treatment (Fig. 6c).


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  		Fig. 6.   Effect of PMA on EGF-, carbachol-, and Tg-induced Ca2+ influx in HSY cells. HSY cells were washed with and resuspended in Ca2+-free PBS. Cells were stimulated with EGF (5 nM), carbachol (100 µM), or Tg (10 nM) as indicated to trigger the release of Ca2+ from the internal stores. PMA (100 nM), an activator of PKC, or vehicle (Me2SO) was added as indicated by the dashed vertical arrows. Extracellular Ca2+ (2 mM) was replenished as indicated to trigger Ca2+ influx in both untreated (dark traces) and PMA-treated (light traces) cells. Changes in [Ca2+]i were measured as described under "Experimental Procedures." Traces from untreated and PMA-treated cells are superimposed for comparison. The traces shown in this figure are representative of three experiments.

Expression of TRPC3 and other transient receptor potential proteins has been shown to increase capacitative and/or other agonist-stimulated Ca2+ entry in various cell types (6). We tested the effect of overexpression of hTRPC3 protein in HSY cells on EGF- and carbachol-responsive Ca2+ entry. If EGF-responsive Ca2+ mobilization involved capacitative pathways, the Ca2+ influx induced by EGF would be enhanced by hTRPC3 overexpression. The results showed that hTRPC3 transfection did not alter EGF- and carbachol-stimulated Ca2+ release measured in Ca2+-free PBS (Fig. 7). Carbachol-mediated Ca2+ influx after extracellular Ca2+ repletion was increased in hTRPC3-transfected cells (Fig. 7b), indicating an enhanced capacitative Ca2+ influx (340/380 ratios: hTRPC3 1.15 ± 0.02 versus vector 0.92 ± 0.03; p = 0.017, n = 8 measurements). In contrast, hTRPC3 transfection did not affect EGF-responsive Ca2+ influx (Fig. 7a) (340/380 ratios: hTRPC3 0.57 ± 0.02 versus vector 0.53 ± 0.01; p = 0.529, n = 5 measurements). These results indicated that transfection of hTRPC3 in HSY cells enhanced muscarinic receptor-activated Ca2+ entry but not EGF-mediated, Ca2+ release-independent Ca2+ influx.


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  		Fig. 7.   Effect of hTRPC3 overexpression on EGF- and carbachol-induced Ca2+ influx in HSY cells. Cultured HSY cells were either transfected with vector pIRESneo or hTRPC3 construct and cultured as described under "Experimental Procedures." The transfected cells were washed with and resuspended in Ca2+-free PBS. Both vector- and hTRPC3-transfected cells were then stimulated with EGF (5 nM) or carbachol (100 µM) as indicated to trigger the release of Ca2+ from the internal stores. Extracellular Ca2+ (2 mM) was replenished to trigger Ca2+ influx as indicated. Changes in [Ca2+]i were measured as described under "Experimental Procedures." Traces obtained from vector- (dark traces) and hTRPC3- (light traces) transfected cells are superimposed for comparison. The effects on hTRPC3 overexpression on EGF- and carbachol-induced Ca2+ influx in multiple experiments were analyzed using Student's t test (EGF-induced Ca2+ influx (mean ± S.E. 340/380 ratios): hTRPC3, 0.57 ± 0.02 versus vector, 0.53 ± 0.01, p = 0.529, n = 5 measurements; carbachol-induced Ca2+ influx: hTRPC3, 1.15 ± 0.02 versus vector, 0.92 ± 0.03, p = 0.017, n = 8 measurements).

Ca2+ depletion of the internal stores is critical for activation of capacitative Ca2+ entry. We therefore examined the effect of EGF on internal Ca2+ stores. Tg-responsive Ca2+ release in the absence of extracellular Ca2+ was used to index the Ca2+ content in the internal stores (9). In HSY cells, 10 nM Tg was sufficient to cause complete depletion of internal store Ca2+ because further addition of Tg (10 nM) did not evoke additional Ca2+ release (Fig. 8A). The cells were stimulated with EGF (5 nM) or carbachol (100 µM) in Ca2+-free PBS to deplete internal stores. After the [Ca2+]i signal induced by EGF or carbachol decayed to basal level, Tg (10 nM) was added to discharge the entire internal Ca2+ store. Under these conditions, Tg still evoked a small Ca2+ release from the internal store (Fig. 8, B and C), indicating incomplete internal store depletion by both EGF and carbachol. Although both EGF and carbachol depleted the internal store to a similar extent, only carbachol activated capacitative Ca2+ influx.


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  		Fig. 8.   Effect of EGF, carbachol, and Tg on internal store Ca2+ in HSY cells. HSY cells were washed with and resuspended in Ca2+-free PBS and then stimulated with EGF (5 nM), carbachol (100 µM), or Tg (10 nM) as indicated. When the [Ca2+]i response decayed back to basal level, Tg (10 nM) was added as indicated to test the Ca2+ content remaining in the internal stores. The traces shown in this figure are representative of three experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although the link between receptor tyrosine kinase pathway-mediated PLC gamma  activation and Ca2+ mobilization is well established, the role of capacitative or store-operated Ca2+ entry in EGF-induced [Ca2+]i signaling has not previously been clarified. The present study provides novel evidence that capacitative Ca2+ entry is not activated after EGF stimulation of PLC gamma  and Ca2+ release from the internal stores in HSY cells, where EGF-responsive Ca2+ influx occurred in the absence of Ca2+ release from the internal stores (Figs. 3 and 4).

It has been shown in Rat-1 fibroblasts and HeLa cells that the [Ca2+]i transient caused by EGF resulted from rac-dependent Ca2+ influx (20). Rac is a small G protein belonging to the rho subfamily of ras-related proteins (21). Introduction of the dominant-negative rac protein in Rat-1 fibroblasts and HeLa cells abolished EGF-mediated Ca2+ mobilization but did not affect other elements of EGF signaling, such as EGF receptor autophosphorylation and extracellular signal-regulated kinase activation. Because EGF activation of PLC gamma  does not involve rac, it is likely that EGF-responsive Ca2+ influx in these two cell types is independent of PLC gamma  activation and Ca2+ release from the internal stores. In HSY cells, EGF-mediated Ca2+ influx occurred at low concentrations of EGF in the absence of Ca2+ release (Fig. 4) and was not blocked by the PLC inhibitor U71322 (Fig. 3). Whether the EGF-responsive Ca2+ influx observed in HSY cells is also rac-dependent as reported in Rat-1 fibroblasts and HeLa cells (20) is currently under investigation. The main difference in EGF-induced [Ca2+]i signaling between HSY and Rat-1 or HeLa cells is that at higher concentrations, EGF also induces PLC-dependent Ca2+ release from the internal stores in HSY cells (Figs. 3 and 4). However, both the present study with HSY cells and the previous study with Rat-1 and HeLa cells (20) demonstrate that EGF does not stimulate capacitative Ca2+ influx, which is activated by G protein-coupled receptors in these cells (Figs. 1-7; Ref. 20).

The coupling between the fall of Ca2+ content in the internal store and activation of capacitative Ca2+ influx has been the subject of extensive studies. Soluble messengers activating Ca2+ entry have been extracted and partially purified from mammalian cells and yeast (22-24). However, the soluble messenger hypothesis will probably not be accepted without definitive identification of the messenger(s). The conformational coupling model suggests that the conformational alteration of IP3 receptors induced by the fall of Ca2+ in the internal stores causes direct interaction of the IP3 receptors with the Ca2+ influx pathway, resulting in activation of Ca2+ entry (25, 26). Although not consistent with the results from studies of IP3 receptor knock-out cells (27, 28), the conformational coupling model has recently gained support from a number of studies from different laboratories (29-31).

Previous studies indicate that analogs of Drosophila melanogaster transient receptor potential proteins could be the molecular candidates for the capacitative influx pathway (4-6, 32-34). Overexpression of different transient receptor potential isoforms in various cells (35-39) and in mouse models (40) has been shown to enhance capacitative Ca2+ influx and capacitative Ca2+ influx-linked cell functions (40). A direct interaction between IP3 receptors and transient receptor potential proteins has been suggested to be essential for activation of capacitative Ca2+ influx (29-31). However, the elimination of IP3 receptors in DT40 lymphocytes (28) and T cells (27) did not prevent activation of capacitative Ca2+ entry by Tg, although both PLC beta - and gamma -mediated Ca2+ release and influx were abolished (28). These studies indicate a distinction between the cellular events involved in capacitative Ca2+ influx responsive to Tg and activation of PLC pathways.

We also observed that treatment of HSY cells with PMA abolished carbachol- but not Tg-mediated capacitative Ca2+ influx (Fig. 6). The reason for the differences between carbachol- and Tg-mediated capacitative Ca2+ entry in HSY and other cells (27, 28) has not yet been determined. It may relate to the different mechanisms of Ca2+ release from internal stores employed by carbachol and Tg. Incomplete depletion of internal Ca2+ stores by carbachol (Fig. 8c) may also contribute to the differences between carbachol- and Tg-mediated capacitative Ca2+ entry in HSY cells (Fig. 6).

Arachidonic acid has been shown to activate Ca2+ influx by a non-capacitative mechanism (41). Moreover, capacitative Ca2+ influx and arachidonic acid-activated Ca2+ influx exhibit mutual antagonism in human embryonic kidney cells (41, 42). Mutual antagonism between EGF-mediated Ca2+ influx and capacitative Ca2+ influx could also occur in HSY cells. Accordingly, in the presence of EGF-mediated Ca2+ influx, capacitative Ca2+ influx would be inhibited even though EGF depletes the internal store Ca2+ (Fig. 8b).

In summary, the present study provides direct evidence that EGF-mediated Ca2+ mobilization in HSY cells does not involve capacitative Ca2+ influx pathways. The EGF-responsive Ca2+ influx occurs via a mechanism that is independent of the EGF effect on internal store Ca2+ and is distinct from the capacitative Ca2+ influx induced by carbachol and Tg.

    ACKNOWLEDGEMENTS

We appreciate Dr. Richard Lin's help in the experiment involving hTRPC3 transient expression and Shuko Lee's assistance with statistical analysis.

    FOOTNOTES

* This study was supported by medical research funds from the Department of Veterans Affairs (to M. S. K. and B.-X. Z.) and by National Institutes of Health Grants DE10756 (to C.-K. Y.) and NS42183 (to M. X. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§§ To whom correspondence should be addressed: 7400 Merton Minter Blvd., San Antonio, TX 78229-4404. Tel.: 210-617-5197; Fax: 210-617-5312; E-mail: katz@uthscsa.edu

Published, JBC Papers in Press, October 3, 2002, DOI 10.1074/jbc.M208077200

    ABBREVIATIONS

The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; PLC, phospholipase C; EGF, epidermal growth factor; Tg, thapsigargin; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline supplemented with 1 mM MgCl2 and 1 mM CaCl2; PKC, protein kinase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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