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Originally published In Press as doi:10.1074/jbc.M411686200 on November 30, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4436-4441, February 11, 2005
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Protein Kinase C Modulates Agonist-sensitive Release of Ca2+ from Internal Stores in HEK293 Cells Overexpressing the Calcium Sensing Receptor*

Amos M. Sakwe{ddagger}, Lars Rask{ddagger}§, and Erik Gylfe¶

From the {ddagger}Department of Medical Biochemistry and Microbiology, Uppsala University, Biomedical Centre, Box 582, SE-751 23 Uppsala, Sweden and the Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23 Uppsala, Sweden

Received for publication, October 14, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
This study examined the mechanism of Ca2+ entry and the role of protein kinase C (PKC) in Ca2+ signaling induced by activation of the calcium sensing receptor (CaR) in HEK293 cells stably expressing the CaR. We demonstrate that influx of Ca2+ following CaR activation exhibits store-operated characteristics in being associated with Ca2+ store depletion and inhibited by 2-aminoethoxydiphenyl borate. Inhibition of PKC with GF109203X, Gö6983, or Gö6976 and down-regulation of PKC activity enhanced the release of Ca2+ from internal stores in response to the polyvalent cationic CaR agonist neomycin, whereas activation of PKC with acute 12-O-tetradecanoylphorbol-13-acetate treatment decreased the release. In contrast, overexpression of wild type PKC-{alpha} or -{epsilon} augmented the neomycin-induced release of Ca2+ from internal stores, whereas dominant negative PKC-{epsilon} strongly decreased the release, but dominant negative PKC-{alpha} had little effect. Prolonged treatment of cells with 12-O-tetradecanoylphorbol-13-acetate effectively down-regulated immunoreactive PKC-{alpha} but had little effect on the expression of PKC-{epsilon}. Together these results indicate that diacylglycerol-responsive PKC isoforms differentially influence CaR agonist-induced release of Ca2+ from internal stores. The fundamentally different results obtained when overexpressing or functionally down-regulating specific PKC isoforms as compared with pharmacological manipulation of PKC activity indicate the need for caution when interpreting data obtained with the latter approach.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Ca2+ receptor (CaR)1 is a G-protein-coupled receptor that senses the extracellular Ca2+ concentration ([Ca2+]o) in parathyroid and other cell types. It is involved in Ca2+ and mineral homeostasis as well as other cellular processes including secretion (1, 2). In parathyroid cells or HEK293 cells stably transfected with the CaR (HEK-CaR), stimulation of the receptor by its agonists triggers the activation of phospholipase C-{beta} that catalyzes the formation of inositol 1,4,5-trisphosphate and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate. Whereas inositol 1,4,5-trisphosphate releases Ca2+ from the endoplasmic reticulum and raises the cytoplasmic Ca2+ concentration ([Ca2+]i), DAG activates protein kinase C (PKC). Elevation of [Ca2+]o to levels that activate the receptor leads to increased cellular levels of these second messengers (3, 4) and consequently a rise of [Ca2+]i (5) and activation of some PKC isoforms (6, 7). Recently, it was suggested that activation of the CaR in HEK-CaR cells stimulates Ca2+ entry independently of store release (8). However, in other cell types Ca2+ influx following the stimulation of several G-protein-coupled receptors occurs by a store-operated Ca2+ entry mechanism (9–13). Depending on the cell type and the receptor that is activated, PKC activation inhibits or facilitates this process (14–16), but it also affects non-store-operated Ca2+ entry (17) and the Ca2+ storage capacity (18).

Most reports on the role of PKC in CaR-mediated processes are based on pharmacological modulation of the activity of the enzyme. In HEK-CaR cells direct activation of PKC with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) reduces the [Ca2+]o sensitivity of the CaR (19, 20). Because PKC phosphorylates the CaR, the activity of this receptor is believed to be modulated by PKC by a feedback mechanism (8, 21). However, it has also been shown that TPA-induced PKC activation affects Ca2+ handling independently of the receptor via activation of plasma membrane Ca2+-ATPases (22, 23). In accordance with such an effect, the amounts of mobilized intracellular Ca2+ were reduced when TPA-treated parathyroid cells were exposed to the Ca2+ ionophore ionomycin (24). Using pharmacological modulators of PKC activity it is therefore difficult to draw conclusions about the role of PKC as a modulator of CaR activity and in CaR-mediated processes including parathyroid hormone secretion.

In the present study, we overexpressed PKC-{alpha} or -{epsilon} in HEK-CaR cells to examine how these DAG-responsive PKC isoforms activated via the CaR or by treatment of HEK-CaR cells with TPA influence receptor-mediated Ca2+ handling. It will be shown that activation of the receptor apart from mobilizing intracellular Ca2+ activated influx of the ion by a pathway inhibited by the store-operated Ca2+ channel blocker 2-aminoethoxydiphenyl borate (2-APB). Based on overexpression of PKC-{alpha} and -{epsilon} in HEK-CaR cells, we found that they differentially affected the release of Ca2+ from intracellular stores. However, pharmacological modulation of PKC activity influenced the release of Ca2+ from internal stores in response to CaR activation in a manner opposite to that obtained when specific PKC isoforms were overexpressed or functionally down-regulated. These results urge for caution when interpreting data obtained with phorbol esters and/or PKC inhibitors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Establishment of PKC-{alpha} or -{epsilon} Overexpressing HEK-CaR Cells—Human embryonic kidney cells stably expressing the human kidney CaR (HEK-CaR cells) or the T888A mutant CaR (herein referred to as mCaR) as well as HEK-CaR cells overexpressing either wild type (wt) or dominant negative (DN) PKC-{alpha} (K368D) or DN PKC-{epsilon} (K437R) were generated as described previously (7). Cells were maintained in Ham's F-12 medium supplemented with 20 mM Hepes, 10% fetal bovine serum and Ca2+ to a final concentration of 1.25 mM. Before use in experiments, cells were serum-starved for 12–16 h in the same medium in which fetal bovine serum was replaced by 0.1% bovine serum albumin.

Measurement of [Ca2+]i—[Ca2+]i measurements on cells in suspension were carried out as described previously (25). Briefly, serum-starved cells grown in 75-cm2 flasks were harvested by trypsinization and resuspended in buffer containing 20 mM Hepes, 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 0.1% bovine serum albumin, 5.5 mM glucose. Approximately 10 mM Na+ was also added when setting the pH to 7.4. The cells were then washed twice in this buffer and incubated for 60 min at 37 °C in the same buffer containing 1 µM fura-2/AM. The cells were subsequently harvested by centrifugation and washed once with 10 ml of similar buffer lacking the indicator and containing 0.5 mM CaCl2 and 0.5 mM MgSO4. The washed cells were resuspended in 1 ml of a Ca2+- and albumin-deficient version of the latter buffer and transferred to a quartz cuvette with continuous stirring and placed in the thermostatically controlled holder of a time-sharing multichannel spectrophotofluorimeter. The excitation fluorescence at 340 and 380 nm as well as the 340/380 ratio were recorded every 10 msec with emission at 510 nm. Activation of CaR was performed by addition of the polyvalent cation neomycin or divalent cations at low [Ca2+]o or in Ca2+-deficient buffer supplemented with the indicated concentrations of EGTA. At the end of each experiment, cells were lysed by addition of 0.1% Triton X-100 to record the maximum 340/380 nm fluorescence ratio as well as the 380 nm fluorescence under both saturating Ca2+ conditions and in the nominal absence of Ca2+ (<1nM). The latter was accomplished by addition of an excess of EGTA as well as Tris to raise pH above 8.3. [Ca2+]i was calculated as described previously (26). In attempts to determine the rate of Ca2+ influx, the Ca2+-buffering capacity of the cytoplasm was increased with the aid of the chelator BAPTA, which was loaded into the cells (5 µM BAPTA-AM) in parallel with fura-2. To determine the effects of PKC inhibition, cells were pretreated with indicated concentrations of GF109203X, Gö6976, or Gö6983 during the fura-2 loading period, whereas acute activation of PKC was accomplished by 5–10 min exposure to TPA prior to initiation of experiments. Prolonged pretreatment with TPA was also used to down-regulate PKC (see below). The amounts of releasable Ca2+ from internal stores in response to maximal CaR activation by neomycin were estimated in Ca2+-deficient medium as the time integrated peak [Ca2+]i response. Statistical analyses were performed by the paired Student's t test.

Subcellular Fractionation and Immunoblotting—HEK-CaR cells were treated with either TPA (200 nM) or Me2SO vehicle (0.03%) for 20 h at 37 °C, 5% CO2 during serum starvation or with 500 nM TPA for 10 min. The cells were harvested in 25 mM Tris-HCl, pH 7.5, 250 mM sucrose, 5 mM MgCl2, 100 mM KCl, and protease inhibitor mixture, disrupted by Dounce homogenization, and fractionated by differential velocity centrifugation as described previously (7). The protein concentration in the cytosolic and detergent-solubilized membranes was estimated using the BCA protein assay reagent, and equal amounts of protein from subcellular fractions (cytosol or detergent-solubilized membrane proteins) were separated in 10% SDS-polyacrylamide gels and blotted onto Hybond C-extra membranes. After blocking overnight at 4 °C in 10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 0.1% Tween 20, 4% bovine serum albumin, the membranes were probed with isoform-specific anti-PKC antibodies followed by the corresponding peroxidase-conjugated secondary antibodies as described previously (7). Blots were visualized by enhanced chemiluminescence. Quantification of the blots was performed using the Fujifilm image gauge software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Pharmacological Inhibition of PKC Augments CaR-mediated Release of Ca2+ from Internal Stores—PKC inhibition has been suggested to increase the sensitivity of the CaR to its agonists (8). We now screened CaR-expressing HEK293 (HEK-CaR) cells for effects of pharmacological PKC modulators on the CaR agonist-induced release of Ca2+ from internal stores. When control cells in Ca2+-deficient medium were first exposed to a modest increase of the Ca2+ concentration (1 mM) there was a small rise of [Ca2+]i with an initial overshoot followed by sustained elevation (Fig. 1, control). Subsequent maximal CaR activation with 200 µM neomycin caused a much more pronounced overshooting of the [Ca2+]i peak and a further rise of the sustained elevation. The overshooting peaks represent intracellular Ca2+ mobilization, and the sustained elevation represents activation of Ca2+ influx. GF109203X, which inhibits most PKC isoforms, or Gö6976, a drug selective for Ca2+-dependent PKC isoforms (27), markedly increased the release of Ca2+ from intracellular pools in response to 1.0 mM Ca2+ and also the sustained elevation. Because the intracellular Ca2+ stores became more depleted in these situations, subsequent maximal stimulation with neomycin gave smaller [Ca2+]i peaks preceding the further rise of the sustained elevation. Acute (10 min) activation of PKC with TPA on the other hand reduced both the Ca2+- and neomycin-induced release of Ca2+ from internal stores (Fig. 1, TPA). These data indicate that PKC inhibition sensitizes the CaR resulting in a left shifted dose-response relationship for activation as suggested previously (8).



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  		FIG. 1.
Effects of pharmacological PKC inhibition/activation on the CaR-activated elevation of [Ca2+] o. Serum-starved HEK293 cells stably expressing the CaR were pretreated with Me2SO vehicle (0.02%; control), GF109203X (2 µM), or Gö6976 (0.5 µM) during fura-2 loading for 60 min. The cells were initially exposed to Ca2+-deficient medium containing 0.5 mM EGTA. The Ca2+ concentration was then increased to 1, 2, and 7 mM, and 200 µM neomycin was present as indicated by horizontal bars.

 
By maximally stimulating the CaR with 200 µM neomycin in Ca2+-deficient medium we next studied the effect of PKC modulators on the amounts of Ca2+, which could be released from intracellular stores. As depicted in Fig. 2, inhibition of most PKC isoforms with GF109203X or Gö6983, or inhibition of Ca2+-dependent PKC isoforms with Gö6976 increased the total amounts of releasable Ca2+ (Fig. 2, A and B), whereas acute activation of PKC with TPA decreased the total amounts. Attempts were made to use the Mn2+ quench technique (12) to estimate the rate of Ca2+ influx induced by CaR activation by neomycin in medium containing low concentration of Ca2+. Unfortunately, this method proved difficult because Mn2+ itself was found to be an effective activator of the CaR. Therefore we tried to measure influx as the rate of the slow gradual rise of [Ca2+]i in response to activation of Ca2+ influx into cells loaded with high concentrations of the Ca2+-buffering chelator BAPTA. With sufficient loading it was possible to completely eliminate the initial [Ca2+]i peak in response to neomycin-induced intracellular mobilization and observe a subsequent slow rise of [Ca2+]i in the presence of extracellular Ca2+. However, it was difficult to obtain consistent results, perhaps because the intracellular concentration of BAPTA varied between experiments after loading the cells with BAPTA-AM. Because of these difficulties we instead used the rise of [Ca2+]i in response to introduction of 1 (Fig. 2) or 6 mM Ca2+ (data not shown) in the presence of 200 µM neomycin as an indirect indicator of Ca2+ influx. This approach did not reveal any significant differences between control cells and cells exposed to modulators of PKC.



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  		FIG. 2.
Effects of pharmacological PKC inhibition/activation on the amounts of Ca2+ released from internal stores after CaR activation. Serum-starved HEK293 cells stably expressing the CaR were pretreated with Me2SO vehicle, GF109203X (2 µM), Gö6983 (2 µM), or Gö6976 (0.5 µM) during fura-2 loading for 60 min. A, the cells were initially exposed to Ca2+-deficient medium containing 0.5 mM EGTA. Neomycin (200 µM) was added, and the Ca2+ concentration was raised to 1 mM as indicated by horizontal bars. At the end of the experiment the Ca2+ concentration was elevated further to 6 mM (data not shown). B, the integrated Ca2+ peaks in response to neomycin (shaded areas in A) were used to quantify the amounts of Ca2+ released in response to maximal CaR activation and are shown as vertical bars± S.E. for 3–4 experiments. *, p < 0.05; ***, p < 0.001 versus Me2SO control.

 
PKC-{alpha} Is More Sensitive to TPA-induced Down-regulation Than PKC-{epsilon} in HEK293 Cells Stably Expressing the CaR—In several types of cells, prolonged treatment with TPA causes depletion of PKC activity. To verify the involvement of PKC in the modulation of [Ca2+]i, cells were pretreated with TPA (200 nM) or Me2SO vehicle for 20 h during serum starvation or acutely treated with TPA (500 nM). As shown in Fig. 3A, PKC-{alpha} was efficiently down-regulated in both the cytosolic (S100) and the detergent-soluble membranes (DSM), whereas PKC-{epsilon}, which is predominantly membrane-associated, was only minimally affected. Although cell type specific, several studies have reported similar effects of chronic treatment of cells with TPA on the expression levels of PKC-{epsilon} (28–32). Consistent with depletion of PKC activity, release of Ca2+ from intracellular stores calculated from the integrated [Ca2+]i peaks in response to neomycin were increased by 34% (p < 0.02) after TPA treatment overnight compared with control cells (Fig. 3B).



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  		FIG. 3.
Effects of prolonged treatment of cells with TPA on the CaR-mediated release of Ca2+ from internal stores and Ca2+ influx. A, HEK-CaR cells were treated with Me2SO vehicle or TPA (200 nM) for 20 h during serum starvation. Cells were harvested and fractionated into cytosolic (S100) and detergent-solubilized membranes (DSM). Equal amounts of proteins were separated in 10% polyacrylamide gels under reducing conditions and analyzed by Western blotting with isoform-specific antibodies to PKC-{alpha} or -{epsilon}. Blots were revealed by enhanced chemiluminescence. B, serum-starved cells treated with TPA or Me2SO vehicle as above were loaded with fura-2 for 60 min. The cells were initially exposed to Ca2+-deficient medium containing 0.5 mM EGTA. Ca2+ (0.5 and 1.5 mM) and 200 µM neomycin were then present as indicated by horizontal bars. TPA overnight, overnight treatment with TPA

 
Co-expression of wt PKC-{alpha} or -{epsilon} with the CaR in HEK293 Cells Increased Whereas Co-expression of Dominant Negative PKC-{epsilon} Decreased Intracellular Ca2+ Release—The relative unspecific pharmacological PKC inhibitors increased the amounts of intracellular Ca2+ released upon receptor activation, and activation of PKC with TPA had the opposite effect (see above). A different picture emerged when manipulating the expression of individual PKC isoforms. Overexpression of wt PKC-{alpha} or -{epsilon} in HEK-CaR cells thus increased intracellular Ca2+ release in response to neomycin, and functional down-regulation of these isoforms (DN mutants) had or tended to have the opposite effect (Fig. 4). Using the rise of [Ca2+]i in response to introduction of 1 mM Ca2+ (Fig. 4A) in the presence of 200 µM neomycin as an indirect indicator of Ca2+ influx no significant differences were found. However, the elevation of [Ca2+]i obtained when subsequently raising Ca2+ to 6 mM (data not shown) was significantly lower in DN PKC-{epsilon} (40% reduction, p < 0.001).



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  		FIG. 4.
Effects of co-expression of PKC-{alpha} or -{epsilon} with the CaR on the amounts of Ca2+ released from internal stores after CaR activation. Serum-starved cells co-expressing the CaR with or without wt or DN PKC-{alpha} or -{epsilon} as well as HEK293 cells overexpressing T888A mutant CaR (mCaR) were loaded with fura-2 for 60 min. A, the cells were initially exposed to Ca2+-deficient medium containing 0.5 mM EGTA. Neomycin (200 µM) was added, and the Ca2+ concentration was raised to 1 mM as indicated by horizontal bars. At the end of the experiment the Ca2+ concentration was elevated further to 6 mM (data not shown). B, the integrated Ca2+ peaks in response to neomycin (shaded areas in A) were used to quantify the amounts of Ca2+ released in response to maximal CaR activation and are shown as vertical bars± S.E. for 5 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus HEK-CaR control cells.

 
PKC-induced phosphorylation of the CaR has been suggested to influence the activity of the receptor (7, 8, 21, 33). To clarify whether the effects of PKC activation were mediated via the receptor, we examined Ca2+ signaling in HEK293 cells overexpressing T888A mutant CaR, which cannot be phosphorylated by PKC at this position. Although a small reduction of the neomycin-induced release of Ca2+ from internal stores in these mCaR cells did not reach statistical significance (Fig. 4) the elevation of [Ca2+]i in response to subsequent rise of extracellular Ca2+ to 6 mM (data not shown) was reduced by about 40% (p < 0.001).

Ca2+ Influx in CaR-expressing Cells Exhibits Store-operated Characteristics—Previous studies have indicated that the CaR activates Ca2+ entry independently of store release (8) and occurs via non-selective Ni2+-sensitive cation channels (7, 34). To further clarify the nature of the Ca2+ influx we used 2-APB, which is a reliable blocker of store-operated Ca2+ entry (35) with slight inhibitory effect on inositol 1,4,5-trisphosphate-induced Ca2+ mobilization (35–38). Consistent with a minor inhibitory effect on inositol 1,4,5-trisphosphate-induced Ca2+ release treatment of HEK-CaR cells with 100 µM 2-APB slightly decreased the magnitude of the initial [Ca2+]i peak in response to 200 µM neomycin (data not shown). However, compared with Me2SO vehicle-treated cells (Fig. 5A), 2-APB completely inhibited the sustained elevation of [Ca2+]i observed after stimulation with neomycin in the presence of 0.5 mM Ca2+ (Fig. 5B). Even the rise of [Ca2+]i observed after increasing the extracellular Ca2+ concentration to 5.5 mM in neomycin-stimulated cells was prevented by 2-APB (cf. Fig. 5, A and B).



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  		FIG. 5.
Ca2+ influx following CaR activation exhibits store-operated characteristics. Serum-starved HEK293 cells stably expressing the CaR were loaded with fura-2 during 60 min. The cells were initially exposed to Ca2+-deficient medium containing 0.5 mM EGTA. The Ca2+ concentration was then raised to 0.5 and 5.5 mM (A–B) and the Ba2+ concentration to 0.5, 1.5, and 6.5 mM (C–D) as indicated. Addition of 200 µM neomycin (A–D) and 100 µM 2-APB (B and D) were as indicated by horizontal bars.

 
To clarify whether the cation influx following activation of the CaR is selective for Ca2+, 0.5 mM Ba2+ was introduced into Ca2+-deficient medium. Such addition caused a small elevation of the fluorescence signal without overshooting the initial peak apparently because of failure to potently activate the CaR (Fig. 5C). The accompanying sluggish rise of the signal is consistent with slow entry of Ba2+. In this situation subsequent maximal CaR activation by neomycin induced the expected initial peak because of mobilization of intracellular Ca2+. However, there was no or little sustained elevation of the fluorescence signal above the extrapolated pre-stimulatory base line indicating failure of Ba2+ to enter through the CaR-activated influx pathway (Fig. 5C). Indeed, 2-APB did not seem to alter the rate of fluorescence increase obtained with elevation of the Ba2+ concentration to 6.5 mM during continued neomycin stimulation (Fig. 5D). Therefore, the influx pathway exhibits specificity for Ca2+ over Ba2+.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The arguments for a direct involvement of PKC in CaR-mediated processes including the Ca2+-regulated secretion of parathyroid hormone from parathyroid cells are based on the effects of TPA and/or PKC inhibitors. Cellular effects resulting from activation of PKC via agonist-induced activation of the CaR have received little attention. The present data indicate that the effects of pharmacological modulation of PKC activity on the CaR-mediated release of Ca2+ from intracellular stores contradict those obtained when overexpressing or functionally down-regulating specific PKC isoforms.

Studies of PKC effects are complicated not only by the existence of at least eight family members that are DAG-dependent and therefore activated by TPA but also by the fact that individual isoforms have special properties and that they are involved in different cellular processes. Treatment of cells with TPA bypasses receptor-mediated physiological activation of the enzyme and may cause a global activation of several DAG-responsive PKC isoforms that directly or indirectly interfere with numerous important processes within the cell. Apart from these complications, it is clear that TPA treatment activates a number of proteins that have only the C1 domain in common with PKC. Among these are Munc-13 proteins (involved in exocytosis), chimaerins (a family of Rac GTPase activating proteins), RasGRPs (exchange factors for Ras/Rap1), and diacylglycerol kinase (39–41). Therefore, it is difficult to separate primary effects of pharmacological manipulation of the activity of these enzymes from secondary or tertiary effects.

One way to support the conclusion that an observed effect of TPA treatment is caused by PKC activation is to try to reverse that effect by prior inhibition of PKC. This has proved effective in many instances, but PKC inhibitors are at best selective, not specific. An alternative way to study possible PKC involvement in a certain signal transduction chain is to overexpress individual wt or DN PKC isoforms and then measure putative effects on the process studied. This approach is more specific because a single isoform can be studied at a time. Moreover, as in this study the effects of a given PKC isoform can be investigated under physiological conditions in response to activation via the relevant receptor. Unfortunately, even this approach is not without flaws. Increased amounts of a certain PKC isoform may cause compensatory changes in the expression levels or activity of other isoforms (42).

PKC activation by TPA has been found to interfere with Ca2+ signaling at several levels by phosphorylating PLC isoforms (43, 44), plasma membrane Ca2+ channels (11, 15, 22, 23), endoplasmic reticulum Ca2+ channels (45, 46), and even the CaR (33). To further elucidate the putative importance of PKC in CaR signaling, we compared neomycin-induced release of Ca2+ from intracellular pools after pharmacological modulation of PKC activity in HEK-CaR cells with the neomycin effect in HEK-CaR cells after overexpressing or functionally down-regulating either of two DAG-responsive PKC isoforms. Inhibition of PKC by pre-treatment of cells with three different inhibitors increased the sensitivity of the CaR to extracellular Ca2+ and enhanced the release of Ca2+ from intracellular stores. Acute treatment of cells with TPA had the opposite effects. We could not observe any significant differences on Ca2+ influx caused by the PKC modulators compared with mock treated cells. CaR-induced Ca2+ influx into CaR expressing HEK cells has been reported to occur independently of intracellular Ca2+ release (8). However, the present data not only indicated association between CaR activation, intracellular Ca2+ store depletion, and activation of Ca2+ influx but also demonstrated that this influx is abolished by 2-APB. Because 2-APB is a potent inhibitor of store-operated Ca2+ influx (12) the simplest interpretation is therefore that the influx is indeed store-operated.

The effects of pharmacological modulators of PKC on the release of Ca2+ from internal stores corroborate the findings reported previously (8, 21). These authors deduced that the effects involve a feedback effect by which treatment of cells with TPA leads to activation of PKC, which in turn phosphorylates the CaR at Thr888 or other potential PKC phosphorylation sites, thereby decreasing the sensitivity of the receptor. TPA has also been shown to drain agonist sensitive intracellular Ca2+ pools by stimulating of Ca2+ efflux after PKC-mediated phosphorylation of the plasma membrane Ca2+ pump (22, 23). In our study, we did not find any significant effect of mutating Thr888 to a nonphosphorylatable residue on neomycin-induced release of Ca2+ from internal stores, but the mutation reduced Ca2+ influx at high [Ca2+]o. However, the Thr888 residue was necessary for full activation of ERK1/2 in response to CaR activation with high [Ca2+]o (7). It is possible that the inhibition of ERK1/2 activation may be related to inhibition of Ca2+ influx into the T888A mutant cells as indicated by the reduced elevation of [Ca2+]i after exposure to high [Ca2+]o.

CaR activation in parathyroid cells leads to elevation of [Ca2+]i and activation of PKC, which are associated with inhibition of parathyroid hormone secretion (47, 48). It is apparent that the elevated [Ca2+]i is an inhibitory signal for secretion (49), but the effect of physiological PKC activation is unclear. Data obtained with pharmacological modulators indicate that PKC exerts a stimulatory action on secretion (50–53), and it has therefore been assumed that physiological CaR activation generates both an inhibitory and a stimulatory signal (2, 54). We previously demonstrated that Ca2+ stimulation of HEK-CaR cells leads to activation of PKC-{alpha} and -{epsilon} (7). The present overexpression of these isoforms and functional down-regulation of PKC-{epsilon} indicate that these isoforms positively modulate the amounts of Ca2+, which are mobilized from internal stores in response to CaR activation. However, pharmacological manipulation of PKC activity gave opposite results suggesting that a less specific activation of PKC has additional and opposing effects on Ca2+ signaling. Therefore it is impossible to excluded that physiological activation of PKC contributes to the rise of [Ca2+]i that serves as an inhibitory messenger for parathyroid hormone secretion. In any case caution is warranted when drawing conclusions about PKC effects based on pharmacological modulators of the enzyme.


   FOOTNOTES
 
* This work was supported by Grants 15029 and 6240 from the Swedish Research Council (Medicine). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Medical Biochemistry and Microbiology Biomedical Centre, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden. Tel.: 4618-4714335; Fax: 4618-4714975; E-mail: Lars.Rask{at}imbim.uu.se.

1 The abbreviations used are: CaR, Ca2+ receptor; DAG, diacylglycerol; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate; 2-APB, 2-aminoethoxydiphenyl borate; HEK, human embryonic kidney; wt, wild type; DN, dominant negative; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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