Phospholipase C-δ1 Is Activated by Capacitative Calcium Entry That Follows Phospholipase C-β Activation upon Bradykinin Stimulation*

To characterize the regulatory mechanism of phospholipase C-δ1 (PLC-δ1) in the bradykinin (BK) receptor-mediated signaling pathway, we used a clone of PC12 cells, which stably overexpress PLC-δ1 (PC12-D1). Stimulation with BK induced a significantly higher Ca2+ elevation and inositol 1,4,5-trisphosphate (IP3) production with a much lower half-maximal effective concentration (EC50) of BK in PC12-D1 cells than in wild type (PC12-W) or vector-transfected (PC12-V) cells. However, BK-induced intracellular Ca2+ release and IP3 generation was similar between PC12-V and PC12-D1 cells in the absence of extracellular Ca2+, suggesting that the availability of extracellular Ca2+ is essential to the activation of PLC-δ1. When PC12-D1 cells were treated with agents that induce Ca2+ influx, more IP3 was produced, suggesting that the Ca2+ entry induces IP3production in PC12-D1 cells. Furthermore, the additional IP3 production after BK-induced capacitative calcium entry was detected in PC12-D1 cells, suggesting that PLC-δ1 is mainly activated by capacitative calcium entry. When cells were stimulated with BK in the presence of extracellular Ca2+, [3H]norepinephrine secretion was much greater from PC12-D1 cells than from PC12-V cells. Our results suggest that PLC-δ1 is activated by capacitative calcium entry following the activation of PLC-β, additively inducing IP3 production and Ca2+ rise in BK-stimulated PC12 cells.

The three-dimensional structure of a PLC-␦1 molecule lacking the pleckstrin homology domain revealed the catalytic domains (X and Y regions), which are tightly associated with two accessory modules, an EF-hand domain and a C2 domain (14), the latter of which was previously suggested to mediate Ca 2ϩdependent binding to lipid vesicles (15). Furthermore, structural studies of the multidomain PLC-␦1 protein suggested that the binding sites for Ca 2ϩ ions and the head group of phosphatidylinositol 4,5-bisphosphate are located both within and outside the catalytic domain (14,15). Other studies of PLC-␦1 also revealed that substances such as Ca 2ϩ ions and inositol 1,4,5-trisphosphate (IP 3 ) could play important roles as positive (16) and negative (17) regulators, respectively.
Although all PLC isozymes are activated by Ca 2ϩ in vitro, PLC-␦ isozymes seem more sensitive to Ca 2ϩ than the other isozymes. An increase in Ca 2ϩ ion concentration within the physiological range (0.1-10 M) was sufficient to stimulate PLC-␦1 but not PLC-␤1 and PLC-␥1 and to hydrolyze cellular inositol lipids present in permeabilized cells (16). An increase in cytosolic Ca 2ϩ to a level sufficient to fix the C2 domain of PLC-␦ might therefore trigger the enzyme's activation. Thus, it has been suggested that the activation of the PLC-␦ isozymes might occur as an event secondary to the receptor-mediated activation of other PLC isozymes or Ca 2ϩ channels (18).

Materials-Bradykinin
Cell Culture and Transfection of PLC-␦1 cDNA-PC12 cells were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated bovine calf serum (Hyclone, Logan, UT), 5% heatinactivated horse serum (Hyclone), and 1% antibiotics (Life Technologies, Inc.) in a humidified atmosphere of 5% CO 2 , 95% air at 37°C. The culture medium was changed every 2 days, and the PC12 cells were subcultured weekly. PLC-␦1 cDNA was cloned in a plasmid vector, pIBI20. The PLC-11/pIBI20 plasmid was then digested with NotI. The 2.8-kilobase pair insert obtained was subcloned into a mammalian expression vector, pZipNeo, which contains a viral promoter and the neomycin resistance gene. The constructed plasmid DNA (PLC-␦1/pZ-ipNeo) or the vector DNA (pZipNeo) alone was transfected into PC12 cells using an electroporator (Bio-Rad, 960 microfarads/250 V). One day after transfection, the cells were selectively grown in the presence of 400 g/ml G418 for a week. The G418-resistant clones were screened for the expression of PLC-␦1 protein by Western blotting and probing with a monoclonal anti-PLC-␦1 antibody using the ECL detection system. Positive clones were then maintained in the presence of 100 g/ml G418.
[Ca 2ϩ ] i Measurement-Cytosolic free Ca 2ϩ concentration ([Ca 2ϩ ] i ) was determined using the fluorescent Ca 2ϩ indicator Fura-2 as reported previously (20). In brief, PC12 cell suspensions were incubated in serum-free RPMI 1640 medium containing Fura-2/AM (3 M) and sulfinpyrazone (250 M) for 40 min at 37°C) with continuous stirring. The cells were then washed with Locke's solution (154 mM NaCl, 5.6 mM KCl, 5.6 mM glucose, 1 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES buffer adjusted to pH 7.4) containing sulfinpyrazone (250 M) and left at room temperature until use. Fluorescence ratios were measured by an alternative wavelength time scanning method (dual excitation at 340 and 380 nm; emission at 500 nm). Calibration of the fluorescent signal in terms of [Ca 2ϩ ] i was performed as described by Grynkiewicz et al. (21).
Mn 2ϩ Quenching of Fura-2 Fluorescence-PC12 cells that had been loaded with Fura-2/AM as described above were stimulated with bradykinin in the presence of 25 M Mn 2ϩ , and changes in fluorescence were measured at an excitation wavelength of 360 nm, which is an isosbestic wavelength, and at an emission wavelength of 500 nm, as described by Lee et al. (22).
Quantification of Inositol 1,4,5-Trisphosphate-IP 3 concentration in the cells was determined by competition assay with [ 3 H]IP 3 as described previously (23). In brief, to determine agonist-evoked IP 3 production, PC12 cells were stimulated with agonists for the indicated periods of time. The reaction was terminated by aspirating the medium off the cells and adding 15% (w/v) ice-cold trichloroacetic acid containing 10 mM EGTA. The cells were left on ice for 30 min to extract the watersoluble inositol phosphates. Trichloroacetic acid was then removed by extraction with diethyl ether. The final preparation was neutralized with 200 mM Tris, and its pH was adjusted to about 7.4. Assay buffer (0.1 M Tris buffer containing 4 mM EDTA and 4 mg/ml bovine serum albumin), [ 3 H]IP 3 (0.1 Ci/ml), and IP 3 -binding protein were added to the cell extract. The mixture was incubated for 15 min on ice and then centrifuged at 2000 ϫ g for 10 min. Water and scintillation mixture were added to the pellet to measure radioactivity. IP 3 concentration in the sample was determined based on a standard curve and expressed as pmol/g of protein in the soluble cell extract. The IP 3 -binding protein was prepared from bovine adrenal cortex according to the method of Challiss et al. (24). Measurement of [ 3 H]NE Secretion-Catecholamine secretion by PC12 cells was measured in 24-well plates following the method reported by Park et al. (25) with some modification. In brief, cells were loaded with [ 3 H]NE (1 Ci/ml; 68 pmol/ml) while incubating in RPMI containing 0.01% ascorbic acid for 1 h at 37°C in 5% CO 2 , 95% air. The cells were washed with Locke's solution twice and incubated in Locke's solution for 15 min to stabilize. Then the cells were incubated in Locke's solution for 10 min during which basal secretion was measured. The cells were subsequently stimulated with the drugs under test for 10 min. After the incubation, the medium was aspirated from each well and transferred to a scintillation vial. Finally, residual catecholamine in the cells was extracted with 10% trichloroacetic acid, and the extract was transferred to a scintillation vial. The radioactivity in each vial was determined in a scintillation counter. The amount of [ 3 H]NE secreted was calculated as percentage of total [ 3 H]NE content. Net secretion was obtained by subtracting basal secretion from the stimulated secretion. In order to study the effect of SK&F 96365 on the BK-induced [ 3 H]NE secretion, the drug was added to both media used to measure basal and stimulated secretion.
Photoaffinity Labeling of G-protein-Photoaffinity labeling of G-protein with [␣-32 P]GTP was carried out by the method of Linse and Mandelkow (26) with minor modifications (27). Samples were photolabeled with 5-10 Ci of [␣-32 P]GTP in the presence of 2 mM MgCl 2 in an ice bath under 254-nm UV irradiation for 5-10 min. After the irradiation, the samples were mixed with Laemmli stopping solution (28) and allowed to stand at room temperature for 1 h. The samples were then subjected to SDS-PAGE using 7.5-12% gels. The gels were dried and exposed to Kodak X-OMAT XAR-5 film using DuPont image-intensifying screens.
Transglutaminase Assay-Transglutaminase activity was determined by quantifying the incorporation of [ 3 H]putrescine into casein as described previously (29). This reaction was carried out in 0.1 ml of buffer containing 50 mM Tris-HCl (pH 8.5), 20% (v/v) glycerol, N,NЈdimethylcasein (1 mg/ml), 250 M putrescine, 1 Ci of [ 3 H]putrescine, 20 mM dithiothreitol, 2 mM MgCl 2 , and the enzyme in the indicated amounts. Where indicated, CaCl 2 (1 mM) and GTP (5 mM) were added in the reaction mixture. Glycerol was included in the buffer, because its presence has been found to stabilize the transglutaminase activity (29). The presence or absence of glycerol in the assay had no effect on the GTP-induced inhibition of guinea pig liver transglutaminase activity. Reaction mixtures were incubated for 1 h at 37°C, and the reaction was stopped by the addition of 0.1 ml of 50% trichloroacetic acid. The precipitate was collected on Whatman GF/C filters and washed three times with 10 ml of 5% trichloroacetic acid. Radioactivity was measured in a liquid scintillation counter.
Immunoblotting and Immunoprecipitation-Cells were grown to confluence and lysed in 500 l of lysis buffer (20 mM HEPES (pH 7.2), 10% glycerol, 1 mM Na 3 VO 4 , 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, 1 g/ml leupeptin, and 1% triton X-100). After sonication, the cell homogenates were centrifuged at 10,000 ϫ g for 10 min. Proteins (50 g) were separated in 7.5-12% (w/v) gels by SDS-PAGE and transferred to Immobilon-P (Millipore Corp., Bedford, MA). The membranes were blocked for 1 h with low detergent blotto (LDB; 80 mM NaCl, 2 mM CaCl 2 , 0.02% NaN 3 , 0.2% (v/v) Nonidet P-40, and 50 mM Tris/HCl (pH 8.0) containing 5% (w/v) nonfat dry milk) at room temperature and then incubated in LDB containing polyclonal antibody against G h ␣ (1:500 dilution) for 1 h at room temperature. For immunoblots probed with monoclonal antibody against PLC-␤1, PLC-␥1, and PLC-␦1, the antibody was diluted 1:2000, and the incubation was overnight. After being washed with LDB, the membranes were incubated with anti-mouse immunoglobulin peroxidase-linked antibody (1:5000 dilution) in high detergent blotto (2% (v/v) Nonidet P-40 in LDB) for 1 h at room temperature. After three washes, the membranes were subjected to the procedures for enhanced chemiluminescence. For immunoprecipitation, cells were lysed in lysis buffer, and each extract (800 g/1000 l) was treated with a preformed complex of Staphylococcus aureus goat anti-mouse IgG (Pansorbin, Calbiochem). After an overnight incubation at 4°C, pellets were obtained by centrifugation at 15,000 ϫ g for 1 min and washed three times with lysis buffer. The pellets were then processed by PAGE and Immunoblotting and probing with anti-G h ␣ antibody, exactly as described above.
Protein Determination-The amount of protein was estimated by the method of Bradford (30) using a Bio-Rad protein determination kit and bovine serum albumin as the standard.
Statistical Analysis-Statistical analysis of the data was done using the unpaired Student's t test in comparison between two experimental groups. Differences were considered significant when probability (p) values were Ͻ0.05.

RESULTS
Overexpression of PLC-␦1 in PC12-D1 Cells-PC12 cells were transfected with a construct containing rat brain PLC-␦1 cDNA. Seven clones were obtained. One clone, PLC␦14, exhibiting the highest level of PLC-␦1 as inferred by Western blot analysis was selected and used under the name PC12-D1 throughout the following experiments. A clone of vector-transfected PC12 cells (PC12-V) was used as a control.
Effect of PLC-␦1 Overexpression on BK-induced [Ca 2ϩ ] i Rise-We investigated the effect of PLC-␦1 overexpression on the BK-induced signaling in PC12 cells. BK induced a much greater [Ca 2ϩ ] i rise in the PC12-D1 cells than in the PC12-W or PC12-V cells ( Fig. 2A). The half-maximal effective concentration (EC 50 ) was much lower for the PC12-D1 cells (ϳ10 nM) than the PC12-W or PC12-V cells (both ϳ100 nM) (Fig. 2B). However, the maximal effective concentrations (EC 100 ) were same for the three kinds of cells, namely 5 M. When the three kinds of PC12 cells were treated with HOE140, an antagonist of B 2 bradykinin receptors, BK-induced [Ca 2ϩ ] i rise was completely blocked (data not shown), suggesting that the BK-induced response is entirely dependent on the B 2 receptors.
We also investigated whether BK-induced [Ca 2ϩ ] i rise is also potentiated in other PC12 clones that overexpress different levels of PLC-␦1. As shown in Fig. 3 ] i rises, although the expression level of PLC-␦1 in ␦14 clone is apparently higher than in ␦15 clone. These results suggest that there is some limitation in the activation of PLC-␦1 when the enzyme is expressed over a certain level.
The BK-induced [Ca 2ϩ ] i rise in PC12 cells occurs via two routes: Ca 2ϩ release from intracellular Ca 2ϩ stores and Ca 2ϩ influx through Ca 2ϩ release-activated calcium channels (31). We tested which route of Ca 2ϩ mobilization contributed to the enhanced [Ca 2ϩ ] i rise after BK treatment in PC12-D1 cells. As shown in Fig. 4A, BK-induced Ca 2ϩ release in the absence of extracellular Ca 2ϩ was not significantly different in the three kinds of PC12 cells. Both EC 50 and EC 100 were similar (Fig.  4B). On the other hand, BK-induced Ca 2ϩ influx after the addition of extracellular Ca 2ϩ , which is thought to occur through Ca 2ϩ release-activated Ca 2ϩ channels, was greater in the PC12-D1 cells than in the PC12-W or PC12-V cells (Fig.  4A). EC 50 was ϳ3 and ϳ30 nM for PC12-D1 and PC12-W or PC12-V cells, respectively (Fig. 4B). However, the EC 100 remained similar (5 M) among the three kinds of cells. The increased BK-induced Ca 2ϩ influx into the PC12-D1 cells was confirmed by Mn 2ϩ quenching experiments. Mn 2ϩ is a good surrogate for Ca 2ϩ ions in these kind of experiments, since it is not pumped out of the cells. Thus, it can be used as a selective tracer for Ca 2ϩ influx (22). As shown in Fig. 5, the fluorescence of Fura-2 was gradually quenched by the presence of Mn 2ϩ . When PC12 cells were stimulated with BK, fluorescence rapidly decreased, suggesting that BK-induced Mn 2ϩ influx had occurred. The fluorescence quenching induced by the BK treatment was greater in the PC12-D1 cells than in PC12-W or PC12-V cells. The results together, therefore, suggest that BKinduced Ca 2ϩ influx through Ca 2ϩ release-activated Ca 2ϩ channels is greatly enhanced in cells overexpressing PLC-␦1.
Effect of PLC-␦1 Overexpression on BK-induced IP 3 Production-Since IP 3 production can be an indicator of PLC activity, BK-induced IP 3 production in PC12-V and PC12-D1 cells was compared. When cells were treated with various concentrations of BK, more IP 3 was formed in the PC12-D1 cells than in the PC12-V cells (Fig. 6A). At 5 M BK concentration, the maximal IP 3 produced occurred 15 s after stimulation, which is in good agreement with our previous result (31) (Fig. 6B). At this time, the PC12-D1 cells produced ϳ1.7 times more IP 3 than the PC12-V cells, suggesting that PLC activity is higher in the PC12-D1 cells. Because PC12-D1 cells overexpress PLC-␦1, the difference in the PLC activity between the PC12-V and PC12-D1 cells can be attributed to the activity of overexpressed PLC-␦1. In the most simple scenario, one could assume that the greater IP 3 production in the PC12-D1 cells subsequently induces a greater Ca 2ϩ release from the intracellular Ca 2ϩ stores. However, the amount of Ca 2ϩ release in PC12-V and PC12-D1 cells was similar, which contradicts the assumption of a greater IP 3 production in PC12-D1 cells. A difference in the experimental conditions may provide a clue for the understanding of this discrepancy. Unlike the IP 3 production experiments, which were done in the presence of extracellular Ca 2ϩ , BKinduced Ca 2ϩ release was determined in the absence of extracellular Ca 2ϩ . Therefore, additional IP 3 production by the overexpressed PLC-␦1 in PC12-D1 cells may depend on the availability of extracellular Ca 2ϩ . This possibility was tested by measuring IP 3 levels under conditions when extracellular Ca 2ϩ was removed and intracellular Ca 2ϩ was chelated with BAPTA. In the absence of any Ca 2ϩ , the IP 3 production in PC12-V and PC12-D1 cells was similar (Fig. 6, C and D), suggesting that Ca 2ϩ is required for the activation of PLC-␦1.
Enhanced Production of IP 3 by Ca 2ϩ Influx-The Ca 2ϩ that is necessary for the activation of PLC-␦1 can be supplied by Ca 2ϩ release from intracellular Ca 2ϩ stores or by Ca 2ϩ influx from the extracellular space. When released Ca 2ϩ can activate PLC-␦1, then the BK-induced Ca 2ϩ release in the PC12-D1 cells should be greater than in PC12-V cells due to the additional IP 3 produced by the overexpressed PLC-␦1. However, released Ca 2ϩ can be ruled out as a prominent candidate for PLC-␦1 activator, considering that the BK-induced Ca 2ϩ release between the PC12-V and PC12-D1 cells was similar (Fig.  4). Therefore, we tested the possibility that Ca 2ϩ could have entered from the extracellular space to activate PLC-␦1. As shown in Fig. 7A, Ca 2ϩ influx-inducing agents such as high K ϩ , thapsigargin, and ionomycin activated additional IP 3 production in PC12-D1 cells but not in PC12-V cells. The additional IP 3 production induced by these agents disappeared in the absence of extracellular Ca 2ϩ (Fig. 7B). The results, therefore, suggest that entry of extracellular Ca 2ϩ activates PLC-␦1.

Activation of PLC-␦1 by BK-induced Capacitative Calcium
Entry-Since the BK-induced Ca 2ϩ influx is generally thought to occur by capacitative calcium entry through Ca 2ϩ releaseactivated Ca 2ϩ channels, it is likely that PLC-␦1 activation after BK treatment is mainly due to capacitative calcium entry. To test this hypothesis, the effect of reintroduction of extracel-lular Ca 2ϩ 30 s after stimulation with BK in the absence of extracellular Ca 2ϩ on IP 3 production was investigated. In contrast to PC12-V cells (Fig. 8B), PC12-D1 cells showed a significant increase in IP 3 after the reintroduction of extracellular Ca 2ϩ (Fig. 8D). In addition, SK&F 96365, an inhibitor of Ca 2ϩ release-activated Ca 2ϩ channels, diminished the additional IP 3 production stimulated by the entry of extracellular Ca 2ϩ into the PC12-D1 cells (Fig. 8D), without affecting the BK-induced IP 3 production into the PC12-V cells (Fig. 8B). These results suggest that, when PC12 cells are stimulated with BK, PLC-␦1 is activated by capacitative calcium entry occurring subsequent to IP 3 production and Ca 2ϩ release after PLC-␤ activation. These results also explain greater Ca 2ϩ influx induced by BK stimulation in PC12-D1 cells as shown in Fig. 4. Capacitative calcium entry in the PC12-D1 cells triggers serial feedback events such as rapid activation of PLC-␦1, more IP 3 production, further depletion of Ca 2ϩ stores, and more capacitative calcium entry.

Effect of PLC-␦1 Overexpression on [ 3 H]NE Secretion-
The effect of PLC-␦1 overexpression on catecholamine secretion, in which Ca 2ϩ increase plays a key role, was also investigated. Like the Ca 2ϩ increase, the BK-induced [ 3 H]NE secretion was much greater in the PC12-D1 cells ( Table I) Lack of Involvement of G h ␣ in PLC-␦1 Activation-PLC-␦1 has been reported to be linked to G h ␣ protein in human myometrium (40,41). In order to elucidate possible involvement of G h ␣ in the BK receptor-mediated signaling, we investigated whether G h ␣ is expressed in PC12 cells. For the examination of the nature of the G-proteins involved in the BK receptor-mediated signal transduction, photoaffinity labeling of G-proteins was carried out. As shown in Fig. 9, a labeling of the 74 -80-kDa protein, G h ␣, was not detected, whereas labeling of the 40 -50-kDa bands was detected. The labeling of these protein bands was specific for guanine nucleotides, since all of these bands could be blocked by unlabeled GTP␥S but not by p(N-H)ppA. These results, therefore, suggest that G h ␣ is not involved in BK receptor signaling. To confirm the above results, we performed a transglutaminase assay, since G h ␣ has transglutaminase activity in addition to GTPase activity. Transglutaminase activity is known to be increased by Ca 2ϩ and blocked by GTP␥S alone or by receptor activation in the presence of GTP␥S (32). As shown in Table II, the transglutaminase activity of purified G h ␣ was enhanced by the addition of Ca 2ϩ , and the enhanced activity was inhibited by GTP. However, there was no detectable transglutaminase activity even in the presence of 1 mM CaCl 2 in the PC12 cells. In addition, immunoblot-ting analysis also revealed that G h ␣ is absent from PC12 cells (data not shown). All of these observations strongly suggest that PLC-␦1 is not coupled to G h ␣ and that Ca 2ϩ ion concentration is the main regulator of PLC-␦1 activity in PC12 cells. Therefore, in PC12 cells activation of PLC-␦1 occurs in a second step after the BK receptor-mediated activation of PLC-␤ isozymes. Furthermore, capacitative calcium entry is important to the activation of PLC-␦1. DISCUSSION Our study clearly demonstrates that in PC12 cells PLC-␦1 is activated not by G-protein, G h ␣, but by Ca 2ϩ ions. More importantly, we found that the activation of PLC-␦1 is mainly dependent upon extracellular Ca 2ϩ ions that enter by capacitative calcium entry via the BK receptor-mediated PLC-␤ pathway. PC12 cells contain at least three immunologically  distinct PLC isozymes, PLC-␤, PLC-␥, and PLC-␦ (33). It has been considered that the BK receptor might be coupled to PLC-␤1 through a family of G-proteins, G q (34). In general, BK can stimulate phosphoinositide hydrolysis in a variety of cell types. However, BK did not lead to production of inositol phosphate in Chinese hamster ovary cells transfected with PLC-␦1 cDNA. This may be due to the absence of PLC-␤1 expression in the host Chinese hamster ovary cells (35). Our results clearly show that the BK-induced IP 3 production and [Ca 2ϩ ] i increase was markedly enhanced in the PLC-␦1-overexpressing PC12-D1 cells as compared with the vector-transfected PC12-V cells. In contrast to previous studies in which permeabilized cells were mainly used to prove that Ca 2ϩ can play the role of PLC-␦1 activator (16,35), our investigations were performed under physiological conditions without permeabilization. It has been suggested that agonist-induced hydrolysis of phosphoinositides is relatively insensitive to the removal of extracellular Ca 2ϩ and that the artificial elevation of Ca 2ϩ does not promote phosphoinositide hydrolysis (36). Banno et al. (37) suggested that in MC3T3-E1 cells, which contain much higher amounts of PLC-␤1 and PLC-␥1 but less PLC-␦1, BK-stimulated IP 3 generation was neither affected by the chelation of extracellular Ca 2ϩ with EGTA nor by intracellular Ca 2ϩ elevation by ionomycin. This is also the case for our wild type PC12 cells. However, in our PC12-D1 cells, cytosolic [Ca 2ϩ ] i rise and IP 3 generation were diminished in the absence of extracellular Ca 2ϩ . Therefore, we suggest that extracellular Ca 2ϩ is necessary to the activation of PLC-␦1. In permeabilized PLC-␦1overexpressing Chinese hamster ovary cells, the [Ca 2ϩ ] i level up to 1 M was sufficient to cause significant IP 3 production, whereas no significant IP 3 production was observed at the same Ca 2ϩ concentration in vector-transfected cells. These results suggest a preferential association of Ca 2ϩ with PLC-␦1 when compared with PLC-␤ in vivo (35). It has been proposed that the initial transient cytosolic [Ca 2ϩ ] i induced by IP 3 resulting from receptor/G-protein-mediated PLC activation may in turn contribute to the prolonged activation of PLC in a positive feedback system (38). Our results strongly support this hypothetical model. The BK receptor-mediated signaling in PC12-D1 cells indicates that the activation of PLC-␤ isozymes leads to a subsequent activation of PLC-␦1. This explains why PLC activity was not affected in PC12-V cells but significantly reduced in PC12-D1 cells in the absence of extracellular Ca 2ϩ . Previous studies of PLC-␦1 have suggested that the presence of Ca 2ϩ ions is sufficient to activate the enzyme. Changes in Ca 2ϩ ion concentration within the physiological range (100 nM to 10 M) selectively stimulated the activity of PLC-␦1 in permeabilized PC12 cells, and the activity of this enzyme was further enhanced in the presence of phosphatidylinositol transfer protein, which could function in supplying and favorably presenting the substrate directly to the enzymes that hydrolyze or modify PIP 2 (16). PLC-␦1 was also reported to directly associate with its receptor through a novel type of G-protein, G h (39). Among the known PLC isozymes, PLC-␤1 and PLC-␥1 were not stimulated by activated G h in a reconstituted system, but a 69-kDa PLC, a proteolytic fragment of PLC-␦1, was found coupled to G h proteins (32,39). When an agonist binds to its receptor, PLC-␦1 is directly activated by GTP-bound G h ␣. The ␣-subunit of this heterodimeric G-protein is characterized by its transglutaminase activity in addition to its GTP binding function. The regulation of PLC-␦1 by G h ␣ seems to be different from the regulation of PLC-␤ isozymes by the subunits of heterotrimeric G-proteins when analyzed in a similar system in vitro (39). ␣ 1B -Adrenergic receptors activate a 69-kDa PLC by coupling to G h ␣ (32). Likewise, PLC-␦1 is an effector of oxytocin receptormediated signaling via G h ␣ in human myometrium (40,41). In these cases, each receptor can independently activate PLC via either G q or G h , just as the thrombin receptor simultaneously and directly couples to G i2 and G q/11 (42). Thus, the same receptor can use multiple G-proteins and effectors to transmit a signal (43,44). To test for a possible coupling of G q and G h with the BK receptors, we investigated whether G h ␣ is expressed in PC12 cells, but we found G h ␣ was not detectable.
Our present study clearly indicates that Ca 2ϩ ions are the main regulators of PLC-␦1 and PLC-␦1 is secondarily activated by the entry of extracellular Ca 2ϩ , in particular by capacitative calcium entry as a downstream effect of PLC-␤ activation during BK receptor-mediated signaling. This regulation of PLC-␦1 has an important physiological meaning as presenting a positive feedback mechanism in that the signaling mediated by PLC-␤-linked receptors can be potentiated and prolonged. This fact explains why the Ca 2ϩ entry was much higher in the PC12-D1 cells than in the PC12-W or PC12-V cells when extracellular Ca 2ϩ was reintroduced after stimulation with BK in the absence of extracellular Ca 2ϩ . Since there are many possible ways in which various PLC isozymes can be activated, this kind of investigation will help to elucidate the role and regulation of PLC-␦1, which still remain an open question in receptor-mediated signaling.  It is interesting that wild type PC12 cells hardly exhibit the Ca 2ϩ entry-mediated activation of PLC-␦1, although they express a significant level of PLC-␦1. Comparative analysis of the correlation between the level of PLC-␦1 expression and the magnitude of BK-induced [Ca 2ϩ ] i increase in the various PC12 clones suggested that PLC-␦1 can be significantly activated by cytosolic calcium ion when the expression level of PLC-␦1 is higher than that of wild type PC12 cells. In addition, similar potentiation of BK-induced [Ca 2ϩ ] i rise was detected in ␦15 and ␦14 clones, although the expression level of PLC-␦1 was different. The results show a saturating effect in the elevation of cytosolic calcium when the enzyme is expressed higher than a certain level. However, the possibility cannot be ruled out that the initial amount of [Ca 2ϩ ] i elevation caused by BK-induced PLC-␤ activation is a limiting factor. In physiological environments, if there is any tissue in which PLC-␦1 is expressed, PLC-␦1 may play an important role in calcium signaling. Therefore, it will be interesting to investigate the expression level of PLC-␦1 and Ca 2ϩ entry-mediated potentiation of phosphoinositide hydrolysis in various tissues and cells.