Regulation of Phospholipase C- (cid:1) Activity by Glycosphingolipids*

Glycosphingolipid-enriched domains are hot spots for cell signaling within plasma membranes and are characterized by the enrichment of glycosphingolipids. A role for glucosylceramide-based glycosphingolipids in phospholipase C-mediated inositol 1,4,5-trisphosphate formation has been previously documented. These earlier studies utilized a first generation glucosylceramide synthase inhibitor to deplete cells of their glycosphingolipids. Recently, more active and specific glucosylceramide synthase inhibitors, including D - threo -ethylendioxyphenyl-2- palmitoylamino-3-pyrrolidinopropanol ( D - t -EtDO-P4), have been designed. D - t -EtDO-P4 has the advantage of blocking glucosylceramide synthase at low nanomolar concentrations but does not cause secondary elevations in cell ceramide levels. In the present study, D - t -EtDO-P4 depleted cellular glucosylceramide and lactosylceramide in cultured ECV304 cells at nanomolar concentrations without obvious cellular toxicity. The expression of several signaling proteins was evaluated in glycosphingo-lipid-depleted ECV304 cells to study the role of glycosphingolipids in phospholipase C-mediated signaling. No difference was observed in the cellular expression of phospholipase C- (cid:1) between controls and glycolipid-depleted cells. Western blot analysis, however, revealed that depletion of endogenous glycosphingolipids in cultured ECV304 cells with D - t -EtDO-P4 induced tyrosine phosphorylation of phospholipase C- (cid:1) in a concentration-dependent manner with maximum induction at 100 n M . The phosphorylation of phospholipase C- (cid:1) induced by D - t -EtDO-P4 was abolished by exogenously added glucosylceramide, consistent with a specific glycosphingolipid-phospholipase C- (cid:1) interaction. The phospholipase C- (cid:1) phosphorylation was maximally enhanced by bradykinin when cells were exposed to 100 n M D - t -EtDO-P4. The meas- urement of cellular activity of phospholipase C- (cid:1) , by myo inositol 1,4,5-trisphosphate radioreceptor assay, demonstrated that depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells with D - t - EtDO-P4 resulted in significantly increased formation of inositol 1,4,5-trisphosphate above base line, and an increased sensitivity of phospholipase C- (cid:1) to bradykinin stimulation. Thus, the activation of phospholipase C- (cid:1) is negatively regulated by membrane glycosphingolipids in ECV304 cells. 1,4,5-trisphosphate; Madin-Darby O

Caveolae are small invaginations in plasma membranes, recognized almost 50 years ago (1,2). These invaginations depend on the expression of caveolin-1, an integral membrane protein. Caveolae are also characterized biochemically as low density membrane domains that are highly enriched in both cholesterol and glycosphingolipids. Cells that lack caveolin-1 also have low density domains, commonly termed lipid rafts or glycosphingolipid-enriched microdomains.
Several investigators have provided support for the hypothesis that these microdomains are hot spots for cell signaling. Support for this view includes the observation that several signaling molecules are concentrated in these lipid domains. These molecules include EGF 1 (3) and PDGF receptors (4), endothelin receptors (5), endothelial nitric-oxide synthase (6), Src family kinases (7), Grb2 (8), Shc (9), mitogen-activated protein kinase (10), and heterotrimeric and low molecular weight G proteins (11). Phosphatidylinositol 4,5-bisphosphate (12,13) and sphingomyelin (14) are concentrated in caveolae. Both lipids are subject to hydrolysis following stimulation of cells with agonists. In addition, other receptors such as angiotensin II (15) and bradykinin (16) are recruited to caveolae following the stimulation of cells with agonists.
Cholesterol appears to be important for the regulation of signal transduction within these microdomains. For example, the depletion of cellular cholesterol with either filipin or lovastatin inhibits PDGF-stimulated kinase activities. Similarly, the depletion of cholesterol with methyl ␤-cyclodextrin inhibits EGF-and angiotensin II-stimulated phosphatidylinositol hydrolysis (17). By contrast, less is known about the potential role of glycosphingolipids in the regulation of signaling events. In earlier work it had been reported that glucosylceramide depletion of MDCK cells with a first generation glucosylceramide synthase inhibitor, PDMP, resulted in the enhanced formation of inositol 1,4,5-trisphosphate following bradykinin stimulation (18). This observation had several limitations. First, the glucosylceramide synthase inhibitor, PDMP, had limited activity and specificity. Glucosylceramide depletion was also accompanied by an increase in cell ceramide levels (19). Second, the mechanism of enhanced phospholipase C activity was not delineated. Third, little was known regarding the microdomains in which phospholipase C activity was present to interpret the significance of this observation.

Materials-Protein
Cell Culture-Human ECV304 cells were routinely maintained in Medium 199 (M199) supplemented with 10% (v/v) newborn calf serum, 2 mM L-glutamine, 4.5 g/liter D-glucose, 100 g/ml streptomycin, and 100 units/ml penicillin. The cells were grown to subconfluence (90%) in either 100-or 150-mm culture dishes at 37°C in a 5% CO 2 -enriched and humidified atmosphere. For treatment of cells with glucosylceramide synthase inhibitors, medium containing 10% serum was aspirated and replaced by serum-free M199 with or without D-t-EtDO-P4 for 48 h before each experiment. ECV304 cells co-incubated with glucosylceramide received 1 M glucosylceramide or lactosylceramide administered as a liposomal preparation (18). Stock solutions of D-t-EtDO-P4 dissolved in 100% Me 2 SO were diluted with serum-free M199 before use. The working solution of D-t-EtDO-P4/M199 contained 1% Me 2 SO, and the final concentrations of Me 2 SO in the incubation media were less than 0.03%. The normal morphology of cells and the total protein content of cultured cells were unaffected by treatment with up to 3 M D-t-EtDO-P4 for 48 h.
Immunoprecipitation and Immunoblotting-ECV304 cell stimulation was conducted at 37°C following treatment with D-t-EtDO-P4. Quiescent cultures of ECV304 cells in 100-mm dishes (90% confluence) were stimulated with bradykinin (2 M) for various times. Following stimulation, plates were washed twice with ice-cold phosphate-buffered saline containing 1 mM Na 3 VO 4 . The cells were lysed by the addition of 1 ml of ice-cold lysis buffer. Lysis buffer consisted of 25 mM Tris-HCl, pH 7.4, 1% Triton X-100, 10% glycerol, 20 mM NaF, 2 mM EDTA, 2 mM Na 3 VO 4 , 137 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin. Plates were then left on ice for 10 min. The cells were harvested by scraping, followed by sonication for 2 s twice with a probe sonicator. The homogenates were clarified by centrifugation at 16,000 ϫ g for 15 min. Samples were normalized for equal amounts of protein using bicinchoninic acid assay (Sigma) with bovine serum albumin as a standard. For immunoprecipitation studies, cell lysates (600 g of protein) were incubated with anti-phosphotyrosine (4 g/ml) for 2 h at 4°C with gentle rotation. After this, protein A-agarose beads (60 l of a 50% suspension) were then added and the samples were rotated at 4°C for an additional 1 h. The immune complexes were recovered by centrifugation at 16,000 ϫ g for 30 s and washed four times with ice-cold buffer containing 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 5 g/ml leupeptin, and 10 g/ml aprotinin. The immunoprecipitates were recovered with Laemmli sample buffer (22) and heated for 5 min at 90°C. The immunoprecipitates or 10 -50 g of protein from total cell lysates were then subjected to a 4 -13% gradient SDS-PAGE and transferred to a nylon membrane (Invitrogen). The membranes were incubated with selected antibodies. Immunoblots were developed and visualized with the enhanced chemiluminescence-plus system (ECL-plus) (PerkinElmer Life Sciences).
Extraction and Measurement of 1,4,5-IP 3 -Following treatment with D-t-EtDO-P4, stimulated cells were exposed to 2 M bradykinin at various times. The incubations were stopped with 100% ice-cold trichloroacetic acid. The dishes were left on ice for 10 min, and the cells were scraped. The cell extracts were centrifuged for 1 min in a microcentrifuge at 12,000 ϫ g. The trichloroacetic acid was removed from extracts by adding 2 ml of trioctylamine/1,1,2-trichloro-1,2,2-trifluoroethane mixture (1:3). After vigorous shaking the supernatants were partitioned, and a clear aqueous upper layer that contained water-soluble 1,4,5-IP 3 was carefully removed and stored on ice for assay. The level of 1,4,5-IP 3 was determined by a competitive ligand-binding assay according to the manufacturer's instructions (Amersham Biosciences).
Lipid Extraction and Analysis-Cells cultured in 150-mm dishes were deprived of serum and incubated in the absence or presence of D-t-EtDO-P4 (concentrations from 0.1 nM to 1 M) for 48 h. Cells were then washed with ice-cold phosphate-buffered saline two times, fixed with 2 ml of ice-cold methanol, and harvested by scraping. The lipids were extracted with chloroform-methanol-water at a ratio of 1:2:0.8 (v/v/v). The samples were sonicated for 15 min in a bath sonicator and centrifuged at 2200 ϫ g for 30 min. The supernatants were transferred into new glass tubes, and pellets were re-extracted with 3 ml of chloroform/methanol (2:1, v/v). After a brief sonication, samples were centrifuged at 2200 ϫ g for another 30 min. The resultant supernatants were pooled with the first extracts and partitioned into aqueous and organic phases by the addition of chloroform and water. The ratio of chloroform/methanol/water was adjusted to 2:1:0.8 (v/v/v). The upper aqueous phase was removed and discarded after centrifugation at 500 ϫ g for 5 min, and the lower organic phase was dried under a stream of nitrogen gas. The residues were resuspended in 600 l of chloroform/methanol (2:1, v/v). After determination of lipid phosphate (23), a portion of the lipids (150 nmol of total phospholipid phosphate) was subjected to base hydrolysis by incubation with 2 ml of chloroform and 1 ml of 0.2 N NaOH in methanol at 37°C for 1 h. The incubation was terminated by the addition of 0.8 ml of 0.3 M acetic acid. The lower organic phase was washed with methanol/water (1:0.8, v/v) two times and evaporated under a stream of nitrogen gas. The residues were dissolved into 60 l of chloroform/methanol (80:20, v/v) and analyzed by high performance thin layer chromatography with a solvent system consisting of chloroform/methanol/water (65:25:4, v/v/v). The glucosylceramide and lactosylceramide levels were determined by densitometric scanning using NIH Image 1.62 software and compared with authentic standards run in parallel on the same plates. The quantitative measurement of ceramide was performed by the diacylglycerol kinase assay (24). Briefly, lipid samples containing 50 nmol of total phospholipid phosphate were evaporated under a steam of nitrogen gas. The ceramide in the lipid extract was converted to [ 32 P]ceramide 1-phosphate by enzymatic reaction with diacylglycerol kinase. A set of ceramide standards was included with each experiment. The products were separated by high performance thin layer chromatography followed by autoradiography. The radioactive spots were scraped and counted by liquid scintillation spectrometry.
For the radiolabeling studies, cells were treated with vehicle or with 100 nM D-t-EtDO-P4 for 12 h at 37°C. The cellular lipids were then radiolabeled by the addition of D-[1-3 H]galactose (0.5 mCi/ml, 6.1 Ci/ mmol) for an additional 24 h before the extraction of total cellular lipids or before the isolation and extraction of the caveolar fractions. The glycosphingolipid-enriched fractions were isolated as previously described (25).

RESULTS
D-t-EtDO-P4 was first solubilized in 100% dimethyl sulfoxide and then diluted in culture medium prior to addition to achieve a final Me 2 SO concentration of less than 0.03%. ECV304 cells grown in the medium containing up to 0.1% Me 2 SO alone for 48 h did not show any significant changes in cell morphology, cell viability, protein, and total phospholipid content compared with control cells (data not shown). The cytotoxicity induced by D-t-EtDO-P4 on the ECV304 cells was measured by trypan blue exclusion and lactate dehydrogenase release. The concentration at which toxicity was observed in 50% of the cultured cells (TC 50 value) was 6 Ϯ 0.25 M (n ϭ 9) after a 48-h incubation with D-t-EtDO-P4.
The concentration-dependent depletion of glucosylceramide and lactosylceramide in ECV304 cells by D-t-EtDO-P4 was next determined (Fig. 1, A and B). Cells were incubated with D-t-EtDO-P4 for 48 h at varying concentrations of inhibitor (0.1, 1, 10, 25, 50, 75, 100, and 150 nM) in serum-free medium. Under these conditions, treatment of cells with 100 nM D-t-EtDO-P4 resulted in maximal decrements of 99.9% glucosylceramide mass and 99.7% lactosylceramide mass, respectively. The concentrations at which half-maximal depletion of glucosylceramide and lactosylceramide occurred were 0.2 Ϯ 0.05 nM and 0.4 Ϯ 0.05 nM, respectively. The ratio of TC 50 /IC 50 or selective index was Ͼ12,000. The toxicities associated with PDMP and earlier homologues of the glucosylceramide synthase inhibitor were associated with elevations in cell ceramide content.
To confirm the high selective index observed with D-t-EtDO-P4 treatment of the ECV304 cells occurred in the absence of changes in cell ceramide levels, ceramide content was determined using the diglyceride kinase assay. At concentrations where D-t-EtDO-P4 was highly effective in depleting glucosylceramide and lactosylceramide, cell ceramide levels were not significantly elevated in ECV304 cells (Fig. 2). Only a 10% increase in ceramide content was seen when ECV304 cells were incubated with 600 nM D-t-EtDO-P4 for 48 h. No changes in sphingomyelin content were detected in cells treated with D-t-EtDO-P4 (data not shown). These results confirm that D-t-EtDO-P4 is both an active and specific inhibitor of glucosylceramide synthase.
The lipid composition of the intact ECV304 cells and the glycosphingolipid-enriched fractions were compared (Table I).
As previously observed in NIH 3T3 cells, a significant enrichment of the major cellular sphingolipids was observed (25). A moderate enrichment of cholesterol was also seen. Triglyceride, on the other hand, was markedly de-enriched in the sphingolipid-enriched fractions. The effect of glucosylceramide synthase inhibition on the glycosphingolipids from these fractions was evaluated by radiolabeling the ECV304 cells with D-[1-3 H]galactose (Fig. 3). The major glycolipids labeled in the intact cells were glucosylceramide, lactosylceramide, globotriaosylceramide, and ganglioside GM3. Globotriaosylceramide and ganglioside GM3 were not detected by charring of the thin layer chromatography plates. Their identification by radiolabeling was aided by the incorporation of the tritiated galactose into both glucose and galactose by the conversion of the radiolabeled substrate, UDP-glucose, to UDP-galactose by a cellular epimerase (18). The radiolabeling of glucosylceramide, lactosylceramide, and globotriaosylceramide was increased in the sphingolipid-enriched fractions. However, ganglioside GM3 was significantly de-enriched in these fractions. The absence of ganglioside GM3 in these fractions is consistent with previous observations (26).
The expression of several signaling proteins including phospholipase C-␥, phospholipase C-␤, phospholipase C-␦, eNOS, c-Raf-1, and Ras as well as annexin II was evaluated by immunoblot analysis following glycosphingolipid depletion with D-t-EtDO-P4 (Fig. 4). Because phospholipase C-␥ has been identified as a key mediator of PDGF-dependent cellular transformation (27), the expression of PDGF-R␤, therefore, was also examined. ECV304 cells were exposed for 48 h to concentrations of D-t-EtDO-P4 ranging from 50 to 300 nM. Cell lysates containing equal amounts of protein were subjected to SDS-PAGE and immunoblotted. No significant differences were observed in the cellular expression of phospholipase C-␥1, phospholipase C-␤1, phospholipase C-␦1, annexin II, eNOS III, c-Raf-1, PDGF-R␤, and Ras between control cells and cells treated with EtDO-P4. These results demonstrate that deple- tion of glucosylceramide-based glycosphingolipids with EtDO-P4 has no effect on the expression of these proteins in cultured ECV304 cells.
Immunoprecipitation using anti-phosphotyrosine antibody followed by Western blot analysis with anti-phospholipase C-␥1 antibody revealed that greater than 99% depletion of glucosylceramide-based glycosphingolipids with 100 nM D-t-EtDO-P4 induced a significant increase in the tyrosine phosphorylation of phospholipase C-␥ (Fig. 5A). The tyrosine phosphorylation was concentration-dependent and paralleled the depletion of glucosylceramide and lactosylceramide. Concentrations of D-t-EtDO-P4 in excess of 100 nM, however, caused a reduction of phospholipase C-␥1 phosphorylation to basal levels. When ECV304 cells were simultaneously incubated with D-t-EtDO-P4 and exogenously added glucosylceramide (1 M), the effect of D-t-EtDO-P4 on the induction of tyrosine phosphorylation of phospholipase C-␥ was abrogated (Fig. 5B), consistent with a functional role of glycosphingolipids in the mediation of transient tyrosine phosphorylation of phospholipase C-␥1.
To determine whether the induction of phospholipase C-␥1 phosphorylation by D-t-EtDO-P4 resulted in a change in phos-pholipase C activity, the hydrolysis of phosphatidylinositol 4,5bisphosphate in the glycosphingolipid-depleted ECV304 cells was measured as inositol 1,4,5-trisphosphate formation. The concentration-and time-dependent effects of EtDO-4 on bradykinin-stimulated inositol 1,4,5-trisphosphate formation were studied (Fig. 6, A-C). The peak formation of 1,4,5-IP 3 formation was observed in cells exposed to 100 nM EtDO-P4. At this concentration of inhibitor, glucosylceramide content was maximally depleted and peak phosphorylation of phospholipase C-␥1 were noted. Cells were also stimulated with 2 M bradykinin. A markedly increased 1,4,5-IP 3 accumulation above base line was observed in bradykinin-stimulated cells treated with inhibitor. The peak effect was noted to occur at 100 nM EtDO-P4 (Fig. 6A).
The relationship between inhibitor concentration and peak 1,4,5-IP 3 formation was determined. The stimulation with bradykinin resulted in a more sustained generation of 1,4,5-IP 3 as the concentration of D-t-EtDO-P4 increased (Fig. 6, B and C). When control cells were stimulated by 2 M bradykinin in the absence of inhibitor pretreatment, a peak of 1,4,5-IP 3 formation  3. Autoradiography of the total cellular and caveolar lipids in the presence or absence of D-t-EtDO-P4. ECV304 cells were treated with 100 nM D-t-EtDO-P4 or vehicle alone for a total of 36 h. After an initial 12-h exposure to inhibitor, the cells were radiolabeled with D-[1-3 H]galactose for an additional 24 h prior to fractionation. Radiolabeled lipids were identified by autoradiography after separation by high performance thin layer chromatography with a solvent system consisting of chloroform/methanol/water (65:25:4, v/v/v). The results are representative of four separate experiments. GlcCer, glucosylceramide; LacCer, lactosylceramide; Gb 3 , globotriaosylceramide; GM 3 , GM3.
The phosphorylation of phospholipase C-␥1 was further assessed to determine whether the change in peak 1,4,5-IP 3 formation corresponded to a change in the tyrosine phosphorylation of the lipase. A peak in tyrosine phosphorylation of the phospholipase was noted to occur at 100 nM EtDO-P4 in the bradykinin stimulated cells (Fig. 7A), the same concentration noted in the unstimulated cells (Fig. 5). No change in total phospholipase C-␥1 was observed. When the phosphorylation was evaluated as a function of time following bradykinin exposure, cells that were not treated with inhibitor demonstrated a peak tyrosine phosphorylation at 30 s (Fig. 7B). In contrast, cells pretreated with 100 nM EtDO-P4 demonstrated a peak of tyrosine phosphorylation at 2 min. The time course was comparable with that observed for 1,4,5-IP 3 formation (Fig. 7C).

DISCUSSION
The present study supports and extends earlier observations reported on the role of glucosylceramide-based glycolipids in the regulation of agonist-stimulated phospholipase C activity. In earlier studies the treatment of MDCK cells with PDMP, a first generation glucosylceramide synthase inhibitor, increased hormone-stimulated 1,4,5-IP 3 levels significantly over base line (18). The effect increased as a function of PDMP exposure and could be replicated in isolated plasma membranes. Furthermore, the increased 1,4,5-IP 3 occurred in membranes exposed to GTP␥S, consistent with an increased activity of phospholipase C. No changes in phosphatidylinositol 4,5-bisphosphate were observed. As in the present study, the PDMP effect was reversed with exogenous glucosylceramide but not galactosylceramide addition. In a subsequent study, increases in endogenous glucosylceramide were shown to have the opposite effect (28). The incubation of MDCK cells with conduritol B epoxide, an inhibitor of ␤-glucocerebrosidase, inhibited phospholipasedependent 1,4,5-IP 3 formation. The conduritol B epoxide effect was both time-and concentration-dependent.
PDMP represents the prototypical glucosylceramide synthase inhibitor. This compound has found widespread application as a tool for the cellular depletion of glucosylceramidebased glycosphingolipids (20). PDMP contains two chiral carbons and thus four enantiomers. Only the D-threo enantiomer of this compound is active conferring a relative high degree of specificity. However, its use is limited in two respects. First, the inhibitory activity of PDMP against the cerebroside synthase is in the middle micromolar range. Second, PDMP treatment of cells results in the accumulation of ceramide, a bioactive sphingolipid. The ceramide elevating effect was originally believed to be secondary to the accumulation of substrate for glucosylceramide formation. However, with the development of more active PDMP homologues, specifically 1-phenyl-2-palmitoyl-3-pyrrolidinopropanol, the ceramide elevation was shown to result from the inhibition of a second enzyme, 1-Oacylceramide synthase (29). This enzyme has phospholipase A 2 activity and catalyzes the transacylation of sn-2 fatty acids from phosphatidylethanolamine or phosphatidylcholine to the 1-hydroxyl of ceramide. Both PDMP and P4 inhibit the transacylase at micromolar concentrations.
The recognition of this second site of PDMP activity and the ability to dissociate ceramide accumulation from glucosylceramide depletion led to the design and synthesis of more active PDMP homologues by Hansch analysis (21). Phenyl group substitutions resulted in two compounds that were significantly more active against glucosylceramide synthase but retained limited activity against the transacylase. These compounds included the ethylendioxyphenyl-and 4Ј-hydroxyphenyl-P4 homologues. Both compounds significantly deplete glucosylceramide-based glycosphingolipids at concentrations between 10 and 100 nM without observable changes in ceramide content. Because the inhibitors have the ability to deplete glycosphingolipids without inducing cell toxicity, they have been proposed for use in the treatment of glucosylceramide-based glycosphingolipidoses, including Fabry disease (30).
In the present study, it has been demonstrated that glucosylceramide depletion in the absence of changes in ceramide content increases the activity of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C␥. The increase in phospholipase activity is the result of changes in tyrosine phosphorylation and not the result of changes in recoverable phospholipase C␥. This conclusion is supported by the close relationships between the biphasic nature of the phosphorylation and the time course of phosphorylation following bradykinin stimulation.
These results extend those of other investigators that have implicated glycosphingolipid containing membrane domains and their unique biochemical composition in the regulation of phosphoinositide signaling. Pike and Casey (12) first demonstrated that phosphatidylinositol 4,5-bisphosphate turnover was localized to caveolae. Subsequently it was demonstrated that EGF-and bradykinin-stimulated phosphatidylinositol turnover was inhibited by cholesterol depletion (31). This basic finding has been confirmed in more recent studies.
The mechanism of glycosphingolipid-mediated regulation of phospholipase C activity remains undefined. Tyrosine phosphorylation of the phospholipase C predictably represents the balance between both kinase and phosphatase activities. Strong support exists for the direct association of ganglioside GD3 with the Src family tyrosine kinase Lyn (7). Whether glucosylceramide-based glycosphingolipids interact with and regulate the activities of one or both of these enzymes or modulate the tyrosine phosphorylation of phospholipase C␥ by direct interaction with the lipase itself will require additional study. FIG. 7. Enhancement of tyrosine phosphorylation of phospholipase C-␥1 by bradykinin (BK) stimulation in glycosphingolipid-depleted human ECV304 cells. A, controls and treated cells (100 nM D-t-EtDO-P4) were stimulated with 2 M bradykinin for 2 min. Following stimulation, cells were lysed, and the cell lysates (600 g of protein) were immunoprecipitated (IP) with anti-phosphotyrosine antibody. The immunoprecipitates (upper panel) and 10 g of cell lysate protein (lower panel) were subjected to a 4 -13% gradient SDS-PAGE. After transfer the membrane was probed with a monoclonal antibody to phospholipase C-␥1. B, control cells were stimulated with 2 M bradykinin for different time intervals. C, treated cells were exposed to 100 nM D-t-EtDO-P4 for 48 h, and were then stimulated with 2 M bradykinin for various times. A blot representative of three independent experiments is shown. WB, Western blot.