Activation of PLC-{delta}1 by Gi/o-coupled receptor agonists

Karnam S. Murthy,1 Huiping Zhou,1 Jiean Huang,1 and Srinivas N. Pentyala2

1Departments of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298; and 2Department of Anesthesiology, School of Medicine, State University of New York, Stony Brook, New York 11794

Submitted 25 May 2004 ; accepted in final form 1 August 2004


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The mechanism of phospholipase (PLC)-{delta} activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca2+ stimulated an eightfold increase in PLC-{delta}1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three Gi/o-coupled receptor agonists (somatostatin, {delta}-opioid agonist [D-Pen2,D-Pen5]enkephalin, and A1 agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-{delta}1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca2+ influx and was not observed in the absence of extracellular Ca2+, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca2+ channels. Treatment of the cells with a Gq/13-coupled receptor agonist, CCK-8, caused only transient, PLC-{beta}1-mediated PI hydrolysis. Unlike Gi/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-{delta}1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G{alpha}13 minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-{delta}1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca2+ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (~25%) and strongly inhibited by SKF-96365 (~75%) and in cells expressing PLC-{delta}1(E341R/D343R). Agonist-independent Ca2+ release or Ca2+ influx via voltage-gated Ca2+ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-{delta}1 antibody or nifedipine. We conclude that PLC-{delta}1 is activated by Gi/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca2+ influx via store-operated Ca2+ channels.

phospholipase C; G protein


FOUR FAMILIES OF phosphoinositide-specific phospholipases (PI-PLC), containing a total of eleven distinct isozymes (PLC-{beta}1, -{beta}2, -{beta}3, -{beta}4; PLC-{gamma}1, -{gamma}2; PLC-{delta}1, -{delta}2, -{delta}3, -{delta}4; and PLC-{epsilon}) have been cloned and characterized (13, 19, 41, 42). All the isozymes contain an NH2-terminal pleckstrin homology (PH) domain for protein-protein interaction, a central catalytic domain with several highly conserved residues (His311, His356, and Glu341), and a COOH-terminal, Ca2+-dependent, membrane-targeting C2 domain (10–13, 16, 42). A long COOH-terminal tail is a characteristic feature of PLC-{beta} isozymes and mediates G{alpha}-dependent binding and activation of PLC-{beta}1 (13, 22, 42, 46). PLC-{beta}2 and PLC-{beta}3 are activated via binding of their PH domains to G{beta}{gamma} subunits, particularly G{beta}{gamma}i (5, 6, 20, 29, 31, 33, 39). PLC-{epsilon} contains two COOH-terminal Ras-associating domains and a Ras guanine nucleotide exchange factor motif and is variously activated by Ras, Rap, G{alpha}12, G{beta}{gamma}, and RhoA (27, 47). PLC-{gamma} isozymes are activated by tyrosine phosphorylation of two conserved residues (Tyr771 and Tyr783) via receptor tyrosine kinases (13, 42).

The PH domain is essential for activation of PLC-{delta}, and its deletion abolishes PLC-{delta} activity (2, 8, 15). The PH domain of PLC-{delta} binds phosphatidylinositol 4,5-bisphosphate (PIP2), which anchors the enzyme to the plasma membrane and enhances its catalytic activity (15). Although the PH domain of PLC-{delta} binds G{beta}{gamma}, the binding is too weak to induce activation of PLC-{delta} (15, 44). Four specific receptors ({alpha}1B- and {alpha}1D-adrenoceptors, {alpha}-thromboxane receptors, and oxytocin receptors) can activate PLC-{delta} via coupling to the atypical G protein (G{alpha}h), also known as transglutaminase-II (3, 18, 40). The mechanism of PLC-{delta} activation via G{alpha}h is not known. A recent study suggests that PLC-{delta} is activated by hypotonicity in some neurons; the effect is mediated by neuromodulin, a neuronal, membrane-anchored osmosensory protein, but the mechanism has not been determined (7).

PLC-{delta}1, the most abundant and widely expressed isoform of PLC-{delta}, is typically activated by Ca2+ in the range of 0.1–10 µM (1, 26, 28). Despite its sensitivity to Ca2+ (~100-fold higher than that of PLC-{beta} or PLC-{gamma}), PLC-{delta} is not activated by agonists that mobilize intracellular Ca2+, possibly because PLC-{delta} is inactivated by PLC-{beta}-dependent generation of protein kinase C (PKC) and inositol trisphosphate (IP3); the latter competes with PIP2 for binding to PLC-{delta} (1, 4). Ca2+ influx induced by Ca2+ ionophores or via voltage-gated Ca2+ channels (e.g., depolarizing concentrations of KCl) causes moderate PI hydrolysis, but the specific isozyme mediating this effect has not been identified (21, 43). In a recent study, activation of PLC-{delta} by capacitative Ca2+ influx induced by thapsigargin or bradykinin was observed only after overexpression of PLC-{delta} (23).

In this study, we have identified a unique mechanism for activation of PLC-{delta}1 via Gi/o-coupled receptors. The inability of Gq/13-coupled receptors to activate PLC-{delta}1 reflected concurrent activation of RhoA.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of dispersed and cultured muscle cells. Smooth muscle cells were isolated from the circular muscle layer of rabbit stomach by sequential enzymatic digestion, filtration, and centrifugation, as described previously (32, 3638). In some experiments, cells were also isolated from the circular or longitudinal muscle layers of small intestine. The cells were resuspended in enzyme-free medium consisting of 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4, 2 mM CaCl2, 0.6 mM MgCl2, 25 mM HEPES, 14 mM glucose, and 2.1% Eagle's essential amino acid mixture. The cells were harvested by filtration through 500-µm Nitex mesh and centrifuged two times at 350 g for 10 min. In experiments with blocking antibodies, the cells were permeabilized with saponin (35 µg/ml) in a medium containing 100 nM Ca2+ and resuspended in 1.5 mM ATP and an ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine phosphokinase). In some experiments, the muscle cells were cultured in DMEM containing 10% FBS until they attained confluence and were used in the first passage.

Assay of PLC activity. Inositol phosphate formation was measured as described previously using anion exchange chromatography (29, 3133). Measurements were made in the absence of Li3+. Freshly dispersed muscle cells (106 cells/ml) were labeled with myo-[3H]inositol for 3 h and cultured muscle cells for 24 h. The cells were then centrifuged at 350 g for 10 min to remove excess [3H]inositol and resuspended in 10 ml fresh HEPES medium. The reaction was terminated by the addition of 940 µl chloroform-methanol-HCl (50:100:1). The samples were extracted with 340 µl chloroform and 340 µl H2O and centrifuged at 1,000 g for 15 min. The upper aqueous phase was applied to a DOWEX AG-1 column, and [3H]inositol phosphates were eluted with 0.8 M ammonium formamate-0.1 M formic acid. Radioactivity was determined by liquid scintillation and expressed as counts per minute per milligram of protein.

Expression of G{alpha}13 minigene, dominant-negative PLC-{delta}1 and RhoA, and constitutively active RhoA in cultured smooth muscle cells. G{alpha}13 minigene (MGLHDNLKQLMLQ), dominant-negative PLC-{delta}1(E341R/D343R) and RhoA(T19N), and constitutively active RhoA(G14V) were subcloned into the multiple cloning site (EcoRI) of the eukaryotic expression vector (PLC-{delta}1: pcDNA3; RhoA: pEXV), and a myc-tag incorporated in the NH2 terminus. Recombinant plasmid cDNA (2 µg) was transiently transfected in cultured smooth muscle cells treated with Lipofectamine Plus reagent for 48 h. The cells were cotransfected with 1 µg pGreen Lantern-1 to monitor expression. Control cells were cotransfected with 2 µg vector (pEXV or pcDNA3) and 1 µg pGreen Lantern-1 DNA. Transfection efficiency (~80%) was monitored by the expression of green fluorescent protein using FITC filters (36, 38).

Association of PLC-{delta}1 and activated RhoA. Muscle cells were lysed by incubation for 30 min at 4°C in 10 mM Tris (pH 7.5), 50 mM NaCl, 1% Triton X-100, and 60 mM octylglucoside, and the lysate was centrifuged at 15,000 g for 30 min. The supernatant was precleared by incubation with 0.1% albumin-coated protein A-Sepharose for 6 h at 4°C and then incubated overnight with polyclonal PLC-{delta}1 antibody at a final concentration of 2 µg/ml. Protein A-Sepharose was then added for 2 h, and the mixture was centrifuged for 5 min. The PLC-{delta}1 immunoprecipitates were washed four times with lysis buffer and boiled in Laemmli buffer. Samples were separated by SDS-PAGE in 12% acrylamide gel, electrotransferred to nitrocellulose paper, and probed with antibody to RhoA or PLC-{delta}1. After incubation with secondary antibody conjugated with horseradish peroxidase, the proteins were visualized using enhanced chemiluminescence. The intensity of the protein band on Hyperfilm-ECL was determined by densitometry.

Assay for activated RhoA. Activated RhoA was measured in freshly dispersed muscle cells by a technique using Rhotekin, as described previously (37). Muscle cell lysates (100 µg protein) were incubated with glutathione-agarose slurry of Rhotekin at 4°C for 45 min. The beads were washed three times with the washing buffer containing 50 mM Tris·HCl (pH 7.2), 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. GTP-bound RhoA was solubilized in Laemmli sample buffer and analyzed by 15% SDS-PAGE followed by Western blot and chemiluminescence.

Materials. Myo-[3H]inositol was obtained from New England Nuclear (Boston, MA); polyclonal antibodies to RhoA, PLC-{delta}1, and various isoforms of PLC-{beta} were from Santa Cruz Biotechnology (Santa Cruz, CA); pGreen Lantern-1 and Lipofectamine Plus reagent were from Life Technologies GIBCO-BRL (Rockville, MD); SKF-96365 was from Biomol (Plymouth Meeting, PA); and all other reagents were from Sigma. RhoA cDNA was a gift of Dr. Andrea Todisco (University of Michigan).


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of PLC-{delta}1 by Ca2+. Ca2+ concentrations in the range 0.1–1.0 µM stimulated PI hydrolysis (PLC activity) in permeabilized gastric smooth muscle cells in a concentration-dependent fashion [maximal 8- to 10-fold increase in PLC activity with 1 µM Ca2+ (5,021 ± 468 cpm/mg protein above basal); Fig. 1A]. The isoform of PLC activated by Ca2+ in smooth muscle was identified by functional blockade with specific antibodies. Our previous studies had shown that all PI-specific PLC isozymes, including PLC-{beta}1, -2, -3, and -4, PLC-{gamma}1, and PLC-{delta}1, were expressed in gastrointestinal smooth muscle (29, 32). Preincubation of permeabilized muscle cells for 1 h with PLC-{delta}1 antibody (10 µg/ml) inhibited maximal Ca2+-stimulated PLC activity by 84 ± 2%, whereas preincubation with PLC-{beta}1, PLC-{beta}2, PLC-{beta}3, PLC-{beta}4, or PLC-{gamma}1 antibody had no effect (Fig. 1B). The pattern of inhibition by PLC antibodies implied that Ca2+ specifically activated PLC-{delta}1.



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Fig. 1. Ca2+-induced stimulation of phospholipase C (PLC)-{delta}1 in smooth muscle. A: concentration-dependent stimulation of phosphoinositide (PI) hydrolysis by Ca2+. Muscle cells labeled with myo-[3H]inositol for 3 h were permeabilized (35 µg/ml for 5 min) and incubated with various concentrations of Ca2+ for 1 min. B: dispersed muscle cells labeled with [3H]inositol were permeabilized and incubated for 1 h with 10 µg/ml antibody to PLC-{beta}1, -{beta}2, -{beta}3, -{beta}4, -{gamma}1, or -{delta}1 and then treated with 1 µM Ca2+ for 1 min. PI hydrolysis was measured, in the absence of Li3+, using anion exchange chromatography and expressed as cpm per mg protein of total inositol phosphates. Ca2+ stimulated PLC activity in a concentration-dependent fashion, and the stimulation was selectively inhibited by preincubation of muscle cells with PLC-{delta}1 antibody. Values are means ± SE of 4–5 experiments. **Significant inhibition (P < 0.001) of Ca2+-stimulated PI hydrolysis.

 
Dual activation of PLC-{beta}3 and PLC-{delta}1 by Gi-coupled receptors. Previous studies in these smooth muscle cells had shown that Gi/o-coupled receptor agonists elicit an initial, transient (~2 min) increase in PI hydrolysis mediated by G{beta}{gamma}i/o-dependent activation of PLC-{beta}3 (29, 31, 33, 36). As shown in Fig. 2, treatment of cultured smooth muscle cells with a Gi-2-coupled receptor agonist ([D-Pen2,D-Pen5]enkephalin or DPDPE) caused an initial PLC-{beta}3-dependent increase in PI hydrolysis followed by a delayed increase (~8-fold above basal levels). The delayed increase in PI hydrolysis was virtually abolished in cells expressing dominant-negative PLC-{delta}1(E341R/D343R), whereas the initial increase was not affected. The delayed increase in PI hydrolysis was also inhibited by expression of a constitutive active RhoA. The significance of this observation is discussed subsequently.



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Fig. 2. Time course of PI hydrolysis mediated by Gi-coupled receptors. Cultured smooth muscle cells expressing vector alone, dominant-negative PLC-{delta}1(E341R/D343R), or constitutively active RhoA(G14V) were labeled with myo-[3H]inositol for 24 h, and the cells were treated with [D-Pen2,D-Pen5]enkephalin (DPDPE, 1 µM) for 10 min. The reaction was terminated at different time points, and PI hydrolysis was measured using anion exchange chromatography, as described in EXPERIMENTAL PROCEDURES. PI hydrolysis was expressed as cpm per mg protein of total inositol phosphates. DPDPE caused a biphasic increase in PI hydrolysis only in control cells expressing vector alone. The delayed increase in PI hydrolysis was strongly inhibited in cells expressing PLC-{delta}1(E341R/D343R) and RhoA(G14V). Inset: Western blot analysis of RhoA and PLC-{delta}1 in cells expressing vector alone (lanes 1) and overexpressing PLC-{delta}1(E341R/D343R) or RhoA(G14V) (lane 2). Values are means ± SE of 6 experiments.

 
A similar 8- to 10-fold increase in PI hydrolysis was elicited by Gi-1- and Gi-3-coupled receptor agonists [somatostatin and the A1 agonist cyclopentyl adenosine (CPA), respectively; Fig. 3A]. The increase induced by these agonists was strongly inhibited in cells expressing PLC-{delta}1(E341R/D343R) (Fig. 3A). The delayed increase in PI hydrolysis was not observed in the absence of extracellular Ca2+ and was virtually abolished by SKF-96365, an inhibitor of store-operated and voltage-gated Ca2+ influx (Fig. 3A).



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Fig. 3. Activation of PLC-{delta}1 by Gi/o-coupled receptor agonists. A: cultured muscle cells expressing dominant-negative PLC-{delta}1(E341R/D343R) or vector alone were labeled with myo-[3H]inositol for 24 h. Cells were incubated for 5 min in the absence of extracellular Ca2+ with the Gi/o-coupled receptor agonists somatostatin (SST), DPDPE, or cyclopentyl adenosine (CPA; 1 µM each) to deplete Ca2+ stores. Ca2+ (2 mM) was then added to elicit capacitative Ca2+ influx. In some experiments, the cells were incubated with 1 µM SKF-96365 for 10 min before addition of Ca2+. B: freshly dispersed muscle cells labeled with myo-[3H]inositol were incubated for 5 min in the absence of extracellular Ca2+ with the Gi/o-coupled receptor agonists somatostatin, DPDPE, or CPA (1 µM each) to deplete Ca2+ stores. Ca2+ (2 mM) was then added to elicit capacitative Ca2+ influx. In some experiments, the cells were incubated with the store-operated Ca2+ channel blocker SKF-96365 (1 µM) or with voltage-gated Ca2+ channel blocker nifedipine (1 µM) for 10 min before addition of Ca2+. PI hydrolysis was expressed as total inositol phosphates (cpm/mg protein). Agonist-induced PI hydrolysis at 5 min was strongly inhibited in cells expressing PLC-{delta}1(E341R/D343R) or by SKF-96365 and slightly inhibited by nifedipine. Inset: Western blot analysis of RhoA and PLC-{delta}1 in cells expressing vector alone (lane 1) and overexpressing PLC-{delta}1(E341R/D343R) (lane 2). Values are means ± SE of 3 experiments. A: **significant inhibition (P < 0.01) of PI hydrolysis by SKF-96365 or by overexpression of PLC-{delta}1 (E341R/D343R). B: *significant inhibition (P < 0.05) of PI hydrolysis by nifedipine; **significant inhibition (P < 0.01) by SKF-96365.

 
An identical pattern was obtained in freshly dispersed muscle cells with all three Gi-coupled receptor agonists (Fig. 3B). The agonists caused a delayed 8- to 10-fold increase in PI hydrolysis similar in magnitude to that induced by 1 µM Ca2+ in permeabilized muscles. The response was not observed in the absence of extracellular Ca2+ (data not shown), was virtually abolished by SKF-96365, and was only slightly inhibited by nifedipine (16–30%), implying that PI hydrolysis was dependent on capacitative Ca2+ influx via store-operated Ca2+ channels and, to a lesser extent, via voltage-gated Ca2+ channels (Fig. 3B).

Selective activation of PLC-{beta} by Gq-coupled receptors. Treatment of dispersed muscle cells with CCK-8 elicited a transient stimulation of PI hydrolysis (Fig. 4A). The initial increase was not followed by a delayed increase in PI hydrolysis, was inhibited by 83 ± 5% in muscle cells preincubated for 60 min with PLC-{beta}1 antibody (10 µg/ml), but was not affected in cells preincubated with PLC-{gamma}1 or PLC-{delta}1 antibody (Fig. 4B). Earlier studies had shown that CCK-stimulated PI hydrolysis in permeabilized muscle cells was blocked by incubation with G{alpha}q antibody (32). The effectiveness of G protein and PLC-{beta} antibodies in blocking agonist-induced, G protein-dependent PI hydrolysis in permeabilized smooth muscle was characterized in previous studies (29, 3134, 36).



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Fig. 4. PI hydrolysis mediated by Gq-coupled receptors. A: freshly dispersed smooth muscle cells were labeled with myo-[3H]inositol for 3 h and then treated with CCK-8 (1 nM) for 10 min. The reaction was terminated at different time points, and PI hydrolysis was measured using anion exchange chromatography, as described in EXPERIMENTAL PROCEDURES. B: freshly dispersed, permeabilized (35 µg/ml for 5 min) smooth muscle cells were incubated for 60 min with PLC-{beta}1, PLC-{delta}1, or PLC-{gamma}1 antibody (10 µg/ml) and then treated with CCK-8 (1 nM) for 1 min. PI hydrolysis was measured in the absence of Li3+ and expressed as total inositol phosphates (cpm/mg protein). CCK caused a transient increase PI hydrolysis that was selectively inhibited by PLC-{beta}1 antibody. Values are means ± SE of 4 experiments.

 
Suppression of PLC-{delta}1 stimulation by activated RhoA. We postulated that the difference in the ability of Gi/o- and Gq-coupled receptor agonists to elicit activation of PLC-{delta}1 could reflect the difference in their ability to stimulate RhoA activity. Previous studies in smooth muscle cells had shown that Gq-coupled receptors (e.g., CCKA, m3) that activate PLC-{beta}1 also activate G{alpha}13 and RhoA (37, 38). In contrast, Gi-coupled receptors (somatostatin receptor 3, {delta}-opioid, A1, m2) that activate PLC-{beta}3 also activate Cdc42/Rac1 and PI 3-kinase but not G{alpha}13 or RhoA (36, 48).

CCK-8 induced RhoA:PLC-{delta}1 association (Fig. 5A) and stimulated RhoA activity in a time-dependent fashion (Fig. 5B). In contrast, somatostatin, DPDPE, and CPA did not stimulate RhoA activity (Fig. 5C). Treatment of freshly dispersed muscle cells with the RhoA inhibitor, C3 exoenzyme, inhibited CCK-induced RhoA:PLC-{delta}1 association (Fig. 5A) and RhoA activity (Fig. 5C) and unmasked a delayed increase in PI hydrolysis (Fig. 6). The initial increase measured in the first minute and mediated by PLC-{beta}1 as shown in Fig. 4B was not affected by C3 exoenzyme. The delayed increase (measured 5 min after addition of CCK-8) coincided with the period of capacitative Ca2+ influx and was not observed in the absence of extracellular Ca2+. Neither the initial nor the delayed increase in PI hydrolysis was affected by the PKC inhibitor bisindolylmaleimide (1 µM) or the Rho kinase inhibitor Y-27632 (1 µM; data not shown).



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Fig. 5. Selective activation of RhoA and stimulation of RhoA:PLC-{delta}1 association by Gq/13-coupled receptor agonist. A: freshly dispersed muscle cells were treated for 5 min with various agonists [CCK-8, somatostatin, {delta}-opioid agonist DPDPE ({delta}), and CPA]. Muscle cells were lysed and treated with PLC-{delta}1 antibody for 12 h to obtain PLC-{delta}1 immunoprecipitates (IP). PLC-{delta}1 immunoprecipitates were separated by SDS-PAGE and probed with RhoA antibody to examine the association of PLC-{delta}1:RhoA. B: freshly dispersed smooth muscle cells were treated with CCK-8 (1 nM) for various intervals. Muscle cell lysates were incubated with glutathione-agarose slurry of Rhotekin, and GTP-bound RhoA (RhoA activity) was analyzed by SDS-PAGE followed by Western blot analysis. C: freshly dispersed muscle cells were treated with various agonists [CCK-8, somatostatin, {delta}-opioid agonist DPDPE ({delta}), and CPA] for 5 min, and RhoA activity was determined. In some experiments, the cells were preincubated for 3 h with the RhoA inhibitor C3 exoenzyme (C3E). CCK, but not somatostatin, DPDPE, or CPA, stimulated RhoA activity and induced association of PLC-{delta}1:RhoA. CCK-induced RhoA activity and association of PLC-{delta}1:RhoA were blocked by C3 exoenzyme. In all experiments, maximally effective concentrations were used (1 nM for CCK-8 and 1 µM for other agonists). Values are means ± SE of 4–6 experiments.

 


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Fig. 6. Activation of PLC-{delta}1 by Gq/13-coupled receptor agonists after inactivation of RhoA. Freshly dispersed muscle cells labeled with myo-[3H]inositol were treated with CCK-8 (1 nM) for 1 or 5 min in the absence of extracellular Ca2+. Capacitative Ca2+ influx was elicited by the addition of 2 mM Ca2+ at 5 min. PI hydrolysis was measured using anion exchange chromatography and expressed as total inositol phosphates (cpm/mg protein). In some experiments, freshly dispersed muscle cells were preincubated for 3 h with the RhoA inhibitor C3 exoenzyme. Inhibition of RhoA had no effect on CCK-induced initial increase in PI hydrolysis but unmasked a delayed increase in PI hydrolysis. Values are means ± SE of 4 experiments.

 
A delayed increase in PI hydrolysis induced by CCK-8 was also unmasked in cultured smooth muscle cells expressing dominant-negative RhoA (T19N; Fig. 7A) or G{alpha}13 minigene (Fig. 7B). The delayed PI hydrolysis caused by CCK-8 reflected activation of PLC-{delta}1, since it could not be elicited in cultured smooth muscle cells coexpressing RhoA(T19N) and PLC-{delta}1(E341R/D343R) or coexpressing G{alpha}13 minigene and PLC-{delta}1(E341R/D343R) (Fig. 7, A and B). Expression of G{alpha}13 minigene inhibited RhoA:PLC-{delta}1 association (Fig. 8A) and RhoA activity (Fig. 8B). The early phase of PI hydrolysis mediated by PLC-{beta}1 was not affected by expression of RhoA(T19N), G{alpha}13 minigene, or PLC-{delta}1(E341R/D343R) (data not shown).



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Fig. 7. Activation of PLC-{delta}1 by Gq/13-coupled receptor agonist in cells expressing dominant-negative RhoA (T19N) or G{alpha}13 minigene. A: cultured muscle cells expressing dominant-negative RhoA (T19N) or coexpressing dominant-negative PLC-{delta}1 (E341R/D343R) were labeled with myo-[3H]inositol for 24 h. The cells were treated with CCK-8 for 5 min in the absence of extracellular Ca2+ to deplete Ca2+ stores. Ca2+ (2 mM) was then added to elicit capacitative Ca2+ influx. In some experiments, muscle cells were treated with the store-operated Ca2+ channel blocker SKF-96365 (1 µM) for 10 min before addition of 2 mM Ca2+. B: parallel studies were done in cultured muscle cells expressing dominant-negative G{alpha}13 minigene or coexpressing G{alpha}13 minigene and PLC-{delta}1(E341R/D343R). PI hydrolysis was measured at 5 min using anion exchange chromatography and was expressed as total inositol phosphates (cpm/mg protein). Expression of RhoA (T19N) or G{alpha}13 minigene unmasked a delayed increase PI hydrolysis in response to CCK, and this delayed increase was inhibited by SKF-96365 or in cells expressing PLC-{delta}1(E341R/D343R). Insets: Western blot analysis of RhoA and PLC-{delta}1 in cells expressing vector alone (lane 1) and overexpressing RhoA(T19N) or PLC-{delta}1(E341R/D343R) (lane 2). Values are means ± SE of 4 experiments. **Significant stimulation (P < 0.01) of sustained PI hydrolysis induced by overexpression of RhoA(T19N) (A) or G13 minigene (B). ##Significant inhibition (P < 0.001) of PI hydrolysis in cells coexpressing PLC-{delta}1(E341R/D343R) with RhoA(T19N) or G{alpha}13 minigene compared with cells overexpressing RhoA(T19N) or G{alpha}13 minigene alone.

 


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Fig. 8. Inhibition of RhoA:PLC-{delta}1 association and RhoA activity induced by Gq/13-coupled receptor agonist in cells expressing G{alpha}13 minigene. Cultured muscle cells expressing vector alone or overexpressing G{alpha}13 minigene were treated with CCK-8 for 5 min. Muscle cells were lysed and treated with PLC-{delta}1 antibody for 12 h to obtain PLC-{delta}1 immunoprecipitates. PLC-{delta}1 immunoprecipitaes were separated by SDS-PAGE and probed with RhoA antibody to examine the association of RhoA:PLC{delta}1. RhoA activity (B) was determined using GST-Rhotekin, as described in EXPERIMENTAL PROCEDURES. RhoA:PLC{delta}1 association and RhoA activity were measured before (a and c) after (b and d) treatment with CCK. Expression of G{alpha}13 minigene inhibited CCK-induced RhoA:PLC{delta}1 association and RhoA activity. Values are means ± SE of 4–6 experiments. **Significant inhibition (P < 0.01) of CCK-stimulated RhoA activity.

 
Further evidence that association of PLC-{delta}1 with activated RhoA prevents activation of PLC-{delta}1 is provided in Fig. 2, where the delayed increase in PI hydrolysis induced by the Gi-coupled receptor agonist DPDPE was virtually abolished in cells expressing a constitutively active RhoA(G14V).

Activation of PLC-{delta}1 by agonist-independent capacitative Ca2+ influx. The relative importance of Ca2+ influx via store-operated and voltage-gated Ca2+ channels in stimulating PLC-{delta}1 activity was examined further using thapsigargin and KCl to activate preferentially store-operated and voltage-gated Ca2+ channels, respectively. Treatment of muscle cells for 20 min with thapsigargin in the absence of Ca2+ followed by addition of 2 mM Ca2+ caused an eightfold increase in PI hydrolysis, similar in magnitude to that elicited by Gi-coupled receptor agonists (Fig. 9A). No increase in PI hydrolysis was observed in the absence of extracellular Ca2+ (data not shown). PI hydrolysis induced by thapsigargin was inhibited strongly (82 ± 6%) by SKF-96365 (Fig. 9A) and minimally by nifedipine (13 ± 2%). PI hydrolysis induced by thapsigargin in cultured muscle cells (4,039 ± 479 cpm/mg protein) was strongly inhibited (75 ± 5%) in cells overexpressing a PLC-{delta}1(E341R/D343R) mutant (Fig. 9B), implying that it was mediated by PLC-{delta}1.



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Fig. 9. Activation of PLC-{delta}1 by agonist-independent capacitative Ca2+ influx. Freshly dispersed muscle cells (A) and cultured muscle cells expressing vector alone or overexpressing dominant-negative PLC-{delta}1(E341R/D343R) (B) were incubated with thapsigargin (TG, 2 µM) in the absence of extracellular Ca2+ for 30 min to deplete intracellular Ca2+; 2 mM Ca2+ were then added to elicit capacitative Ca2+ influx. In some experiments, the cells were incubated with the store-operated Ca2+ channel blocker SKF-96365 (SKF; 1 µM) for 10 min before addition of Ca2+. PI hydrolysis was measured using anion exchange chromatography and expressed as total inositol phosphates (cpm/mg protein) in cells prelabeled with [3H]inositol. Thapsigargin-induced PI hydrolysis was inhibited by SKF-96365 or in cells expressing PLC-{delta}1 (E341R/D343R). Inset: Western blot analysis in cells expressing vector alone (lane 1) or overexpressing PLC-{delta}1(E341R/D343R) (lane 2). Values are means ± SE of 3 experiments. **Significant inhibition (P < 0.001) of PI hydrolysis by SKF-93635. ##Significant inhibition (P < 0.001) of PI hydrolysis in cells overexpressing PLC-{delta}1(E341R/D343R).

 
In contrast, Ca2+ influx induced by treatment of intact muscle cells with a depolarizing concentration of KCl (20 mM) or with {alpha},{beta}-methylene ATP, a P2X receptor agonist, caused only a moderate two- to threefold increase in PI hydrolysis that was abolished by nifedipine (Fig. 10A). Previous studies in these cells had shown that activation of the P2X receptor, a ligand-gated cationic channel, depolarizes smooth muscle cells and stimulates Ca2+ influx via voltage-gated Ca2+ channels (34).



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Fig. 10. Activation of PLC-{delta}1 by Ca2+ release and Ca2+ influx. A: dispersed muscle cells labeled with [3H]inositol were treated for 1 min with the ligand-gated P2X receptor agonist {alpha},{beta}-methylene ATP (1 µM) or with 20 mM KCl for 1 min in the presence or absence of the Ca2+ channel blocker nifedipine (1 µM). B: dispersed intestinal muscle cells labeled with [3H]inositol were permeabilized and then treated with 1 µM inositol trisphosphate (IP3, circular muscle cells) or 1 µM cADP ribose (longitudinal muscle cells) for 30 s to elicit Ca2+ release. In some experiments, muscle cells were preincubated with PLC-{delta}1 antibody (10 µg/ml) for 1 h. PI hydrolysis was expressed as cpm/mg protein of total inositol phosphates. Values are means ± SE of 3–6 experiments. **Significant inhibition (P < 0.01) of PI hydrolysis by PLC-{delta}1 antibody or nifedipine.

 
Activation of PLC-{delta}1 by agonist-independent Ca2+ release. As noted above, PI hydrolysis induced by CCK-8 in permeabilized muscle cells was not affected by preincubation with PLC-{delta}1 antibody (Fig. 4), suggesting either that Ca2+ release did not activate PLC-{delta}1 or that activation of PLC-{delta}1 was offset by concomitant inhibition by IP3 and PKC. In the absence of agonist, treatment of permeabilized intestinal circular muscle cells with IP3 (1 µM) caused a significant twofold increase in PI hydrolysis that was abolished in cells preincubated for 60 min with PLC-{delta}1 antibody (Fig. 10B). It is possible that the response to Ca2+ release induced by IP3 might have been attenuated by the inhibitory effect of IP3 on PLC-{delta}1 activity. To obviate the confounding effect of IP3, the experiments were repeated in intestinal longitudinal muscle cells, which express predominantly IP3-insensitive, ryanodine receptors/Ca2+ channels that are highly sensitive to Ca2+ and cADP ribose (24, 25). Treatment of permeabilized intestinal longitudinal muscle cells with cADP ribose caused a somewhat higher threefold increase in PI hydrolysis, which was inhibited by 73 ± 5% in cells preincubated with PLC-{delta}1 antibody.

The pattern that emerges from comparison of PLC-{delta}1 activity induced by Gi/o-dependent or -independent capacitative Ca2+ influx with PLC-{delta}1 activity induced by G protein-independent Ca2+ influx (KCl; {alpha},{beta}-methylene ATP) or Ca2+ release (IP3 and cADP ribose) is that the mechanism of Ca2+ entry rather than the magnitude of Ca2+ entry or Ca2+ release is the main determinant of PLC-{delta}1 activity.


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This study provides the first evidence that Gi/o-coupled receptors can activate PLC-{delta}1 and that activation is preferentially mediated by capacitative Ca2+ influx. The study also shows that the inability of Gq/13-coupled receptors to activate PLC-{delta}1 reflects concurrent activation of RhoA, which inactivates PLC-{delta}1 by binding directly to RhoA or indirectly via RhoGAP (17). A link between PI-PLC activity and capacitative Ca2+ influx was suggested by earlier studies showing an increase in PI-PLC activity in cells overexpressing PLC-{delta}1, but the specific requirements of coupling to Gi/o and absence of RhoA activity were not addressed (23).

Previous studies in smooth muscle cells had shown that Gi/o-coupled receptor agonists, including all three agonists used in this study, cause an initial G{beta}{gamma}i-dependent stimulation of PLC-{beta}3 activity, resulting in IP3-dependent Ca2+ release and contraction (25, 29, 31, 33). Here we show that these agonists also induce a delayed PI hydrolysis that is virtually abolished in smooth muscle cells expressing a dominant-negative PLC-{delta}1(E341R/D343R) or a constitutively active RhoA(G14V), implying that PI hydrolysis was mediated by PLC-{delta}1 and blocked by activated RhoA. Conversely, Gq/G{alpha}13-coupled agonists that activate RhoA did not cause delayed PI hydrolysis. Inactivation of RhoA with C3 exoenzyme or by expression of dominant-negative RhoA(T19N) unmasked a delayed PI hydrolysis mediated by PLC-{delta}1. Coexpression of dominant-negative RhoA(T19N) or G{alpha}13 minigene with dominant-negative PLC-{delta}1(E341R/D343R) suppressed delayed PI hydrolysis, implying that it was mediated by PLC-{delta}1.

The initial increase in PI hydrolysis resulting from Gq-dependent activation of PLC-{beta}1 or G{beta}{gamma}i-dependent activation of PLC-{beta}3 induces IP3-dependent Ca2+ release followed by capacitative Ca2+ influx triggered by depletion of Ca2+ stores. Here we show that capacitative Ca2+ influx is the proximate stimulus of PLC-{delta}1 activity. Delayed PI hydrolysis initiated by Gi-coupled receptor agonists (or by Gq/13-coupled agonists after inactivation of RhoA) was not observed in the absence of extracellular Ca2+, was only slightly decreased by blockade of voltage-gated Ca2+ channels with nifedipine, but was virtually abolished by blockade of both voltage-gated and store-operated Ca2+ channels with SKF-96365.

The importance of Ca2+ influx via store-operated channels was evident in the relative effects of thapsigargin, KCl, and {alpha},{beta}-methylene ATP. Ca2+ influx via voltage-gated Ca2+ channels induced by depolarizing concentrations of KCl or by {alpha},{beta}-methylene ATP caused only a moderate increase in PLC-{delta}1 activity, whereas Ca2+ influx via store-operated Ca2+ channels, induced by depletion of Ca2+ stores with thapsigargin, caused a maximal increase in PLC-{delta}1 activity that was abolished by SKF-96365. PI hydrolysis induced by thapsigargin was virtually abolished in cells expressing PLC-{delta}1(E341R/D343R), corroborating the identity of the PLC isozyme activated by capacitative Ca2+ influx.

The profound effect on PLC-{delta}1 activity of Ca2+ influx via store-operated Ca2+ channels (inhibition by SKF-96365) relative to that of Ca2+ influx via voltage-gated Ca2+ channels (inhibition by nifedipine) or to that of Ca2+ release suggests a close association of PLC-{delta}1 and store-operated Ca2+ channels. A similar association has been observed between these channels and membrane-bound Ca2+-sensitive adenylyl cyclases that leads to inhibition of adenylyl cyclase type V/VI and activation of adenylyl cyclase type VIII (9, 14, 35). Preliminary studies show association of PLC-{delta}1, adenylyl cyclase V/VI, A-kinase anchoring protein, and activated RhoA with caveolin that could act as a membrane-bound scaffolding protein (Murthy KS, unpublished observations). Recent studies suggest an association between caveolin and Trp4 channels involved in capacitative Ca2+ influx (45).

The exquisite sensitivity of PLC-{delta}1 to Ca2+ relative to the sensitivity of PLC-{beta}1 or PLC-{gamma}1 previously demonstrated by in vitro assay (1) was corroborated in vivo in this study (Fig. 1). Even Ca2+ release induced by exogenous IP3 or cADP ribose caused a moderate increase in PLC-{delta}1 activity. The identity of the PLC isozyme activated by Ca2+ release was confirmed by blockade with PLC-{delta}1 antibody. A potential increase in PLC-{delta}1 activity that might result from agonist-induced Ca2+ release would be offset by concurrent inhibition of PLC-{delta}1 by IP3 and PKC.

Although this study identifies a physiological mechanism for activation of PLC-{delta}1 by Gi-coupled receptor agonists, the functional role of PLC-{delta}1 and delayed PI hydrolysis has yet to be elucidated. Conceivably, delayed generation of IP3 could alter the dynamics of Ca2+ release and Ca2+ uptake in the cell and sarcoplasmic stores, which could result in a more sustained cellular response. Because Gi-coupled receptor agonists do not activate RhoA or its downstream effectors PLD and PKC, PLC-{delta}1 activity becomes the main source of sustained PKC activity in response to these agonists. The effect of sustained PKC activity from this source has not been determined. This notion, however, sheds light on our earlier observation that the initial contractile response of permeabilized smooth muscle cells to Ca2+ was mediated by Ca2+/calmodulin-dependent myosin light chain (MLC) kinase, whereas the sustained response was dependent on PKC (30). Activation of PKC was probably a consequence of PI hydrolysis mediated by Ca2+-stimulated PLC-{delta}1, resulting in PKC-mediated inhibition of MLC phosphatase (36). Consistent with this notion, the sustained contraction induced by exogenous Ca2+ was suppressed in cells treated with PLC-{delta}1 antibody. Accordingly, studies of "Ca2+ sensitization," in which permeabilized smooth muscle is exposed sequentially to Ca2+-free and Ca2+-containing media, should take into account the probability that Ca2+-mediated activation of PLC-{delta}1 would generate signaling molecules that could influence the response.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15564.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. S. Murthy, Dept. of Physiology, Medical College of Virginia Campus, Virginia Commonwealth Univ., Richmond, VA 23298 (E-mail: skarnam{at}hsc.vcu.edu)

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.


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