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
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ABSTRACT |
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phospholipase C; G protein
The PH domain is essential for activation of PLC-, and its deletion abolishes PLC-
activity (2, 8, 15). The PH domain of PLC-
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-
binds G
, the binding is too weak to induce activation of PLC-
(15, 44). Four specific receptors (
1B- and
1D-adrenoceptors,
-thromboxane receptors, and oxytocin receptors) can activate PLC-
via coupling to the atypical G protein (G
h), also known as transglutaminase-II (3, 18, 40). The mechanism of PLC-
activation via G
h is not known. A recent study suggests that PLC-
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-1, the most abundant and widely expressed isoform of PLC-
, is typically activated by Ca2+ in the range of 0.110 µM (1, 26, 28). Despite its sensitivity to Ca2+ (
100-fold higher than that of PLC-
or PLC-
), PLC-
is not activated by agonists that mobilize intracellular Ca2+, possibly because PLC-
is inactivated by PLC-
-dependent generation of protein kinase C (PKC) and inositol trisphosphate (IP3); the latter competes with PIP2 for binding to PLC-
(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-
by capacitative Ca2+ influx induced by thapsigargin or bradykinin was observed only after overexpression of PLC-
(23).
In this study, we have identified a unique mechanism for activation of PLC-1 via Gi/o-coupled receptors. The inability of Gq/13-coupled receptors to activate PLC-
1 reflected concurrent activation of RhoA.
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EXPERIMENTAL PROCEDURES |
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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 G13 minigene, dominant-negative PLC-
1 and RhoA, and constitutively active RhoA in cultured smooth muscle cells.
G
13 minigene (MGLHDNLKQLMLQ), dominant-negative PLC-
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-
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-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-
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-
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-
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-1, and various isoforms of PLC-
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).
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RESULTS |
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Selective activation of PLC- 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-
1 antibody (10 µg/ml), but was not affected in cells preincubated with PLC-
1 or PLC-
1 antibody (Fig. 4B). Earlier studies had shown that CCK-stimulated PI hydrolysis in permeabilized muscle cells was blocked by incubation with G
q antibody (32). The effectiveness of G protein and PLC-
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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CCK-8 induced RhoA:PLC-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-
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-
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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Activation of PLC-1 by agonist-independent capacitative Ca2+ influx.
The relative importance of Ca2+ influx via store-operated and voltage-gated Ca2+ channels in stimulating PLC-
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-
1(E341R/D343R) mutant (Fig. 9B), implying that it was mediated by PLC-
1.
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The pattern that emerges from comparison of PLC-1 activity induced by Gi/o-dependent or -independent capacitative Ca2+ influx with PLC-
1 activity induced by G protein-independent Ca2+ influx (KCl;
,
-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-
1 activity.
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DISCUSSION |
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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 Gi-dependent stimulation of PLC-
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-
1(E341R/D343R) or a constitutively active RhoA(G14V), implying that PI hydrolysis was mediated by PLC-
1 and blocked by activated RhoA. Conversely, Gq/G
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-
1. Coexpression of dominant-negative RhoA(T19N) or G
13 minigene with dominant-negative PLC-
1(E341R/D343R) suppressed delayed PI hydrolysis, implying that it was mediated by PLC-
1.
The initial increase in PI hydrolysis resulting from Gq-dependent activation of PLC-1 or G
i-dependent activation of PLC-
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-
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 ,
-methylene ATP. Ca2+ influx via voltage-gated Ca2+ channels induced by depolarizing concentrations of KCl or by
,
-methylene ATP caused only a moderate increase in PLC-
1 activity, whereas Ca2+ influx via store-operated Ca2+ channels, induced by depletion of Ca2+ stores with thapsigargin, caused a maximal increase in PLC-
1 activity that was abolished by SKF-96365. PI hydrolysis induced by thapsigargin was virtually abolished in cells expressing PLC-
1(E341R/D343R), corroborating the identity of the PLC isozyme activated by capacitative Ca2+ influx.
The profound effect on PLC-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-
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-
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-1 to Ca2+ relative to the sensitivity of PLC-
1 or PLC-
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-
1 activity. The identity of the PLC isozyme activated by Ca2+ release was confirmed by blockade with PLC-
1 antibody. A potential increase in PLC-
1 activity that might result from agonist-induced Ca2+ release would be offset by concurrent inhibition of PLC-
1 by IP3 and PKC.
Although this study identifies a physiological mechanism for activation of PLC-1 by Gi-coupled receptor agonists, the functional role of PLC-
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-
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-
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-
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-
1 would generate signaling molecules that could influence the response.
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GRANTS |
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FOOTNOTES |
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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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