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Am J Physiol Gastrointest Liver Physiol 280: G381-G388, 2001;
0193-1857/01 $5.00
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Vol. 280, Issue 3, G381-G388, March 2001

Sequential activation of heterotrimeric and monomeric G proteins mediates PLD activity in smooth muscle

K. S. Murthy, H. Zhou, J. R. Grider, and G. M. Makhlouf

Departments of Medicine and Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The identity of G proteins mediating CCK-stimulated phospholipase D (PLD) activity was determined in intestinal smooth muscle cells. CCK-8 activated Gq/11, G13, and G12, and the monomeric G proteins Ras-homology protein (RhoA) and ADP ribosylation factor (ARF). Activation of RhoA, but not ARF, was mediated by G13 and inhibited by Galpha 13 antibody. CCK-stimulated PLD activity was partly mediated by RhoA and could be inhibited to the same extent (47 ± 2% to 53 ± 6%) by 1) a dominant negative RhoA mutant, 2) RhoA antibody or Galpha 13 antibody, and 3) Clostridium botulinum C3 exoenzyme. PLD activity was also inhibited by ARF antibody, and the effect was additive to that of RhoA antibody or C3 exoenzyme. PLD activity was inhibited by calphostin C, bisindolylmaleimide I, and a selective protein kinase C (PKC)-alpha inhibitor; the inhibition was additive to that of ARF and RhoA antibodies and C3 exoenzyme. In contrast, activated G12 was not coupled to RhoA or ARF, and Galpha 12 antibody augmented PLD activity. Thus agonist-stimulated PLD activity is mediated additively by G13-dependent RhoA and by ARF and PKC-alpha and is modulated by an inhibitory G12-dependent pathway.

G12; protein kinase C; phospholipase D; intestinal smooth muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A PHOSPHOLIPASE D (PLD) GENE superfamily characterized by a number of conserved structural domains and sequence motifs has been identified in various species (6, 7, 12). PLD is widely distributed in mammalian tissues and located in cellular membranes and the cytosol. The specific substrate for the main mammalian isoforms, PLD1a and PLD1b, is phosphatidylcholine (PC). Phosphatidic acid (PA) and choline, the primary products of PC hydrolysis by PLD, are rapidly converted to diacylglycerol (DAG) and phosophocholine by phosphatidate phosphohydrolase and choline kinase, respectively (9, 19). Dephosphorylation of PA is a major source of agonist-stimulated, sustained DAG production in various tissues, including smooth muscle, resulting in sustained activation of protein kinase C (PKC) (9, 26). PLD also catalyses transphosphatidylation, in which a phosphatidyl group of PC is transferred to glycerol or a primary alcohol (9, 26). This PLD-specific reaction has facilitated analysis of the regulation of PLD.

We (26) have previously shown that in intestinal smooth muscle, agonist-stimulated PLD activity is preceded by, but independent of, phosphatidylinositol (PI) hydrolysis via PLC-beta . PLD activity was modulated by PKC but did not require an increase in resting intracellular Ca2+ levels (26). Sustained PLD and PKC activities were abolished by guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S) and appeared to be regulated by a G protein(s) distinct from members of the Gq or Gi/o families (25, 26). In various cells, PLD activity can be modulated by phosphatidylinositol 4,5-bisphosphate and/or fatty acids, such as oleate (9, 21, 31).

Recent studies (3, 5, 13, 18-20, 22, 23, 31, 34) have examined the upstream pathways linking the receptor to PLD activity, in particular the participation of heterotrimeric and monomeric G proteins. Two monomeric G proteins, the Ras-homology protein (RhoA) and the ADP ribosylation factor (ARF), have been identified that vary in their ability to activate PLD in different tissues (3, 5, 18-20, 31, 34). In the resting state, RhoA, like other Rho family G proteins, is bound to a GDP-dissociation inhibitor in the cytoplasm. Agonist-receptor binding activates a specific guanine nucleotide exchange factor (Rho-GEF) that promotes dissociation of the inhibitor from RhoA, translocation of RhoA to membranes, and activation of RhoA by exchange of GTP for GDP (13). A similar process promotes the translocation of ARF to membranes and its activation by GTP/GDP exchange (22, 23). There is also substantial evidence that either or both G13 and G12 are involved in mediating receptor-dependent activation of RhoA (10, 18, 28). The involvement of either G protein is often cell and agonist specific. Microinjection of Galpha 12 or Galpha 13 into Swiss 3T3 fibroblasts stimulates the formation of stress fibers, a process mediated by RhoA (4). Thrombin, however, stimulates stress fiber formation via G12 only and lysophosphatidic acid via G13 only (18). In CCL39 fibroblasts, Rho-dependent stimulation of Na+/H+ exchange is activated by G13 but inhibited by G12 (17).

The existence of pathways involving sequential coupling of heterotrimeric (G13 and G12) and monomeric (RhoA and ARF) G proteins to activation of PLD in smooth muscle has not been determined. In a recent study of cultured vascular smooth muscle, PLD activity induced by angiotensin II was shown to be partly inhibited by RhoA and Galpha 12 antibodies, as well as by Gbeta and pp60src antibodies, suggesting involvement of Gbeta gamma -Src and G12-RhoA pathways (34). In the present study, we examined the roles of G13 and G12 in activation of RhoA and ARF and of both monomeric G proteins in activation of PLD. CCK-8 was shown to stimulate PLD activity additively via RhoA, ARF, and PKC-alpha . CCK activated both G13 and G12, but only G13 was coupled to activation of RhoA, Rho kinase (ROK), and PLD. ARF activation was not mediated by either G13 or G12, and activation of G12 resulted in inhibition of PLD.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dispersion of intestinal smooth muscle cells. Smooth muscle cells were isolated from the circular muscle layer of rabbit intestine by sequential enzymatic digestion, filtration, and centrifugation as described previously (24-26). Muscle strips were incubated for 60 min at 31°C in 15 ml of HEPES medium containing 0.1% collagenase (type II) and 0.1% soybean trypsin inhibitor with no added Ca2+. The composition of the medium was 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4, 0.6 mM MgCl2, 25 mM HEPES, 14 mM glucose, and 2.1% Eagle's essential amino acid mixture. The partly digested tissue was washed with 100 ml of enzyme-free medium and reincubated for 40-60 min to allow spontaneous dispersion of muscle cells. The cells were harvested by filtration through 500-µm Nitex mesh, centrifuged twice for 10 min at 350 g, and resuspended in HEPES medium containing 2 mM Ca2+. In some experiments, the cells were permeabilized by incubation for 5 min with saponin (35 µg/ml) in a low-Ca2+ (100 nM) medium as described previously (24, 25) and resuspended in saponin-free medium with 1.5 mM ATP and ATP-regenerating system (5 mM creatine phosphate and 10 U/ml creatine phosphokinase).

Identification of receptor-activated G proteins in membranes. G proteins selectively activated by CCK-8 were identified by an adaptation of the method of Okamoto et al. (29) as described previously (24). Muscle cells were homogenized in 20 mM HEPES medium (pH 7.4). After centrifugation at 25,000 g for 15 min, the membranes were solubilized at 4°C in 20 mM HEPES medium (pH 7.4) and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The membranes were incubated with 60 nM 35S-labeled guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgamma S) in a medium containing 10 mM HEPES (pH 7.4), 100 µM EDTA, and 10 mM MgCl2 for 20 min at 37°C in the presence or absence of CCK-8 (1 nM). The reaction was stopped with 10 vol of 100 mM Tris · HCl medium (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP, and the solubilized membranes were incubated for 2 h on ice in wells precoated with specific antibodies to Galpha q/11, Galpha 13, and Galpha 12. The wells were washed three times with phosphate buffer containing 0.05% Tween 20, and the radioactivity in each well was counted.

Measurement of expression, translocation, and activation of RhoA and ARF. Muscle cells were homogenized in a solution containing 10 mM Tris · HCl (pH 7.5), 5 mM MgCl2, 2 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml aprotinin. The suspension was centrifuged at 100,000 g for 30 min at 4°C, and the supernatant was collected as the cytosolic fraction. Pellets were resuspended, and proteins were extracted by incubation for 30 min in the homogenization buffer containing 1% Triton X-100 and 1% sodium cholate. The extract was centrifuged at 1,000 g for 10 min, and the supernatant was collected as the particulate fraction. Proteins (80-100 µg) were resolved by 12% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. After incubation in 5% nonfat dry milk to block nonspecific antibody binding, the blots were incubated first with antibodies to RhoA or ARF and then with secondary antibodies conjugated with horseradish peroxidase. The bands were identified by enhanced chemiluminescence.

RhoA and ARF activities were measured in muscle cells incubated for 3 h in low-phosphate (0.12 mM NaH2PO4) buffer containing 10 mM HEPES, 2.5 mM glucose, 1% BSA, and 10 mCi of 32PO4. Aliquots (2 × 106 cells) were treated with CCK-8 (1 nM) for 10 min, and the reaction was stopped with lysis buffer containing 20 mM Tris · HCl (pH 7.4), 250 mM sucrose, 150 mM NaCl, 2 mM EGTA, 10 mM MgCl2, 1 mM Na2P2O7, 1 mM NaF, 1 mM Na3VO4, 1% Triton X-100, 0.5% Nonidet, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 20 µg/ml aprotinin. RhoA and ARF were immunoprecipitated separately, using specific antibodies, washed three times with lysis buffer, and boiled for 20 min at 68°C in buffer containing 5 mM EDTA, 2 mM DTT, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP. GTP and GDP were separated on polyethylene-cellulose plates developed with 1 M KH2PO4 (pH 3.4) and measured by autoradiography.

Assay for PLD activity. PLD activity was determined by the formation of phosphatidylethanol (PEt), a specific product of PLD activity in the presence of ethanol. Muscle cells (2 × 106 cells/ml) were incubated with [3H]myristic acid (2 µCi/ml) for 3 h and then with 150 mM ethanol for 15 min at 31°C in HEPES medium. The cells were then centrifuged at 350 g for 10 min to remove excess [3H]myristic acid and resuspended in fresh medium. CCK-8 (1 nM) was added for 10 min, and the reaction was terminated by the addition of 1.8 ml of chloroform-methanol-HCl (100:200:2, vol/vol/vol) and extracted by the method of Bligh and Dyer (2) as described previously (26). The organic phase was dried under N2 and analyzed for [3H]PEt by TLC on silica gel plates (dipped in 1% potassium oxalate), with ethyl acetate-2,2,4-trimethylpentane-acetic acid-water (13:2:3:10) as a running solvent. [3H]PEt was identified using unlabeled standards, which were sprayed with 0.1% 1,2-dichlorofluorescein in isopropyl alcohol and visualized under ultraviolet light at 357 nm. The spots corresponding to PEt were scraped and counted by liquid scintillation.

Transfection of dominant negative RhoA cDNA into cultured smooth muscle. Dominant negative RhoA cDNA was subcloned into the multiple cloning site (EcoR I) of the eukaryotic expression vector pEXV. A myc tag was incorporated into the NH2 terminus. Recombinant plasmid DNAs were transiently transfected into the muscle cells in primary culture using Lipofectamine Plus reagent. Cells were cotransfected with 2 µg of pEXV-myc tag RhoA dominant negative and 1 µg of pGreen Lantern-1 for 48 h. Control cells were cotransfected with 2 µg pEXV vector and 1 µg of pGreen Lantern-1 DNA. Transfection efficiency was monitored by the expression of the green fluorescent protein using FITC filters. In the RhoA dominant negative mutant, asparagine was substituted for serine at position 19 (N19RhoA).

Materials. [3H]myristic acid (22.4 Ci/mmol) and carrier-free [32P]Pi were obtained from NEN Life Science Products (Boston, MA). Collagenase type II and soybean trypsin inhibitor were from Worthington Biochemicals (Freehold, NJ). Polyclonal antibodies to Galpha 13, Galpha 12, Galpha q/11, RhoA, and ARF were from Santa Cruz Biotechnology, (Santa Cruz, CA), and all other chemicals were from Sigma Chemical (St. Louis, MO). Dominant negative RhoA cDNA was a gift of Dr. Andrea Todisco, University of Michigan. Myristoylated pseudosubstrate peptide inhibitors of PKC isoforms were a gift from Drs. A. Dartt and D. Zoukhri, Harvard Medical School.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression and receptor-mediated activation of G13 and G12 in intestinal smooth muscle. Previous studies (24) have shown that several G proteins (Gq/11, Gs, Gi-1, Gi-2, and Gi-3) are expressed in intestinal smooth muscle, where they are coupled to various receptors. Western blot analysis in the present study showed that G13 and G12 are also expressed in intestinal smooth muscle cells (Fig. 1). CCK-8, a ligand previously shown to activate Gq/11, also activated G13 and G12, significantly increasing the binding of [35S]GTPgamma S to Galpha q/11, Galpha 13, and Galpha 12 by 75 ± 4% (P < 0.01), 86 ± 17% (P < 0.01), and 102 ± 14% (P < 0.01), respectively (Fig. 1).


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  		Fig. 1.   Expression and activation of Galpha 13, Galpha 12, and Galpha q/11 in intestinal smooth muscle. A: Western blot analysis was performed in homogenates prepared from dispersed intestinal circular muscle cells using specific antibodies to Galpha 13, Galpha 12, and Galpha q/11. B: agonist-induced activation of G proteins was measured by the increase in binding of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) to Galpha 13, Galpha 12, and Galpha q/11. Intestinal muscle cell membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and incubated with 60 nM [35S]GTPgamma S in the presence or absence of 1 nM CCK for 20 min. Aliquots were added to wells precoated with Galpha 13, Galpha 12, or Galpha q/11 antibody for 2 h, and bound radioactivity was measured and expressed as counts/min (cpm)/mg protein. CCK-8 caused a significant increase in the binding of [35S]GTPgamma S to Galpha 13, Galpha 12, and Galpha q/11. Values are means ± SE of 4 experiments. ** P < 0.01.

Receptor-mediated translocation and activation of RhoA and ARF. Western blot analysis showed that the monomeric G proteins RhoA and ARF were present mainly in the cytosolic fraction in the resting state, but increased significantly in the membrane fraction after stimulation of the muscle cells with CCK-8 (Figs. 2 and 3).


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  		Fig. 2.   CCK-induced translocation and activation of Ras-homology protein (RhoA) in intestinal smooth muscle. A: Western blot analysis using RhoA antibody was performed on cytosolic (C) and particulate (M) fractions prepared from muscle cell homogenates before and after treatment with 1 nM CCK-8. An increase in binding of RhoA to membranes was observed after treatment of the cells with CCK-8. B: activation of RhoA was measured in permeabilized muscle cells labeled for 3 h with 32PO4 and treated for 10 min with CCK-8 (1 nM). Experiments were done in control cells and in cells incubated for 1 h with antibody (Ab) to Galpha 13 or Galpha 12. [32P]GTP binding in RhoA immunoprecipitates was determined by TLC followed by autoradiography. Results are expressed as %GTP incorporation. GTP binding to RhoA was blocked by pretreatment of cells with antibody to Galpha 13 but not Galpha 12. Values are means ± SE of 4-5 experiments. ** P < 0.01.



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  		Fig. 3.   CCK-induced translocation and activation of ADP ribosylation factor (ARF) in intestinal smooth muscle. A: Western blot analysis using ARF antibody was performed on cytosolic and particulate fractions prepared from muscle cell homogenates before and after treatment with 1 nM CCK-8. An increase in binding of ARF to membranes was observed after treating the cells with CCK-8. B: activation of ARF was measured in permeabilized muscle cells labeled for 3 h with 32PO4 and treated for 10 min with CCK-8 (1 nM). Experiments were done in control cells and in cells incubated for 1 h with antibody to Galpha 13 or Galpha 12. [32]GTP binding in ARF immunoprecipitates was determined by TLC followed by autoradiography. Results are expressed as %GTP incorporation. GTP binding to ARF was not blocked by pretreatment of cells with Galpha 13 or Galpha 12 antibody. Values are means ± SE of 4-5 experiments. ** P < 0.01.

CCK-induced translocation of RhoA and ARF to the membrane was accompanied by a significant increase in the activities of both G proteins as indicated by the increase in the incorporation of [32P]GTP (Figs. 2 and 3). RhoA activity was inhibited 74 ± 9% (P < 0.01) by preincubation of permeabilized muscle cells for 1 h with Galpha 13 antibody (5 µg/ml), whereas ARF activity was not affected (Figs. 2 and 3). Preincubation with Galpha 12 antibody had no effect on either RhoA or ARF activity (Figs. 2 and 3).

G13-dependent activation and G12-dependent inhibition of PLD. CCK caused a sustained fourfold increase in PLD activity as determined by the formation of [3H]PEt [basal: 455 ± 71 counts/min (cpm)/106 cells; CCK-8: 2,415 ± 158 cpm/106 cells]. CCK-stimulated PLD activity was inhibited in a concentration-dependent fashion by the PLD inhibitor PCCG-16 with an EC50 of 0.1 µM. PLD activity was also inhibited in a concentration-dependent fashion by preincubation of permeabilized muscle cells for 1 h with Galpha 13 antibody (0.1-10 µg/ml); a maximal inhibition of 47 ± 2% (P < 0.001) was elicited with 5 µg/ml of antibody (Fig. 4). In contrast, preincubation with Galpha 12 antibody (10 µg/ml) increased PLD activity by 33 ± 2% (P < 0.001). Preincubation with antibodies to Galpha q/11, Gialpha 1-2, Gialpha 3, and Gbeta had no effect on PLD activity.


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  		Fig. 4.   Inhibition of CCK-stimulated phospholipase D (PLD) activity in intestinal smooth muscle by Galpha 13 and RhoA antibodies. Muscle cells were labeled with [3H]myristic acid for 3 h, and CCK (1 nM)-induced PLD activity was determined by the formation of phosphatidylethanol ([3H]PEt) in the presence of ethanol. Results are expressed as cpm/106 cells. Preincubation of permeabilized muscle cells for 1 h with Galpha 13 antibody or RhoA antibody (0.1-10 µg/ml) inhibited PLD activity in a concentration-dependent fashion. Values are means ± SE of 4 experiments.

RhoA- and ARF-dependent activation of PLD. The ability of RhoA to activate PLD was examined 1) in cultured smooth muscle cells transfected with a dominant negative mutant of RhoA (N19RhoA), 2) in permeabilized, freshly dispersed smooth muscle cells incubated for 1 h with RhoA antibody, and 3) in intact smooth muscle cells incubated for 3 h with the Clostridium botulinum C3 exoenzyme.

Transfection of cultured muscle cells with a dominant negative RhoA mutant inhibited CCK-stimulated PLD activity by 49 ± 8% (P < 0.01; n = 5) (Fig. 5). Preincubation of freshly dispersed permeabilized smooth muscle cells with RhoA antibody (0.1-10 µg/ml) inhibited CCK-stimulated PLD activity in a concentration-dependent fashion with a maximal inhibition of 53 ± 6% (P < 0.01) at 5 µg/ml (Fig. 4). Preincubation of freshly dispersed intact smooth muscle cells with C3 exoenzyme (0.2-2 µg/ml), which inactivates RhoA by ADP ribosylation of Asn41, inhibited CCK-stimulated PLD activity in a concentration-dependent fashion with a maximal inhibition of 52 ± 3% (P < 0.001) at 2 µg/ml (Fig. 6). The inhibition was similar to that elicited by RhoA antibody or by transfection of the dominant negative RhoA mutant. A combination of RhoA antibody (5 µg/ml) and C3 exoenzyme (2 µg/ml) was not additive (54 ± 5% inhibition). A similar degree of inhibition by Galpha 13 antibody and RhoA antibody or C3 exoenzyme was consistent with the ability of G13 to stimulate PLD by activating only RhoA. HA-1077, which preferentially inhibits RhoA kinase activity, inhibited CCK-stimulated PLD activity by 29 ± 3% (P < 0.01) when used at an EC50 of 10 µM. At this concentration, HA-1077 has only a minimal effect on PKC activity (<10%) (33). PLD activity stimulated by GTPgamma S (100 µM) was inhibited 49 ± 4% (P < 0.01) by RhoA antibody (5 µg/ml) and to the same extent (53 ± 4%; P < 0.01) by C3 exoenzyme. A combination of C3 exoenzyme and RhoA antibody did not elicit greater inhibition (54 ± 6%). PLD activity stimulated by phorbol 12-myristate 13-acetate (1 µM) was not affected by pretreating the cells with either C3 exoenzyme or RhoA antibody.


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  		Fig. 5.   Inhibition of CCK-stimulated PLD activity in cultured smooth muscle cells expressing a dominant negative mutant of RhoA (N19RhoA). Muscle cells were labeled with [3H]myristic acid for 3 h, and PLD activity induced by CCK-8 (1 nM) was determined by the formation of [3H]PEt in the presence of ethanol. Results are expressed as cpm/106 cells. In muscle cells transfected with vector only, basal and CCK-stimulated PLD activities were similar to those in freshly dispersed muscle cells. CCK-stimulated PLD activity was inhibited 49 ± 8% in cells expressing N19RhoA. Values are means ± SE of 5 experiments. ** P < 0.01, significant inhibition.



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  		Fig. 6.   Inhibition of CCK-stimulated PLD activity in intestinal smooth muscle by Clostridium botulinum C3 exoenzyme. Muscle cells were labeled with [3H]myristic acid for 3 h, and PLD activity induced by CCK-8 (1 nM) was determined by the formation of [3H]PEt in the presence of ethanol. Results are expressed as cpm/106 cells. Preincubation of muscle cells for 3 h with C3 exoenzyme inhibited PLD activity in a concentration-dependent fashion. Maximal inhibition was similar to that obtained with RhoA and Galpha 13 antibodies (see Fig. 4). Values are means ± SE of 4 experiments.

Preincubation of permeabilized smooth muscle cells for 1 h with ARF antibody (5 µg/ml) inhibited CCK-stimulated PLD activity by 29 ± 9% (P < 0.05) (Fig. 7). The inhibition was additive to that elicited by RhoA antibody (5 µg/ml) or C3 exoenzyme (2 µg/ml) (78 ± 3% and 75 ± 6% inhibition, respectively) (Fig. 7).


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  		Fig. 7.   Inhibition of CCK-stimulated PLD activity in intestinal smooth muscle by combinations of ARF antibody with RhoA antibody or C3 exoenzyme (C3). PLD activity was measured as described in the Fig. 4 legend, and the results are expressed as cpm/106 cells. A combination of ARF antibody (5 µg/ml) with either RhoA antibody (5 µg/ml) or C3 exoenzyme (2 µg/ml) caused additive inhibition. A combination of C3 exoenzyme and RhoA antibody was not additive (see RESULTS). Values are means ± SE of 5-6 experiments. ** P < 0.01, significant inhibition from control CCK response; ## P < 0.01, significantly different from inhibition by each agent alone.

PKC-dependent activation of PLD. Calphostin C, which blocks the DAG-binding site of PKC, and bisindolylmaleimide I, which blocks the ATP-binding site, inhibited CCK-stimulated PLD activity by 30 ± 3% and 30 ± 2%, respectively (P < 0.01) (Fig. 8). A selective myristoylated pseudosubstrate peptide inhibitor of PKC-alpha and a common inhibitor of PKC-alpha ,beta ,gamma inhibited PLD activity to the same extent as calphostin C (32 ± 3% and 29 ± 2%, respectively; P < 0.01); a selective pseudosubstrate inhibitor of PKC-epsilon had no effect (1 ± 2%) (Fig. 8). A combination of calphostin C with either PKC-alpha or PKC-alpha ,beta ,gamma inhibitors was not additive (30 ± 4% and 31 ± 5%, respectively). The pattern implied that PKC-dependent activation of PLD was mediated by PKC-alpha .


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  		Fig. 8.   Inhibition of CCK-stimulated PLD activity in intestinal smooth muscle by selective protein kinase C (PKC) inhibitors. PLD activity was measured as described in the Fig. 4 legend, and the results are expressed as cpm/106 cells. PLD activity was significantly inhibited by bisindolylmaleimide (Bis, 1 µM), calphostin C (CalC, 1 µM), and myristoylated pseudosubstrate peptide inhibitors of PKC-alpha and PKCalpha ,beta ,gamma (1 µM). Values are means ± SE of 4 experiments. ** P <=  0.01, significantly different from control CCK response.

The ability of PKC to activate PLD was additive to that of either RhoA or ARF. A combination of calphostin C with RhoA antibody or C3 exoenzyme inhibited CCK-stimulated PLD activity by 75 ± 5% and 73 ± 4%, respectively, and a combination with ARF antibody inhibited PLD activity by 58 ± 3% (Fig. 9). Combining calphostin C with both RhoA and ARF antibodies inhibited PLD activity by 83 ± 2% (Fig. 9), whereas a combination of calphostin C with both C3 exoenzyme and ARF antibody inhibited PLD activity by 86 ± 2%.


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  		Fig. 9.   Inhibition of CCK-stimulated PLD activity in intestinal smooth muscle by combinations of calphostin C with ARF and RhoA antibodies. PLD activity was measured as described in the Fig. 4 legend, and the results are expressed as cpm/106 cells. Inhibition induced by RhoA or ARF antibody was additive to that of calphostin C. Values are means ± SE of 5-6 experiments. ** P < 0.01, significant inhibition from control CCK response; ## P < 0.01, significantly different from inhibition by each agent alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that agonist-induced, sustained activation of PLD involves a distinct set of heterotrimeric and monomeric G proteins. The pathways involved are depicted schematically in Fig. 10. CCK-8 activated the heterotrimeric G proteins G13 and G12 and the monomeric G proteins RhoA and ARF. The alpha -subunit of G13, but not G12, was coupled to sequential activation of RhoA and PLD. RhoA was the dominant activator of PLD, accounting for 50% of the response, and its effect appeared to be mediated by ROK. ARF also activated PLD, but its effect was not mediated by either G13 or G12. The effects of RhoA and ARF were additive to those of PKC-alpha , the specific isoform that mediates activation of PLD by PKC. Unexpectedly, the activation of G12 by CCK-8 resulted in inhibition of PLD. The evidence is summarized as follows.


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  		Fig. 10.   Schema depicting the role of heterotrimeric G proteins (G13 and G12), monomeric G proteins (RhoA and ARF), and PKC-alpha in the regulation of agonist-stimulated sustained PLD activity in intestinal smooth muscle. Receptor-linked agonists (e.g., CCK-8) activate both G13 and G12. Galpha 13 initiates a cascade involving sequential activation of a Rho-specific, guanine nucleotide exchange factor (Rho-GEF), RhoA, Rho-associated kinase (ROK), and PLD. Galpha 12 mediates an inhibitory pathway that attenuates agonist-induced activation of PLD; steps in the pathway initiated by G12 have not been identified. Agonist-induced activation of ARF enhances PLD activity; steps in the pathway involving ARF have not been identified. Dephosphorylation of the phosphatidic acid (PA), the primary product of PLD, yields diacylglycerol (DAG), which activates Ca2+-dependent and -independent PKC isozymes, one of which, the Ca2+-dependent PKC-alpha , acts to enhance PLD activity. Thus agonist-stimulated, sustained PLD activity is mediated additively by G13-dependent RhoA and by ARF and PKC-alpha and is modulated by an inhibitory G12-dependent pathway (stimulation and inhibition denoted by + and -, respectively).

Receptor-mediated activation of G13 and G12 and its relation to PLD activity. CCK-8 activated three heterotrimeric G proteins (Gq/11, G13, and G12), only one of which, G13, was coupled to sustained activation of PLD. Galpha 13 antibody inhibited CCK-stimulated PLD activity, whereas Galpha 12 antibody increased CCK-stimulated PLD activity, suggesting that G12 mediated an inhibitory pathway. Antibodies to the alpha -subunits of Gq/11, Gi1-2, Gi3, and Gs, and a common antibody to Gbeta had no effect.

Coupling of G13 to RhoA but not ARF. CCK-8 induced translocation of RhoA and ARF to the membrane and activated both monomeric G proteins as indicated by the increase in GTP binding to RhoA and ARF. Galpha 13 antibody but not Galpha 12 antibody inhibited activation of RhoA; neither Galpha 13 nor Galpha 12 antibody had any effect on ARF. Thus in intestinal smooth muscle, only G13 and RhoA were sequentially coupled.

Activation of PLD via RhoA and ARF. Agonist-stimulated PLD activity was mediated additively by RhoA and ARF. RhoA antibody, the Clostridium botulinum C3 exoenzyme, and a dominant negative RhoA mutant transfected into cultured muscle cells inhibited agonist-stimulated PLD activity to the same extent (49% to 53%). The extent of inhibition was similar to that obtained with Galpha 13 antibody (47%), consistent with sequential activation of G13, RhoA, and PLD. ARF antibody inhibited PLD activity to a lesser extent (29%), and its effect was additive to that of RhoA antibody or C3 exoenzyme (75% to 78%), suggesting that RhoA and ARF activate PLD via distinct mechanisms. The effect of RhoA appeared to be mediated by ROK and was inhibited by HA-1077, a preferential inhibitor of ROK (30, 33).

Neither the mechanism by which CCK induced activation of ARF and its translocation to membranes nor the mechanism by which ARF activated PLD was identified. As noted above, Galpha 12 and Galpha 13 antibodies did not block receptor-mediated activation of ARF. It seemed unlikely that Gbeta gamma subunits that can bind to ARF were involved in its activation, because a common Gbeta antibody had no effect on CCK-stimulated PLD activity.

Activation of PLD by PKC. Activation of PLD by PKC has been demonstrated in various cell types and represents a feedback mechanism, because DAG, the main activator of PKC, is largely generated by dephosphorylation of PA, the primary product of PLD activity (1, 9, 32). Previous studies (26) on intestinal circular and longitudinal smooth muscle have shown that agonist-stimulated PLD activity was partly inhibited by calphostin C. The present study confirmed that PLD activity was partly inhibited by calphostin C, as well as by selective inhibitors of PKC-alpha . The involvement of PKC-alpha , and to a lesser extent PKC-beta I and -beta II, has been demonstrated in other cell types (1, 9, 32). In the present study, the inhibition of PLD activity by calphostin C was additive to that of RhoA or ARF antibodies, applied separately or in combination.

The mechanism by which PKC activates PLD has not been fully established. In vitro studies suggest that the stimulatory activity of PKC resides in its regulatory domain, because PKC fragments devoid of catalytic domain retain their stimulatory activity in vitro, and inhibitors of catalytic activity appear to be ineffective (8). In vivo, however, as in the present study, blockers of the regulatory and catalytic domains of PKC inhibited PLD activity (1, 8, 9). Because no evidence exists for direct phosphorylation of PLD by PKC, the effectiveness of both types of inhibitors suggests that PKC may act indirectly on PLD via an intermediate susceptible to stimulatory phosphorylation.

A recent study by Ushio-Fukai et al. (34) showed that angiotensin II-stimulated PLD activity in cultured vascular smooth muscle was inhibited by antibodies to Galpha 12, Gbeta , RhoA, and c-src, suggesting involvement of Galpha 12-RhoA and Gbeta gamma -Src pathways. However, the roles of G13, ARF, or PKC and their interplay with the RhoA/ROK pathway were not examined. In intestinal smooth muscle, G12, unlike G13, mediated an inhibitory PLD response, whereas in cultured vascular smooth muscle, G12 mediated a stimulatory response. Differential involvement of G12 and G13 in activation of monomeric G proteins has been reported (4, 10, 17, 18, 28) in other cell types, and as noted earlier, appears to be both cell and agonist specific.

The functional significance of agonist-stimulated, sustained activation of PLD resides in the ability of its primary product, PA, to generate DAG and thus activate PKC. We and others (15, 16, 25, 35) have provided evidence that specific isoforms of PKC are involved in sustained contraction of vascular and visceral smooth muscle. Sustained contraction of intestinal smooth muscle induced by G protein-coupled agonists is mediated by the Ca2+-independent isoform, PKC-epsilon , whereas sustained contraction induced by phorbol esters and growth factors (e.g., epidermal growth factor) is mediated by PKC-alpha , and possibly other Ca2+-dependent isoforms (25). Preliminary evidence suggests that agonist-stimulated PKC-epsilon activity and sustained contraction of intestinal smooth muscle are mediated by a pathway involving sequential activation of G13, RhoA, and PLD and could be inhibited by GDPbeta S, Galpha 13 and RhoA antibodies, and by PLD and PKC inhibitors (27). Recent studies (11, 14, 16, 33) have provided further evidence of a functional linkage between RhoA, PKC, and sustained muscle contraction; activation of ROK inhibits myosin light chain phosphatase via the PKC target protein CPI-17, resulting in phosphorylation of myosin light chain and sustained contraction.


    ACKNOWLEDGEMENTS

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: G.M. Makhlouf, PO Box 980711, Medical College of Virginia, Virginia Commonwealth Univ., Richmond, Virginia 23298-0711 (E-mail: makhlouf{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.

Received 13 July 2000; accepted in final form 11 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 280(3):G381-G388
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