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(Circulation Research. 2000;87:221.)
© 2000 American Heart Association, Inc.

Cellular Biology

Role for G₁₂/G₁₃ in Agonist-Induced Vascular Smooth Muscle Cell Contraction

Antje Gohla, Günter Schultz, Stefan Offermanns

From the Institut für Pharmakologie (A.G., G.S., S.O.), Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany, and Pharmakologisches Institut (S.O.), Universität Heidelberg, Heidelberg, Germany. Dr Gohla is now at The Scripps Research Institute, La Jolla, Calif.

Correspondence to Dr Stefan Offermanns, Pharmakologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. E-mail stefan.offermanns{at}urz.uni-heidelberg.de

	Abstract

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Abstract—Receptor-induced vascular smooth muscle cell contraction is mediated by dual regulation of myosin light chain (MLC₂₀) phosphorylation through Ca²⁺-dependent stimulation of myosin light chain kinase and Rho/Rho-kinase–mediated inhibition of myosin phosphatase. Although myosin light chain kinase regulation is initiated by the coupling of receptors to G proteins of the G_q family, G_q and G₁₁, it is not known how receptors regulate the Rho/Rho-kinase–mediated pathway. In vascular smooth muscle cells, receptor-mediated MLC₂₀ phosphorylation and cell contraction was blocked by inhibitors of each of the pathways. Receptors of various vasocontractors were found to couple to G_q/G₁₁ and G₁₂/G₁₃, and constitutively active forms of G₁₂ and G₁₃ induced a pronounced contraction of vascular smooth muscle cells that could be blocked by C3 exoenzyme, by inhibition of Rho-kinase, and by stable analogues of cGMP and cAMP. Receptor-mediated smooth muscle cell contraction was strongly inhibited by dominant-negative forms of G₁₂ and G₁₃. These data indicate that a G₁₂/G₁₃-mediated Rho/Rho-kinase–dependent pathway operates in smooth muscle cells and that dual regulation of MLC₂₀ phosphorylation by vasocontractors is initiated by the dual coupling of their receptors to G proteins of the G_q and G₁₂ families.

Key Words: smooth muscle • vasocontractors • G proteins • Rho-kinase • myosin light chain phosphorylation

	Introduction

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Contraction and relaxation of smooth muscle cells are primarily regulated by the phosphorylation and dephosphorylation of the regulatory light chain of myosin (MLC₂₀).^{1 2} The phosphorylation state of MLC₂₀ is under dual control by myosin light chain (MLC) kinase (MLCK) and myosin phosphatase.^{3 4} The classical pathway through which contracting stimuli induce MLC₂₀ phosphorylation is initiated by coupling of their receptors to members of the G_q family of heterotrimeric G proteins. This results via activation of ß isoforms of phospholipase C (PLC) and formation of inositol-1,4,5-trisphosphate in an increase of the free cytosolic Ca²⁺ concentration. The complex of Ca²⁺ and calmodulin then activates MLCK, leading to increased MLC₂₀ phosphorylation.¹

The role of Ca²⁺ in the regulation of smooth muscle contraction is well established, and it has long been known that various physiological stimuli can also induce smooth muscle contraction in the absence of an increase in the free cytosolic Ca²⁺ concentration.^{5 6 7} During recent years, it has become clear that this Ca²⁺-independent regulation occurs through the inhibition of myosin phosphatase and involves the monomeric GTP-binding protein RhoA.^{8 9 10 11} Activation of RhoA leads to the stimulation of Rho-kinase. Rho-kinase, in turn, phosphorylates the regulatory myosin-binding subunit of myosin phosphatase, which results in the inhibition of the enzyme.^{12 13} This Ca²⁺-independent pathway mediates, at least in part, the tonic contraction induced by various stimuli,¹⁴ and evidence has been provided that this Rho/Rho-kinase–mediated mechanism plays an important role in the maintenance of increased vascular tone under pathological conditions.^{15 16} However, it remains unclear how activated receptors regulate the Ca²⁺-independent Rho-mediated pathway in smooth muscle cells.

In the present study, we used isolated vascular smooth muscle cells to gain insight into the mechanism by which receptors regulate smooth muscle cell contraction via the Ca²⁺-independent Rho-mediated pathway. We demonstrate that G₁₂/G₁₃ can induce vascular smooth muscle cell contraction through a Rho/Rho-kinase–mediated pathway, and we provide evidence that receptors couple to G proteins of the G_q as well as of the G₁₂ family to efficiently induce cell contraction via dual regulation of MLC₂₀ phosphorylation.

	Materials and Methods

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The Materials and Methods section is available online at http://www.circresaha.org.

	Results

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It is generally believed that vasocontractors lead to an increase in the free cytosolic Ca²⁺ concentration through the receptor-mediated activation of G proteins of the G_q family. To determine the G proteins potentially involved in stimulus-induced vasocontraction, we studied the coupling of various vasocontractor receptors to heterotrimeric G proteins in plasma membranes of aortic smooth muscle cells. Photolabeling of receptor-activated G proteins with the hydrolysis-resistant GTP analogue GTP-azidoanilide and subsequent immunoprecipitation of individual G-protein subunits confirmed that endothelin, vasopressin, and angiotensin II receptors couple to G_q/11 (Figure 1). Although the ₁-adrenergic receptor agonist phenylephrine did not induce a significant increase in photolabeling of G_q/11, G_12/13, or G_i (data not shown), incubation of membranes with endothelin, vasopressin, and angiotensin II resulted in markedly increased photolabeling of the subunits of G₁₂, G₁₃, and G_i (Figure 1). This suggests that agonist-induced vascular smooth muscle contraction is not solely mediated by G_q/11-dependent pathways but may also involve other G proteins, such as G₁₂ and G₁₃.

View larger version (76K): [in this window] [in a new window]   Figure 1. Receptor-mediated activation of G proteins in membranes of aortic smooth muscle cells. Membranes from bovine aortic smooth muscle cells were photolabeled with [-32P]GTP azidoanilide in the absence (-) or presence of 1 µmol/L angiotensin II (AT II), 1 µmol/L vasopressin (VP), or 1 µmol/L ET-1. Membranes were solubilized, and G-protein subunits were immunoprecipitated with antisera recognizing G12, G13, Gq/11, and Gi1-3. Precipitated proteins were subjected to SDS-PAGE. Shown are autoradiograms of dried gels.

To evaluate whether G₁₂ and G₁₃ are able to induce a contraction of smooth muscle cells, we expressed constitutively active forms of G₁₂ and G₁₃ in isolated vascular smooth muscle cells by intranuclear injection of respective expression plasmids. The expression of constitutively active G₁₂ (G₁₂Q229L) and G₁₃ (G₁₃Q226L) led to a marked smooth muscle cell contraction that eventually resulted in the rounding of cells (Figures 2A and 3). Contraction could be observed 3 hours after intranuclear injection of the plasmids, a time point that coincided with the appearance of the protein (data not shown). Cells injected with control plasmid or with plasmids carrying constitutively active forms of G_q (G_qR183C) and G_i2 (G_i2Q205L) did not show a morphological change (Figures 2A and 3). To test whether the G₁₂/G₁₃-induced cell contraction could be influenced by physiological relaxing mechanisms, cells were treated with the cAMP analogue Sp-5,6-DCl-cBIMPS and the cGMP analogue 8-pCPT-cGMP. Both cyclic nucleotides markedly blocked the effect of constitutively active G₁₂ and G₁₃ mutants (Figures 2A and 3).

View larger version (138K): [in this window] [in a new window]   Figure 2. A, Smooth muscle cell contraction induced by constitutively active mutants of G12 and G13. A through D, Eukaryotic expression plasmids carrying constitutively active mutants of Gq (A), Gi2 (B), G12 (C), and G13 (D) were coinjected with the lacZ gene to identify cells expressing the injected cDNAs. Six hours after injection of plasmids, cells were fixed and stained for ß-galactosidase activity. E through H, Cells expressing constitutively active G12 (E and G) or G13 (F and H) and ß-galactosidase were incubated with 0.1 mmol/L of the cAMP analogue Sp-5,6-DCl-cBIMPS (E and F) or 1 mmol/L of the cGMP analogue 8-pCPT-cGMP (G and H) 30 minutes before fixation and staining. Bars=5 µm. B, Involvement of Rho and Rho-kinase in G12- and G13-induced smooth muscle cell contraction. Cells were injected with plasmids carrying the lacZ gene and constitutively active forms of G12 (A, C, E, and G) or G13 (B, D, F, and H). Cells were coinjected with C3 exoenzyme (C3, A and B) and a dominant-negative form of Rho-kinase (RB, G and H) or were incubated in the presence of 10 µmol/L Y-27632 (Y, E and F). Six hours after injection, cells were fixed and stained for ß-galactosidase activity. Bars=5 µm.

View larger version (71K): [in this window] [in a new window]   Figure 3. Statistical evaluation of effects of constitutively active G protein subunits. Plasmids carrying constitutively active forms of Gq (qRC), Gi2 (i2QL), G12 (G12QL), or G13 (G13QL) were coinjected with a plasmid carrying the lacZ gene into the nucleus of cells. Cells injected with active forms of G12 (dark gray) or G13 (light gray) were treated with Sp-5,6-DCl-cBIMPS (cBIMPS), 8-pCPT-cGMP (cGMP), Y-27632, and C3 exoenzyme (C3) or were coinjected with dominant-negative Rho-kinase (dnROCK) as described in legends to Figures 2A and 2B. Thereafter, cells were fixed and stained for ß-galactosidase activity. Shown is the average cell length as percentage of control cells. Cells expressing only lacZ on the same slide served as controls. Data represent mean±SD of at least 150 cells from 3 independent experiments.

G₁₂ and G₁₃ have been shown to activate Rho.^{17 18 19 20} Therefore, we tested whether the G₁₂/G₁₃-induced smooth muscle cell contraction was mediated by Rho and Rho-kinase. Injection of the C3 exoenzyme of Clostridium botulinum, which ADP-ribosylates and inactivates Rho, completely blocked the effect of G₁₂QL and G₁₃QL (Figures 2B and 3). Similarly, incubation of cells with the Rho-kinase inhibitor Y-27632 as well as coexpression of the dominant-negative Rho-kinase mutant RB/PH(TT)²¹ blocked G₁₂QL- and G₁₃QL-induced cell contraction (Figure 2B). Incubation of cells with tyrosine kinase inhibitors, such as genistein or tyrphostin 25, was without effect (data not shown). The data indicate that constitutively active forms of G₁₂ and G₁₃ can induce vascular smooth muscle cell contraction in a Rho/Rho-kinase–dependent manner.

Smooth muscle cell contraction is primarily regulated by the phosphorylation state of MLC₂₀. We tested various vasocontractors, namely, endothelin-1 (ET-1), angiotensin II, vasopressin, and the thromboxane A₂ mimetic U46619, for their ability to induce MLC₂₀ phosphorylation by using the anti–phospho-MLC antiserum pp2b.²² Incubation of cells with ET-1 produced the strongest phosphorylation of MLC₂₀. Preincubation of cells with the PLC inhibitor U73122 markedly reduced the effect of ET-1, whereas an inactive analogue, U73343, was without effect (Figure 4A). Similarly, inhibition of MLCK by ML-7 blocked ET-1–induced MLC₂₀ phosphorylation (Figure 4B). The Rho-kinase inhibitor Y-27632 and C3 exoenzyme were used to determine the contribution of the Rho/Rho-kinase pathway in receptor-dependent MLC₂₀ phosphorylation. Both agents were without effect on ET-1–induced elevation of free cytosolic Ca²⁺ (data not shown). Treatment of the cells with Y-27632 markedly reduced basal phosphorylation of MLC₂₀ and completely prevented ET-1–dependent MLC₂₀ phosphorylation (Figure 4B). C3 exoenzyme did not affect basal phosphorylation of MLC₂₀ but also blocked the effect of ET-1 (Figure 4C), whereas pretreatment of cells with pertussis toxin had no effect (Figure 4D). This indicates that both the Ca²⁺-dependent pathway involving PLC and MLCK as well as the Rho/Rho-kinase–mediated pathway are involved in receptor-dependent MLC phosphorylation in smooth muscle cells and that both pathways are required for efficient regulation of MLC₂₀ phosphorylation through receptors.

View larger version (28K): [in this window] [in a new window]   Figure 4. MLC20 phosphorylation in vascular smooth muscle cells. Smooth muscle cells were preincubated for 15 minutes with 10 µmol/L of U73122 or U73343 (A) or with 10 µmol/L of ML-7 or 10 µmol/L of Y-27632 (B) or were pretreated for 8 hours with 10 µg/mL C3 exoenzyme and 10 mg/mL lipofectamine (C) or for 18 hours with 100 ng/mL of pertussis toxin (PTX, D). Thereafter, cells were incubated for 5 minutes in the absence (-) or presence of 0.1 µmol/L ET-1 (E). MLC phosphorylation was determined as described in the online Materials and Methods (available at http://www.circresaha.org). For each condition, a representative experiment of at least 3 independently performed experiments is shown.

Because the Rho/Rho-kinase–mediated pathway is involved in agonist-induced MLC phosphorylation and because G₁₂ and G₁₃, which are activated by vasocontractors, are able to induce Rho/Rho-kinase–mediated cell contraction, we next examined whether G₁₂ and G₁₃ are involved in agonist-induced smooth muscle cell contraction. ET-1 led to contraction of 90% of smooth muscle cells within 15 minutes (Figure 5). ET-1–induced smooth muscle cell contraction lasted for 30 minutes. Thereafter, the cells reexpanded, indicating that agonist-dependent contraction was a reversible process (data not shown). To inhibit G₁₂ and G₁₃ function, we expressed dominant-negative forms of G₁₂ (G₁₂G228A) and G₁₃ (G₁₃G225A) via intranuclear injection of respective expression plasmids in vascular smooth muscle cells. Both mutants have been shown to block receptor-induced G₁₂/G₁₃-mediated stress fiber formation in fibroblasts.¹⁹ Cells successfully injected were identified by fluorescence of coinjected fluoro-emerald–labeled dextran. In contrast to uninjected cells or to cells injected with a control plasmid, >90% of cells expressing a mixture of dominant-negative G₁₂ and G₁₃ did not show ET-1–induced contraction (Figures 5 and 7). Expression of G₁₂G228A and G₁₃G225A alone had no measurable effect on ET-1–induced smooth muscle cell contraction. Calyculin A, a PP1/2A-type phosphatase inhibitor, which inhibits myosin phosphatase, induced contraction of injected and uninjected smooth muscle cells, indicating that expression of dominant-negative G₁₂ and G₁₃ did not unspecifically prevent smooth muscle cell contraction (see Figure 5). These data show that ET-1–induced smooth muscle contraction involves G₁₂/G₁₃.

View larger version (173K): [in this window] [in a new window]   Figure 5. Effect of dominant-negative G12 (12GA) and G13 (13GA) mutants on ET-1–induced smooth muscle cell contraction. A through D, Cells injected with a control plasmid (A and B) or with plasmids carrying dominant-negative forms of 12GA and 13GA (C and D) were incubated for 6 hours and challenged with 0.1 µmol/L ET-1. Photographs were taken before (A and C) or 15 minutes after (B and D) the addition of agonist. To detect injected cells, cells were coinjected with a fluorescent dye as described in the online Materials and Methods (available at http://www.circresaha.org). E, Fluorescence image is of cells shown in panel C. Arrows point to cells injected with dominant-negative 12GA and 13GA; arrowheads indicate uninjected cells. F, After the experiment, cells shown in panels C and D were treated for 15 minutes with 100 nmol/L calyculin A. Bars=5 µm.

View larger version (15K): [in this window] [in a new window]   Figure 7. Statistical evaluation of inhibitory effects on ET-1–induced cell contraction. Cells were pretreated under the following conditions: intranuclear injection of expression plasmids carrying a control plasmid (lacZ), dominant-negative forms of G12 and G13 (12/13GA), and a dominant-negative form of Rho-kinase (dnROCK); cytoplasmic injection of C3 exoenzyme (C3) or incubation of cells with 10 µmol/L ML-7, 10 µmol/L U73122, and U73343; or incubation of cells with 100 ng/mL of PTX for 18 hours. Thereafter, cells were incubated in the presence of 0.1 µmol/L ET-1. Photographs were taken before and 15 minutes after addition of the stimulus to determine agonist-induced cell contraction. Shown is the percentage of cells responding with contraction to ET-1. Responding cells were defined as those showing at least a 10% reduction in cell length in response to ET-1. Data represent means of the results from at least 100 cells for each condition obtained in 3 independent experiments.

We evaluated the role of the Ca²⁺-dependent and the Rho/Rho-kinase–mediated pathway in ET-1–induced smooth muscle cell contraction analogously to the experiments shown in Figure 4. Inhibition of PLC by U73122 and inhibition of MLCK by ML-7 completely blocked the effects of ET-1 on cell contraction (Figures 6 and 7). Similarly, ET-1–induced cell contraction was prevented by injection of the cells with C3 exoenzyme and by expression of dominant-negative Rho-kinase (Figures 6 and 7), whereas pertussis toxin had no effect on cell contraction induced by ET-1 (Figure 7). Thus, receptor-mediated vascular smooth muscle contraction and MLC₂₀ phosphorylation require a functional Ca²⁺/MLCK-mediated pathway as well as a Rho/Rho-kinase–dependent pathway.

View larger version (106K): [in this window] [in a new window]   Figure 6. ET-1–induced smooth muscle cell contraction. Vascular smooth muscle cells were pretreated for 15 minutes with 10 µmol/L of U73122 (A and B) and 10 µmol/L of ML-7 (C and D) or were injected cytoplasmically with 100 µg/mL of C3 exoenzyme (C3, E and F) or intranuclearly with an expression plasmid carrying a dominant-negative Rho-kinase (RB, G and H). To detect injected cells, cells were coinjected with a fluorescent dye as shown in Figure 4. Injected cells are marked with arrows. Fifteen minutes after addition of U73122 and ML-7, 30 minutes after injection of C3, and 6 hours after injection of RB, cells were incubated with 0.1 µmol/L ET-1. Shown are cells before (A, C, E, and G) or 15 minutes after (B, D, F, and H) the addition of ET-1. Bars=5 µm.

	Discussion

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Vascular smooth muscle contraction by most physiological stimuli involves the activation of G protein–coupled receptors, resulting in the phosphorylation of MLC₂₀. It is now well established that activated receptors can influence the phosphorylation state of MLC₂₀ by regulating the rate of MLC₂₀ phosphorylation through MLCK as well as the rate of dephosphorylation of MLC₂₀ through myosin phosphatase. Stimulation of MLCK via Ca²⁺/calmodulin is induced by the coupling of activated receptors to G proteins of the G_q family. Inhibitory regulation of myosin phosphatase in contrast does not seem to require an elevated Ca²⁺ concentration but involves the Rho/Rho-kinase–mediated pathway. It is not known how activated receptors lead to stimulation of Rho/Rho-kinase–mediated MLC₂₀ phosphorylation in smooth muscle cells.

Among various vasocontractors tested, ET-1 most effectively induced MLC₂₀ phosphorylation in bovine aortic smooth muscle cells. The effect of ET-1 could be blocked by an inhibitor of PLC as well as by the MLCK inhibitor ML-7 (Figure 4). Receptor-induced phosphorylation of MLC₂₀ was also inhibited by the preincubation of cells with C3 exoenzyme and the Rho-kinase inhibitor Y-27632. Inhibition of Rho-kinase by Y-27632 markedly reduced the basal levels of MLC₂₀ phosphorylation, suggesting that myosin phosphatase was under tonic inhibition by Rho-kinase also in the absence of an exogenously added receptor agonist. ET-1–induced smooth muscle cell contraction exhibited a similar dependence on both G_q/11/PLC-ß–mediated Ca²⁺-dependent MLCK regulation and Rho/Rho-kinase–mediated myosin phosphatase regulation (Figures 6 and 7). These data are consistent with the well-established role of Ca²⁺-dependent MLCK activation in agonist-induced MLC₂₀ phosphorylation and smooth muscle cell contraction. The data also agree with several reports showing that especially the tonic phase of receptor-mediated contraction of intact smooth muscle is strongly inhibited after the inactivation of Rho or the inhibition of Rho-kinase.^{10 11 15 23} The sensitivity of agonist-induced MLC₂₀ phosphorylation and cell contraction to inhibition of each pathway suggests that there is a considerable level of basal phosphorylation and dephosphorylation of MLC₂₀ and that a coordinated induction of Ca²⁺-dependent MLCK activation and of Rho/Rho-kinase–mediated inhibition of myosin phosphatase is required for agonist-induced MLC₂₀ phosphorylation and tonic contraction of smooth muscle cells.

In permeabilized smooth muscle preparations, evidence has been provided that agonists differ in their efficacy to induce Ca²⁺-dependent and Ca²⁺-independent pathways,^{1 6} suggesting that signaling pathways induced by activated receptors bifurcate at a level relatively upstream in the signaling cascade. However, at present, it is not clear at which level the agonist-induced signaling pathways diverge. G proteins of the G₁₂ family have been shown to regulate Rho/Rho-kinase–dependent signaling processes.^{17 18 24 25 26} Therefore, we decided to study the hypothesis that agonist-induced vasocontraction of smooth muscle cells via dual regulation of MLC₂₀ phosphorylation is initiated by the coupling of receptors to G proteins of the G_q and G₁₂ families.

Many G protein–coupled receptors that are able to activate G proteins of the G_q family also couple to G₁₂ and G₁₃.^{19 27} In membranes of aortic smooth muscle cells, we could demonstrate that receptors for ET-1, angiotensin II, and vasopressin couple to G_q/11 as well as to G₁₂ and G₁₃, supporting the notion that G₁₂/G₁₃ is involved in the responses of smooth muscle cells to vasocontractors. We also observed an activation of G_i-type G proteins by these stimuli. Activation of G_i results in the inhibition of adenylyl cyclase but may also contribute to receptor-mediated activation of PLC ß isoforms and other effectors through ß subunits released from the activated heterotrimer.²⁸ Inactivation of G_i-type G proteins by pretreatment of cells with pertussis toxin did not affect receptor-induced MLC₂₀ phosphorylation and cell contraction (Figures 4 and 7), suggesting that G_i is not involved in these acute responses of smooth muscle cells.

Although receptor-mediated smooth muscle cell contraction required activation of the Rho/Rho-kinase as well as the Ca²⁺-dependent pathway, expression of the constitutively active mutants of G₁₂ and G₁₃ alone resulted in a pronounced contraction of isolated vascular smooth muscle cells (Figure 2A). This effect appeared to be specific, inasmuch as cells injected with a control plasmid or with plasmids carrying active forms of G_q and G_i2 showed no morphological change. Cell contraction induced by activated G₁₂ and G₁₃ was blocked by C3 exoenzyme, Y-27632, and by dominant-negative Rho-kinase (Figures 2B and 3). These data demonstrate that a pathway involving G₁₂/G₁₃, Rho, and Rho-kinase operates in smooth muscle cells and that activation of this pathway by constitutively active G₁₂/G₁₃ results in smooth muscle contraction. Analogues of cyclic nucleotides cAMP and cGMP blocked G₁₂/G₁₃-induced cell contraction (Figures 2B and 3), indicating that the G₁₂/G₁₃-induced signaling pathway is subject to inhibitory regulation by cGMP- and cAMP-dependent processes. This is in line with data showing that cAMP inhibits Rho/Rho-kinase–mediated processes in various cells.^{24 29 30} Although the exact mechanism for cAMP-dependent inhibition of the pathway is currently unclear, there is evidence that cGMP can accelerate MLC₂₀ dephosphorylation by myosin phosphatase^{31 32} and that this effect involves a direct interaction of cGMP-dependent protein kinase with the regulatory subunit of myosin phosphatase³³ and/or a telokin-mediated mechanism.³⁴

Because receptors of various vasocontractors are able to couple to G₁₂/G₁₃ and because active forms of G₁₂/G₁₃ induce smooth muscle cell contraction in a manner depending on Rho and Rho-kinase, we tested whether dominant-negative active forms of G₁₂ and G₁₃ are able to interfere with receptor-mediated smooth muscle cell contraction. Coexpression of G₁₂G228A and G₁₃G225A blocked agonist-induced smooth muscle contraction (Figures 5 and 7). This inhibitory effect was comparable to that observed after inhibition of Rho or Rho-kinase (Figures 6 and 7). Our data clearly indicate that G proteins of the G₁₂ family are involved in receptor-dependent smooth muscle contraction. We propose a model for agonist-induced phosphorylation of MLC₂₀ in which the dual regulation of MLC₂₀ phosphorylation through Ca²⁺-dependent MLCK activation and Rho/Rho-kinase–mediated myosin phosphatase inhibition is initiated by the dual coupling of receptors to G proteins of the G_q and G₁₂ families (Figure 8). A very similar scenario has been described for stimulus-induced MLC₂₀ phosphorylation in platelets.²⁴ The mechanism by which G₁₂/G₁₃ activates Rho in smooth muscle cells remains to be clarified. Tyrosine kinases have been involved in G₁₂/G₁₃-induced Rho activation in fibroblasts and neuronal cells.^{18 26} However, G₁₂/G₁₃-induced Rho/Rho-kinase–mediated smooth muscle cell contraction was insensitive to tyrosine kinase inhibitors (data not shown), indicating that similar to the situation in platelets,²⁴ this pathway apparently does not involve tyrosine kinases. Regulation of Rho by G₁₂/G₁₃ may be mediated by a Rho-specific guanine nucleotide exchange factor (GEF). Genetic studies in Drosophila have demonstrated that the Drosophila RhoGEF protein DRhoGEF2 is under control of the concertina gene product, a homologue of G₁₂/G₁₃.³⁵ Related mammalian RhoGEF proteins, such as p115RhoGEF and PDZ-RhoGEF, have recently been shown to interact with G₁₂ and G₁₃.^{25 36}

View larger version (22K): [in this window] [in a new window]   Figure 8. Model of receptor-mediated regulation of MLC20 phosphorylation in smooth muscle cells. PLC-ß indicates ß isoforms of PLC, PKC, protein kinase C; IP3, inositol-1,4,5-trisphosphate; DAG, diacylglycerol; CaM, calmodulin; and ROCK, Rho-kinase. For details, see text.

In vascular smooth muscle cells, we show that receptors of vasocontractors couple to G₁₂/G₁₃, that G₁₂/G₁₃ is able to induce cell contraction in a Rho/Rho-kinase–dependent manner, and that efficient agonist-induced cell contraction involves G₁₂/G₁₃. These data clearly indicate that the contractile response of smooth muscle cells to stimuli depends not only on a G_q/G₁₁-mediated pathway (resulting in MLCK activation) but also on a G₁₂/G₁₃-mediated pathway (leading to Rho/Rho-kinase–dependent inhibition of myosin phosphatase). Thus, dual regulation of MLC₂₀ phosphorylation through MLCK and myosin phosphatase by receptor agonists appears to be induced by the dual coupling of activated receptors to G_q/G₁₁ and G₁₂/G₁₃. Our data also suggest that differences in the efficacy of vasocontractors to induce Ca²⁺-dependent and Rho-Rho-kinase–mediated signaling pathways may be due to different abilities of their receptors to activate G proteins of the G_q and G₁₂ family, respectively.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

Received March 27, 2000; accepted June 8, 2000.

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