Agonist-induced Ca2+ Sensitization in Smooth Muscle

Background: Multiple RhoGEFs regulate agonist-induced Ca2+-sensitized force. Results: PDZRhoGEF and LARG are functionally redundant, translocate to the cell membrane, and form hetero- and homodimers to mediate Gα12/13-dependent RhoA activation. Conclusion: Ca2+-sensitized force is induced by parallel signaling through RhoGEFs, which are rate-limiting due to their slow recruitment and activation. Significance: Signaling through RhoGEFs suggests new therapeutic targets for diseases of smooth muscle. Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca2+]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca2+ sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca2+-sensitized force are not well understood. Using permeabilized blood vessels from PRG(−/−) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca2+-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5′-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca2+ transient and phasic force components and the onset of Ca2+-sensitized force.

Contraction of smooth muscle (SM) 2 is responsible for critical bodily functions, including the maintenance of proper blood pressure within the vasculature. It is well established that agonists such as angiotensin, epinephrine, endothelin-1 (ET-1), thromboxane A2 (TXA2), etc. induce SM contraction through G-protein-coupled receptors (GPCRs) and G␣ subunits of the heterotrimeric G-proteins (1). Stimulation of the receptors initiates two parallel signaling pathways: an influx of Ca 2ϩ , both through dedicated channels and release from intracellular storage, and activation of the RhoA/RhoA-activated kinase (ROCK) pathway, which amplifies the Ca 2ϩ -induced contractile force (also known as Ca 2ϩ sensitization) (2). The increase in [Ca 2ϩ ] i leads to activation of the myosin light chain kinase by the Ca 2ϩ ⅐calmodulin complex, and subsequent phosphorylation of the myosin regulatory light chain (RLC 20 ) (3). ROCK phosphorylates and inhibits the MYPT1 subunit of the myosin light chain phosphatase (MLCP), increasing the phosphorylation level of RLC 20 and consequently amplifying the contractile force (4,5). The physiology of these events is of profound medical significance because misregulation of RhoA signaling results in abnormalities of SM function such as hypertension, asthma, cerebral and coronary vasospasm, preterm labor, disturbed gut motility, and erectile dysfunction as reviewed elsewhere (6).
Although the mechanism of Ca 2ϩ -mediated induction of force is relatively well understood, the molecular pathways involved in Ca 2ϩ sensitization are less clear largely because of the degenerate nature of the signaling routes. Agonist-activated GPCRs are coupled to various G␣ subunits, including G␣ 12 and G␣ q/11 , which in turn interact with and activate a range of guanine nucleotide exchange factors for Rho family GTPases (RhoGEFs) that catalyze the GDP to GTP exchange on RhoA (7). Once loaded with GTP, RhoA is capable of binding to and activating ROCK (6). Of the 74 known Dbl homology GEFs found in the human genome, a significant number are known to act on RhoA (8), and a number of them are expressed in SM cells, but it is not known exactly how many are functional and how they are coupled to GPCRs. One possibility is that by acting on specific GPCRs different agonists initiate signaling along unique pathways mediated by specific RhoGEFs (1). Thus, for example, the leukemia-associated RhoGEF (LARG), which acts through G␣ 12/13 , was implicated uniquely to control salt-induced hypertension but not the maintenance of basal blood pressure (9). Also, we and others have discovered that G␣ q/11 activates p63RhoGEF and that this route may be important for agonists acting on GPCRs coupled to G␣ q/11 such as ET-1 and angiotensin (10 -12). However, it is possible that two or more RhoGEFs respond to agonist stimulation either through the same or different G␣ subunits. Each agonist may stimulate one or more GPCRs, and this may potentially lead to activation of various RhoGEFs with different catalytic activities and potentially different activation times. If this is true, then the balance of the Ca 2ϩinduced and Ca 2ϩ -sensitized components of the contractile response may vary considerably between agonists, and the same could be true of the time course of the Ca 2ϩ -sensitized response.
In this study, we probed both the potential redundancy of RhoGEFs downstream of the G␣-mediated signaling in SM and the nature of the time lag associated with the onset of Ca 2ϩ -sensitized force following Ca 2ϩ -induced transient phase.
We focused on a family of RhoGEFs known to be activated by G␣ 12/13 that includes LARG (13), PDZRhoGEF (hereafter referred to as PRG) (14), and p115RhoGEF (15), all of which contain the regulator of G-protein signaling-like domain that mediates the interaction (16). The G␣ 12/13 subunits are coupled to several GPCRs that are activated by potent vasoconstricting agonists such as TXA2 and ET-1, and so in principle each agonist could activate all three RhoGEFs. However, it has been established that SM of p115RhoGEF(Ϫ/Ϫ) mice develops normal contraction when stimulated by ET-1 and TXA2 (17), and so we pursued the question as to whether PRG and LARG are activated together. Unfortunately, the combined PRG(Ϫ/Ϫ) and LARG(Ϫ/Ϫ) knock-out is lethal (18), so we took advantage of a unique procedure that allowed us to silence LARG in organ-cultured blood vessels derived from both normal and PRG(Ϫ/Ϫ) mice (18). To our knowledge, this is the first study of this kind to be reported for SM vessels. Using canonical contractility assays we determined that SM tissue samples derived from the doubly genetically modified vessels show much more deficient Ca 2ϩ sensitization than either the PDZ(Ϫ/Ϫ) or LARG knockdown (LARG kd ) SM samples. Moreover, we present data supporting the notion that this functional redundancy is caused by both direct activation of the two RhoGEFs and their co-recruitment due to heterodimerization. Finally, tissues deficient in either PRG or LARG showed significantly delayed onset of ET-1-induced Ca 2ϩ -sensitized force and required a much longer time to reach maximum contraction. We explored this further by directly measuring times to activation using photol-ysis of caged phenylephrine, caged GTP␥S in the bath solution, and caged GTP␥S or caged GTP loaded on the RhoA⅐RhoGDI complex or on the G14V RhoA⅐RhoGDI complex, respectively, to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca 2ϩ transient and phasic force components and the onset of Ca 2ϩ -sensitized force. Our data strongly suggest that that activation of RhoGEFs is rate-limiting for Ca 2ϩ sensitization of SM.

EXPERIMENTAL PROCEDURES
All procedures using animals were carried out according to protocols approved by the Animal Care and Use Committee at the University of Virginia.
Generation of PRG(Ϫ/Ϫ) Mice-The generation, genotyping, and characterization of the PRG(Ϫ/Ϫ) knock-out mice have been described in detail elsewhere (18). The lack of PRG expression is documented in supplemental Fig. 1. Other details are provided in the supplemental Experimental Procedures.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Screen for RhoGEF mRNA-mRNA was purified from primary mouse aortic cells and SM tissues, and quantitative RT-PCR was performed with primers shown in supplemental Table 1 as described in the supplemental Experimental Procedures.
Adenoviral Transfection of siRNAs into Tissues in Organ Culture and Cultured SM Cells-The construction of shRNA plasmids for suppression of LARG and their characterization are detailed in the supplemental Experimental Procedures. Using an adenoviral construct we developed a method to deliver shRNA to the SM cells in blood vessels. We used a plasmidbased system designated as pMighty in which a specific LARG sequence was inserted downstream of the H1 promoter (19). Replication-deficient adenoviruses encoding the LARG shRNA were generated by the Gene Transfer Vector Core (University of Iowa). A nuclearly targeted LacZ reporter was incorporated into the virus to monitor infection of SM cells using ␤-galactosidase staining. Treatment of the vessels is detailed in the supplemental Experimental Procedures. Murine portal vein and cerebral arteries, but not upper mesenteric artery, showed a high level of viral infection in SM cells throughout the vessel media ( Fig. 1). Portal vein was chosen for silencing LARG and for contractility assays in preference to cerebral arteries because of experimental difficulties when working with small vessels. The effectiveness of the pAd5-Mighty LARG shRNAcontaining virus was first tested on primary cultures of mouse and rat aortic SM cells, which showed a suppression of LARG expression by ϳ70 and ϳ60%, respectively (supplemental Figs. 2 and 3). Next, we conducted infection of WT and PRG-null mouse portal veins in organ culture and in both cases observed a reduction in LARG levels by ϳ65% (Fig. 2). The ability of the contractile apparatus in intact vessels to develop force in response to a depolarizing stimulus with high [K ϩ ] was not different from wild-type portal veins, indicative of normal depolarization-induced Ca 2ϩ release and RLC 20 phosphorylation (supplemental Fig. 4).
Tissue Preparation and Force Measurements-Mouse or rabbit portal veins were dissected, denuded of endothelium, cut into small strips (150 -250 m wide, 2-3 mm long), and mounted on a bubble plate or on a wire myograph system for force measurements (20) or in a muscle trough for photolysis experiments (21). The magnitude of contraction with 154 mM K ϩ was measured prior to stimulation with different agonists (20). Following force measurements, muscle strips were pooled for biochemical analysis. For Ca 2ϩ sensitization experiments, mouse portal veins were permeabilized with ␣-toxin (1,500 units/ml) and treated with 10 M A23187 and with NO synthase inhibitor L-N G -nitroarginine methyl ester. Ca 2ϩ -buffered solutions are detailed in the supplemental Experimental Procedures. Muscle strips were exposed to Ca 2ϩ clamped at pCa 6.7-6.3, and once the force reached a plateau, strips were Ca 2ϩ -sensitized by an appropriate agonist. Force was normalized to a maximum Ca 2ϩ -induced force (pCa 4.5), taken as 100% of potential contractile force. For photolysis experiments, muscles were permeabilized with 75 M ␤-escin in G1 solution as detailed in the supplemental Experimental Procedures. Experiments were performed at 22°C.
Apparatus and Photolysis Techniques-The UV laser for laser flash photolysis, a computer-controlled muscle trough system for solution exchange, the force transducer, and the data collection system have been described in detail previously (21,22). Protocols for Ca 2ϩ measurements, loading of the AM ester of Fluo3 into intact portal vein, pretreatment with cyclopiazonic acid, and introduction of caged compounds and their photolysis are described in the supplemental Experimental Procedures. The rate constant of decay of the aci-nitro intermediate of 1-(2-nitrophenyl)ethyl esters of nucleotides (23) following a laser flash was used as a measure of the rate of photolysis of the complexes of caged nucleotides with RhoA⅐RhoGDI (supplemental Fig. 5). The apparatus was modified as described in the supplemental Experimental Procedures.
Caged Nucleotide-RhoA⅐RhoGDI Complex-Human RhoA or the constitutively active G14V RhoA mutant were co-expressed with the cytosolic inhibitory protein RhoGDI in Saccharomyces cerevisiae. The expression and purification were described in detail elsewhere (24). Caged GTP or GTP␥S was exchanged into the complexes as described previously for GTP (24). In the case of caged GTP, the R-diastereoisomer was used for the exchange (25,26). Bound nucleotides were determined by HPLC and quantified as described in the supplemental Experimental Procedures. Final concentrations of caged nucleotides in complexes with RhoA⅐RhoGDI were in the range of 100 -300 M with 50 -75% of the desired nucleotide exchanged into the complex. Following photolysis, 30 l of the solution was collected, and the fraction of photolyzed nucleotide was quantified by HPLC. Under the experimental conditions used here, an average 8% photolysis yield (range, 3.5-16%) was determined for the caged GTP, caged GTP␥S, and both caged nucleotide-loaded RhoA⅐RhoGDI complexes.

FIGURE 2. Reduction of LARG protein in WT and PRG-null portal veins following infection with an adenoviral LARG-targeted shRNA construct.
LARG protein expression was significantly suppressed with shRNA targeting LARG but not with a non-targeting control in the portal vein strips pooled from three to four mice following contractile measurements. The errors bars correspond to S.E.; n ϭ 3-4.
Cell Culture and Cell Transfection-Culture and transfection of HEK 293T and rat aortic SM cells and isolation of WT and PRG(Ϫ/Ϫ) mouse aortic SM cells are detailed in the supplemental Experimental Procedures.
MLCP and RLC 20 Phosphorylation-Portal veins treated with control or LARG shRNA, permeabilized, and stimulated with ET-1 (100 nM) or U46619 (300 nM) were analyzed for phosphorylation of MYPT1 at Thr-696/853 and of RLC 20 at Ser-19 as described previously (28) and shown in supplemental Fig. 6.
Tissue Screen, Western Blots, and Immunofluorescence Staining-SM tissues and cultured cells were lysed in radioimmune precipitation assay buffer or rhotekin assay lysis buffer and used in SDS-PAGE and Western blot. Procedures and antibodies used are given in the supplemental Experimental Procedures. For immunofluorescence microscopy of PRG(ϩ/Ϫ) mouse cerebral arteries, see the supplemental Experimental Procedures.
Statistical Analysis-Mean values and errors (S.E.) were obtained from three to eight independent measurements for each experiment. The statistical significance of group differences was assessed using Student's t test. A p value of Ͻ0.05 was considered significant. A p value of Ͼ0.05 was considered not significant.

Ca 2ϩ -sensitized Force in Normal and Genetically Modified
Portal Veins-To assess the physiological role of the LARG and PRG exchange factors in G␣ 12/13 -mediated Ca 2ϩ sensitization in SM, we conducted canonical measurements of the contractile force induced by the agonists ET-1 and U46619, a stable analog of TXA2, using normal and genetically modified samples from mouse portal veins. The genetically altered samples included those obtained from the PRG(Ϫ/Ϫ) mouse, normal samples in which LARG was knocked down (LARG kd ) by ϳ65%, and PRG(Ϫ/Ϫ) samples in which LARG was knocked down. To confirm that the PRG(Ϫ/Ϫ) mouse did not show compensating changes in the expression patterns of other prevalent RhoGEFs that may have biased our results, we tested transcription levels of GEF-H1, p63RhoGEF, GEF17, Lbc, LARG, and p115RhoGEF. The mRNA levels did not differ from those found in the tissues of normal animals (Fig. 3A). LARG, p115RhoGEF, and p63RhoGEF protein expression levels were also similar in both animals as were those for G␣ q/11 , G␣ 12 , G␣ 13 , and TXA2 and ET-1 receptors (Fig. 3, B and C). We concluded that any differences in Ca 2ϩ -sensitized force between vessels from the two animals are due to the absence of PRG.
In the first series of experiments, we conducted experiments using intact mouse portal veins where agonist stimulation initiates a full physiological response, including Ca 2ϩ -mediated phosphorylation of RLC 20 as well as activation of RhoA/ROCKmediated inhibitory phosphorylation of MLCP (supplemental Fig. 6). Upon stimulation with U46619, knockdown of LARG alone did not affect the contractile force at maximum agonist concentration, whereas the PRG(Ϫ/Ϫ) veins showed ϳ15% reduction under the same condition (supplemental Fig. 7). In the case of ET-1 stimulation, both samples showed ϳ10% reduction in force at maximum dosage. As expected, the LARG kd /PRG(Ϫ/Ϫ) veins were affected the most with maximum force reduced by ϳ30% when U46619 was used and by ϳ20% when ET-1 was used as the agonist (supplemental Fig. 7).
In the second set of experiments, we used permeabilized portal veins in which [Ca 2ϩ ] i was clamped with the EGTA buffer. This allowed us to assess the impact of genetic modifications specifically on Ca 2ϩ sensitization. At pCa 6.3, the magnitude of the Ca 2ϩ -sensitized contractile response to U46619 was virtually unaffected by either the PRG knock-out or LARG knockdown (Fig. 4). However, experiments with the LARG kd /PRG(Ϫ/Ϫ) muscle strips showed a response that was reduced on average to ϳ40% of that observed for the normal muscle strip. A similar effect was observed in experiments where ET-1 was used as an agonist, although in this case, both the PRG(Ϫ/Ϫ) and LARG kd strips had reduced contractile responses (ϳ70 and ϳ75%, respectively, of the response of a normal tissue). The LARG kd /PRG(Ϫ/Ϫ) vein showed only ϳ25% of the response of the normal sample. To ascertain that the observed differences in Ca 2ϩ -sensitized contractility are consistent with the expected biochemical changes, we assayed the levels of phosphorylated RLC 20 and MYPT1. We found that the impairment of Ca 2ϩ -sensitized contractility of vessels from LARG kd /PRG(Ϫ/Ϫ) mice was associated with a significant decrease in phosphorylation of MYPT1 at the ROCK site, Thr-853 (by ϳ30 and ϳ60% in U46619-and ET-1-stimulated vessels, respectively), and of RLC 20 at Ser-19 (ϳ40 and ϳ55%, respectively) (Fig. 5).
LARG and PRG Are Co-recruited in Response to Agonist Stimulation-It is well established that PRG and LARG form homo-and heterodimers in which the binary interactions are mediated by the C-terminal domains, specifically their coiled coil segments (32,33). Dimerization does not affect the nucleotide exchange activity nor does the interaction with G ␣12/13 affect dimerization (33). First, we co-expressed FLAG-tagged full-length PRG and Myc-tagged full-length LARG and performed immunoprecipitation using anti-FLAG antibody-conjugated beads. We observed co-immunoprecipitation of Myctagged LARG (Fig. 6A). We also co-expressed FLAG-tagged full-length PRG and Myc-tagged full-length PRG and performed immunoprecipitation using anti-Myc antibody-conjugated beads. We observed co-immunoprecipitation of FLAGtagged PRG. In both cases, no co-immunoprecipitation occurred in the absence of epitope-tagged partners corresponding to antibody-conjugated beads. We wondered whether the heterodimerization might play a role in Ca 2ϩ -sensitized force. We hypothesized that the isolated dimerization domain from PRG (residues 1207-1553), but not its truncated variant (residues 1207-1453) lacking the C-terminal coiled coil, might interfere with homo-and heterodimerization and affect Ca 2ϩ sensitization. Indeed, a recombinant C-terminal domain, but not a control lacking the coiled coil region, significantly inhibited U46619-and ET-1-induced Ca 2ϩ sensitized force when added to ␤-escin-permeabilized abdominal aorta or portal vein (Fig. 6B). Furthermore, a recombinant C-terminal domain with a double phosphomimic mutation (S1508E/S1510E) was the most potent inhibitor of Ca 2ϩ sensitization (Fig. 6B).
Next, we asked whether LARG and PRG might be co-recruited to the cell membrane following GPCR stimulation by a single agonist. We used mouse cerebral vessels isolated from PRG(ϩ/Ϫ) and PRG(Ϫ/Ϫ) mice in which we were able to image individual SM cells. Immunolabeling of the TXA2 receptors clearly showed their localization at the cell periphery. Immunostaining of both PRG and LARG in PRG(ϩ/Ϫ) mice showed diffuse cytosolic distribution of both proteins. Upon stimulation of the heterozygous cells with U46619, both PRG and LARG translocated from the cytosol to the cell periphery (Fig. 7). As expected, PRG expression was absent in the PRG(Ϫ/Ϫ) cerebral vessels, and LARG expression was unchanged. Moreover, upon stimulation with U46619, LARG relocated to the membrane.

Time Course Evolution of Ca 2ϩ -sensitized Force in Normal
and Genetically Altered SM-To determine how the time course evolution of Ca 2ϩ -sensitized force in RhoGEF-depleted tissues compares with normal SM, we measured the kinetics of activation of Ca 2ϩ -sensitized force in ␣-toxin-permeabilized mouse portal veins induced with U46619 and ET-1 by direct addition of the agonists to the muscle bath. Normal as well as genetically modified SM strips were used. In the case of normal SM strips, the force trace of the kinetics displayed (as expected) a slightly sigmoidal shape with a short lag phase of 32 Ϯ 3 s for ET-1 and 21 Ϯ 3 s for U46619. The t1 ⁄2 was 56 Ϯ 3 s for samples stimulated with ET-1 and 34 Ϯ 3 s for stimulation with U46619.
Although the PRG(Ϫ/Ϫ) and LARG kd in ␣-toxin-permeabilized portal vein strips displayed maximum contraction very similar to that of normal tissues (Fig. 4), they took significantly longer to reach the maximum force, which was most apparent for ET-1 stimulation. For those stimulated by ET-1, the t1 ⁄ 2 was 44 Ϯ 3 s for WT, 61 Ϯ 7 s for PRG(Ϫ/Ϫ), and 103 Ϯ 12 s for LARG kd . Unfortunately, for the PRG(Ϫ/Ϫ)/LARG kd samples, the contractile force was compromised to a degree that did not allow for a reliable estimate of the kinetic parameters.
Kinetics of Ca 2ϩ -sensitized Force in ␤-Escin-permeabilized SM-To better understand the kinetics of the activation of the Ca 2ϩ -sensitized force, we conducted measurements of the time course of the force development after photolysis of caged phen-

FIGURE 5. Loss of excitatory U46619 or ET-1 Ca 2؉ -sensitized contractility of vessels from LARG kd /PRG(؊/؊) mice is accompanied by a decrease in ROCK-mediated phosphorylation of myosin phosphatase at Thr-853 and RLC 20 phosphorylation at Ser-19. A typical Western blot analysis (top) and
summary (bottom) of mouse blood vessels stimulated with 100 nM ET-1 or 300 nM U46619 are shown. The error bars correspond to S.E.; n ϭ 4 -5. FIGURE 6. PRG and LARG co-immunoprecipitate, and a C-terminal PRG peptide suppresses Ca 2؉ -sensitized contractility of mouse vessels consistent with hetero-oligomerization. A, mammalian plasmids expressing FLAG-tagged PRG and Myc-tagged LARG were cotransfected into HEK 293T cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. After 48 h of incubation, the cells were lysed in buffer (0.5% Triton X-100, 150 mM NaCl, 50 mM Tris (pH 7.5), 2 mM EDTA, 2 mM sodium orthovanadate, 50 mM NaF, 1% protease inhibitor mixture (Sigma)) and subjected to EZview Red anti-FLAG M2 affinity gel (Sigma) for immunoprecipitation (IP). Western blot (WB) analysis was performed using anti-Myc antibody. B, muscles were equilibrated with the indicated peptides or their filtrate as a control for 10 min prior to agonist addition. The filtrate was without effect, indicating that the changed contractility was not due to Ca 2ϩ contamination in the peptide solution. The error bars correspond to S.E. AA, S1508A/S1510A; EE, S1508E/S1510E. ylephrine, an ␣ 1 -selective adrenergic agonist coupled to G ␣q/11 , and after photolysis of caged GTP␥S, either free and thereby acting on G␣ subunits or bound to the RhoA⅐RhoGDI complex. The use of caged compounds circumvents diffusional delays that would contribute to the kinetic measurements. Strips from rabbit ␤-escin-permeabilized veins were used throughout except for caged phenylephrine, which was also assayed in ␣-toxin-permeabilized samples for comparison. Full contraction was first induced at pCa 4.5 followed by relaxation in G1 solution (Fig. 8A) and then induction of ϳ50% of maximum contraction at pCa 6.5. The samples were then transferred into the photolysis solution (pCa 6.5) containing the appropriate caged substrate at the required concentration and incubated for 3 min to allow full equilibration of the caged compound throughout the muscle. A 50-ns laser pulse generated the Ca 2ϩsensitized force, the force of which at its maximum was ϳ30% of the maximal force observed. In each case, the lag phase and the time required to reach half the maximum contractile force (t1 ⁄ 2 ) were measured (Table 1  . Importantly, for about 9 s, virtually no force was observed (latent phase) (Fig. 8B). The total time required to reach half the maximum force (t1 ⁄ 2 ) induced by 5-10 M caged phenylephrine was in the range of 40 -50 s.
Experiments involving photolysis of caged GTP␥S were also conducted for a range of GTP␥S concentrations (0.03-6.0 M) liberated from caged GTP␥S with the force reaching a maximum at 4.0 M where the lag phase was estimated at 15.0 Ϯ 1.0 s and with a t1 ⁄ 2 of 61.0 Ϯ 7.0 s. Importantly, these results are very close within experimental error to those obtained for caged phenylephrine (Table 1).
Next, we used the photolysis of the caged GTP␥S-RhoA⅐RhoGDI complex to initiate Ca 2ϩ -sensitized force (Fig. 8B). Under these conditions, photolysis of caged GTP␥S results in the dissociation of the complex and translocation of RhoA to the membrane, thus circumventing the action of RhoGEFs. A typical experimental force trace for contraction induced by photolysis of 25 M caged GTP␥S bound to RhoA⅐RhoGDI is shown in Fig.  8B. Contractions were examined over a range of concentrations of 0.24 -6.0 M photolyzed GTP␥S-RhoA⅐RhoGDI (measured in the postphotolysis solution collected from the muscle trough), and the contractile force reached a maximum at 2.0 M. The lag phase was 7 Ϯ 0.3 s, and the t1 ⁄ 2 was 23 Ϯ 1.5 s (Table  1), revealing significantly faster kinetics and notably the disappearance of the latent phase.
Finally, as GTP is the natural guanosine nucleotide in this system, we initiated Ca 2ϩ -sensitized force by photolysis of caged GTP bound to the constitutively active RhoA G14V⅐RhoGDI over a range of concentrations (0.24 -6.0 M photolyzed GTP-G14V RhoA). Contractile force saturated at 2.0 M. This experiment yielded data very comparable with experiments using the caged GTP␥S-RhoA⅐RhoGDI complex. The lag phase was 8 Ϯ 1.5 s, but the t1 ⁄ 2 was 43 Ϯ 3.9 s presumably due to hydrolysis of GTP (Table  1). A force trace with this complex was reported in a review by us elsewhere (2) but never in a published experimental study (Fig. 3). The commonality of the short lag phase (7-8 s) with both the GTP␥Sand GTP-caged complexes in contrast to that of caged phenylephrine-induced Ca 2ϩ -sensitized force (18 s) further substantiates the presence of a slow rate-limiting step of ϳ10 s (Fig.  8C) upstream of the generation of RhoA-GTP. Based on studies using caged ATP, RLC 20 phosphorylation is known to contribute only 0.2-0.5 s to the 7-8 s lag phase observed with the caged complexes (34). The remaining 6.5-7.5 s lag phase reflects the translocation of RhoA to the cell membrane, activation of ROCK, and inhibitory phosphorylation of MLCP.
Kinetics of Agonist-induced Ca 2ϩ -sensitized Force in Intact SM-To assess whether the experiments involving permeabilized SM samples reflect the kinetics in intact muscles, we conducted experiments using an intact rabbit portal vein and induced full contraction using photolysis of caged phenylephrine. Release of phenylephrine induced a rapid increase in [Ca 2ϩ ] i at t 0 ϩ 0.2 s that preceded force development at t 0 ϩ 1.5 s (Fig. 9). The [Ca 2ϩ ] i subsequently fell and by t 0 ϩ 15 s stabilized just above the basal [Ca 2ϩ ] i . The force fell to a lesser degree and was followed by a secondary rise. Thus, the [Ca 2ϩ ] i /force relationship is no longer maintained with the onset of Ca 2ϩ -sensitized force. Upon pretreatment with cyclopiazonic acid to reduce the rise in [Ca 2ϩ ] i through inhibition of Ca 2ϩ storage in the sarcoplasmic reticulum, the phenylephrine-induced Ca 2ϩ transient and initial force were markedly depressed, whereas the secondary slow rise in tension was maintained (Fig. 9). As expected, the addition of the ROCK inhibitor, Y-27632 completely relaxed the secondary rise in tension (data not shown).

DISCUSSION
Many physiological agonists induce Ca 2ϩ -sensitized force in SM tissues, and although their actions are mediated by specific GPCRs, downstream signaling may occur through degenerate networks as GPCRs can couple to various G␣ subunits of heterotrimeric G-protein, which in turn may potentially activate one or more RhoGEFs, catalyzing nucleotide exchange on RhoA. In this study, we investigated the possibility of concerted, cooperative activity of two such RhoGEFs, i.e. LARG and PRG. Unlike many other studies that use cultured SM cells, we used intact and permeabilized blood vessels that were genetically depleted in PRG and LARG. Because a double knock-out in mice causes embryonic lethality (18), we turned to a different approach of silencing the LARG gene in intact organ-cultured tissues isolated from the PRG(Ϫ/Ϫ) mouse. This technique yielded samples in which LARG was reduced by ϳ65%, whereas PRG was obviously absent. Importantly, mRNA screens and Westerns blots indicated that the knock-out of PRG did not noticeably disrupt the expression patterns of other RhoGEFs that potentially might bias the experiments.
We targeted PRG and LARG because both act downstream of G␣ 12 and G␣ 13 with which they interact through a dedicated domain known as the regulator of G-protein signaling-like domain (35), and both have been found by us and others to be expressed in SM (36,37). We assayed the RhoA-mediated Ca 2ϩ -sensitized component of the SM contractile force induced by two potent vasoconstricting agonists, i.e. ET-1 and U46619, a stable analog of thromboxane A2.
Interestingly, the knock-out of PRG or depletion of LARG through silencing had little effect on the maximum contractile force when compared with normal portal vein SM tissue, and the reduction of force was statistically significant only when ET-1 was used as agonist. In contrast, samples in which PRG was knocked out and LARG was depleted showed a dramatic and reproducible phenotype in which contractile force was reduced to Ͻ50% when U46619 was used and to Ͻ25% with ET-1 stimulation. These results strongly suggest that both RhoGEFs are activated by each of the two agonists, and each RhoGEF is capable of generating a high or maximal level of Ca 2ϩ -sensitized force. This is further supported by our finding An initial maximal contraction is induced by high [Ca 2ϩ ]. Following relaxation in low Ca 2ϩ (G1), a partial contraction is induced by pCa 6.5. Addition of filtrate or the caged complex is without effect. Following a 3-min incubation to allow penetration of the caged complex into the muscle, a 50-ns near-UV laser pulse (Flash) induces photolysis and Ca 2ϩ -sensitized force. Following washout in G1, a rapid return to pCa 6.5 gives an enhanced response, indicating that the muscle has not recovered from Ca 2ϩ sensitization processes, which are relaxed by the Rho kinase inhibitor Y-27632. B, activation of RhoA-mediated Ca 2ϩ sensitization pathway by photolysis of caged GTP␥S-RhoA⅐RhoGDI complex using the protocol shown in A. Upon photolysis at t 0 , 2 M, a saturating concentration of free GTP␥S-RhoA⅐RhoGDI, was released at a rate of 60 s Ϫ1 . Back-extrapolation of the fast rising phase to the prephotolysis base line (inset) shows a lag phase of 7 s. The lag following photolysis of caged ATP in un-prephosphorylated SM leading to RLC 20 phosphorylation and the onset of force accounts for less than 0.5 s (34) (C). Thus, the 6.5-s lag phase triggered by photolysis of the complex reflects the time course of dissociation of RhoA-GDP from GDI, translocation of RhoA-GTP to the membrane, activation of ROCK, and inhibition of MLCP as shown in C. Photolytic release of saturating concentrations of phenylephrine (2 M) displayed much slower kinetics than the GTP␥S-RhoA⅐RhoGDI complex with a long delay (16 s) having a latent phase with no measurable force followed by a lag phase. The rate of force development is also significantly slower: t1 ⁄2 is 37 s versus 23 s for the GTP␥S-RhoA⅐RhoGDI complex. This slow time course reflects the events leading to activation of RhoGEFs as shown in C, including their translocation to the cell membrane and nucleotide exchange on RhoA-GDP⅐RhoGDI. C, agonist-induced signaling cascade for Ca 2ϩ -sensitized force with the measured lag times preceding force development following photolysis of caged phenylephrine, caged GTP␥S, caged GTP␥S-loaded RhoA⅐RhoGDI complex, or caged ATP in the presence of thiophosphorylated RLC 20 . The ϳ10-s difference between the lag times for caged phenylephrine or caged GTP␥S versus GTP␥S-loaded RhoA⅐RhoGDI complex reflects the processes underlying the activation of RhoGEFs. mN, millinewtons. Although in our experiments we targeted RhoGEFs that are expected to be activated primarily by G␣ 12/13 , some caution must be exercised in the interpretation of the results. First, the strictly G q -coupled histamine receptor has been shown to activate LARG (38). Also, it is not clear that TXA2 and ET-1 signal exclusively through G␣ 12/13 . It is widely believed that the Ca 2ϩsensitizing function of TXA2 is mediated by G␣ 12/13 , but it is also known that G q/11 may be activated by the TXA2 receptor in which case the p63RhoGEF could be activated. However, this is unlikely as we have previously shown that the silencing of p63RhoGEF in mouse portal vein had no effect of U46619-induced Ca 2ϩ -sensitized force but did suppress the ET-1 response (11). Similarly, ET-1 has been shown to cause transient Ca 2ϩ -induced contraction through G q -coupled receptors, whereas the Ca 2ϩ -sensitized force is mediated by G␣ 13 coupling (39). Thus, the G q /p63RhoGEF coupling is also possible with ET-1 stimulation. These degenerate signaling networks may easily explain why a significant fraction of sensitized force was still observed after the PRG knock-out and LARG silencing. We also note that the silencing depleted ϳ70% of LARG, so one would expect that the LARG pathway is still active in the modified samples. Whatever the source of the residual force in the PRG/LARG-depleted samples, it is the combined effect of these two RhoGEFs that is responsible for Ca 2ϩ -sensitized force induced by either ET-1 or U46619, and both translocate to the membrane following stimulation by U46619. G␣ 12/13 -LARG has already been implicated in salt-mediated hypertension in mouse models (9). In that study, the authors noted that both PRG and LARG are present in murine SM but discounted the role of PRG due to its lower copy numbers present. Interestingly, a deficiency of both PRG and LARG, but not either alone, results in defective embryonic development, supporting an essential developmental role for both of these GEFs (18). Here we propose, based on our experimental evidence, that PRG is almost equally important for RhoA activation. Is important to note that PRG and LARG are catalytically very potent, and this could compensate for low levels (40,41). Even more significant is the observation that PRG and LARG heterodimerize in cells (33). In concert, we showed that both RhoGEFs, not only LARG, are detectably translocated to the cell membrane in murine cerebral vessels stimulated with U46619. This co-recruitment could be due to direct activation and corecruitment of PRG in a heterodimer with LARG. However, the isolated recombinant C-domain of PRG, which is responsible for heterodimerization with LARG, was able to inhibit the Ca 2ϩ -sensitized force, attesting to the fact that dimerization makes a significant contribution to the co-recruitment.
Another important observation in our study is that the depletion of either PRG or LARG impaired the kinetics of the ET-1induced Ca 2ϩ -sensitized force, significantly extending the halftime required to reach full contraction. This indicates that although either of the RhoGEFs can generate nearly full contraction (especially in the U46619-stimulated samples) the rate by which RhoA activation is saturated becomes the limiting factor. This observation is consistent with the fact that in general terms RhoGEFs are relatively poor catalysts, and the nucleotide exchange reaction may take minutes or even longer in vivo to activate available RhoA.
Given the impact the RhoGEFs have on the kinetics of Ca 2ϩsensitized force development, we wondered whether we could probe more deeply into the process. Unfortunately, murine portal vein and other vessels present considerable challenge for experimental studies, and ␤-escin permeabilization necessary for the introduction of caged complexes fails to produce samples in which Ca 2ϩ -sensitized force can be reliably monitored. We were therefore limited in our studies to larger portal veins obtained from rabbits that give highly reproducible contractions and for which receptor coupling is conserved with ␤-escin permeabilization. We asked what differences could be observed in force kinetics between the Ca 2ϩ -sensitized components induced by stimulation of the vasoconstricting agonist receptor, direct stimulation of G␣ subunits, and direct activation of RhoA, circumventing the RhoGEFs. Laser photolysis was used to activate caged phenylephrine, caged GTP␥S, and the caged GTP␥S-RhoA⅐RhoGDI and caged GTP-G14V RhoA complexes. In this way, we eliminated diffusional effects. Although we used caged phenylephrine (caged ET-1 and U46619 are not available), which is coupled to G␣ q/11 and not G␣ 12/13 , we assumed that the kinetics of the response will be generally the same as for isolated G␣ 12/13 . However, the underlying mechanisms responsible for the slow GEF activation for the different stimuli may differ. For example, G␣ q/11 -coupled p63RhoGEF is palmitoylated and when expressed is found at the cell surface (42,43), whereas we found that LARG and PRG translocated from the cytoplasm to the SM cell surface in cerebral vessels stimulated with U46619 stimulation (Fig. 7). The lag phase and t1 ⁄ 2 for force stimulated by photolysis of phenylephrine and caged GTP␥S are almost identical, consistent with the fact that the signaling between receptors and G␣ proteins is on a subsecond scale. Receptor/G-protein interaction is maximal in about 50 ms (44), and activation of G␣ subunits of GPCRs is also fast with a half-time of activation of G␣ q of 350 ms (45) and of G s and G i of 450 and 690 ms, respectively (46,47). In contrast, we observed a marked difference between the kinetics of the Ca 2ϩsensitized response to photolysis of caged GTP␥S and to the release of GTP␥S-RhoA or GTP-G14V RhoA from the complex with RhoGDI ( Fig. 8C and Table 1). The lag phase was shortened from 15 to 7 s. The shorter t1 ⁄ 2 may in part be an artifact due to the larger amount of active RhoA translocated to the membrane in experiments where the recombinant RhoA⅐RhoGDI complex was introduced as opposed to activation of endogenous RhoA only. The long delay of contraction induced by photolysis of caged phenylephrine (16 s) includes a latent phase with no measurable force (Fig. 8, B and C) and a lag phase, which hints at cooperativity at an early stage of force development. This reflects events leading to the activation of RhoGEFs, including their translocation to the plasma membrane, and nucleotide exchange on RhoA-GDP⅐RhoGDI. The cooperative effect may be due at least in part to the recently discovered positive feedback loop whereby the activated, GTP-bound RhoA binds to the PH (pleckstrin homology) domains of RhoGEFs of the Lbc family, which includes PRG and LARG, increasing turnover of basal RhoA activity by as much as 40-fold (48). Another reason for the sigmoidal curve of the kinetics is concentration enhancement effects. The clustering of receptors and signaling molecules in scaffolds at the membrane is critical for increasing the local concentrations as well as amplification of steps in signal transduction cascades (49).
Clearly, the significantly delayed contraction of SM tissues with depleted PRG or LARG is fully consistent with this picture of Ca 2ϩ -sensitized force kinetics even though the two models used in our studies are not fully comparable. In conclusion, we have shown that both PRG and LARG are activated in SM tissues stimulated with G␣ 12/13 -coupled physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptor agonists ET-1 and U46619 and that RhoGEF activation plays an important role in the delay of Ca 2ϩ -sensitized, sustained force with respect to the initial Ca 2ϩ -induced transient.