Translocation of rhoA Associated with Ca2+ Sensitization of Smooth Muscle*

(Received for publication, September 17, 1996, and in revised form, January 29, 1997)

Ming Cui Gong Dagger , Hideyoshi Fujihara Dagger , Avril V. Somlyo Dagger § and Andrew P. Somlyo Dagger par

From the Departments of Dagger  Molecular Physiology and Biological Physics, § Pathology, and  Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We determined the relationship between the localization of rhoA and Ca2+ sensitization of force in smooth muscle. In alpha -toxin-permeabilized rabbit portal vein at pCa 6.5, the particulate hydrophobic fraction of rhoA (10 ± 1.6% of the total) was significantly increased by phenylephrine to 18 ± 5.5% at 5 min, by AlF4- to 26 ± 8.4% at 20 min, and dose-dependently up to 62 ± 9.5% by guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; 0.3-50 µM). Translocation of rhoA was selective (Rac1 and Cdc42 were not translocated) and was quantitatively correlated (up to ~50%; r = 0.91, p < 0.05) with Ca2+ sensitization; high GTPgamma S concentrations (>= 10 µM) further increased translocation without increasing force. The initial recruitment of rhoA to the membrane paralleled the time course of contraction, but sensitization could be reversed without a decrease in particulate rhoA. High [Ca2+] (pCa 4.5) also increased particulate rhoA to 31 ± 5.8%. Membrane-associated rhoA in unstimulated portal vein was a good substrate for in vitro ADP-ribosylation, whereas the large amount translocated by GTPgamma S was not. We conclude that 1) translocation of rhoA plays a causal role in Ca2+ sensitization, and 2) membrane-bound rhoA can exist in two or more states.


INTRODUCTION

Phosphorylation of the regulatory light chain of myosin (MLC20)1 by a calcium/calmodulin-dependent protein kinase is the primary determinant of force developed by smooth muscle. However, this phosphorylation can also be increased ("Ca2+ sensitization") at constant [Ca2+] by a G-protein-coupled mechanism (1-4) that inhibits the trimeric phosphatase (SMPP-1M) (5-7) that dephosphorylates MLC20. The Ca2+-sensitizing effect of recombinant p21rhoA and the inhibition of agonist-induced Ca2+ sensitization by selective ADP-ribosylation of p21rhoA (either recombinant or endogenous) have implicated this monomeric G-protein in Ca2+ sensitization (8-11). Because bacterially expressed p21rhoA that lacks the prenylated C terminus required for membrane association (12) did not show significant Ca2+-sensitizing activity and even active (geranylgeranylated) p21rhoA failed to Ca2+-sensitize preparations heavily permeabilized with Triton X-100 (11), we suggested that association of p21rhoA with the plasma membrane may be required for its Ca2+-sensitizing effect (11). Recruitment of cytosolic proteins to the membrane is an important component of several other signaling systems, such as the Raf-Ras pathway (13) and protein kinase C cascades (conventional and novel) (14). The purpose of this study was to determine whether p21rhoA signaling of Ca2+ sensitization also involves its translocation to the cell membrane in vivo. We now show that GTPgamma S-induced translocation of p21rhoA is quantitatively and kinetically associated with Ca2+ sensitization of smooth muscle and provide evidence of more than one conformational state of membrane-associated p21rhoA.


MATERIALS AND METHODS

Isometric Tension Measurement

Small strips (200 µm wide and 3 mm long) of rabbit portal vein and ileum longitudinal smooth muscle were dissected, and isometric tension was measured as published (15-17).

Separation of Particulate and Cytosolic Fractions

A minimum of 10 small (200 µm wide and 3 mm long) strips of rabbit portal vein or ileum longitudinal smooth muscle were used to provide sufficient protein for reliable separation of cytosolic and particulate fractions. Stimulated and control strips were homogenized in ice-cold homogenization buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)bezenesulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml aprotinin) and centrifuged at 100,000 × g for 30 min at 4 °C (OptimaTM TLX ultracentrifuge, TLA 120.1 rotor, Beckman Instruments), and the supernatant was collected as the cytosolic fraction. Pellets were resuspended, and membrane proteins were extracted by incubation for 30 min in homogenization buffer containing 1% Triton X-100 and 1% sodium cholate or only 2% Triton X-114. The latter buffer was used to avoid the increase in the cloudy point of Triton X-114 by a second detergent. The extract was centrifuged at 800 × g for 10 min. The supernatant was collected and is referred to as the particulate fraction, and the pellet was collected and is referred to as the detergent-insoluble particulate fraction. Cytosolic, particulate, and detergent-insoluble particulate fraction proteins were separated by SDS-polyacrylamide gel electrophoresis. Only the cytosolic and particulate p21rhoA proteins are shown in most of the figures, as no immunoblot-detectable p21rhoA was found in the detergent-insoluble particulate fraction. The absence of p21rhoA in the detergent-insoluble particulate fraction verified the completion of the extraction of membrane p21rhoA proteins and completion of homogenization. Prompt termination of the reaction in homogenization buffer was verified by the absence of translocation of p21rhoA when control strips were homogenized in GTPgamma S (50 µM)-containing homogenization buffer.

Phase Separation by Triton X-114

Precondensed Triton X-114 stock solution was added to tissue homogenates or cytosolic fractions to a final concentration of ~2%, and proteins were extracted by incubation for 30 min on ice with occasional mixing (18). The mixture was centrifuged at 10,000 × g for 10 min at 4 °C; the pellet was solubilized in sample buffer; and proteins were separated by SDS-polyacrylamide gel electrophoresis to determine cellular proteins insoluble in nonionic detergent. The supernatant was collected in a fresh tube and warmed to 37 °C in a water bath until the solution became cloudy (for ~5 min). Phase separation was achieved by centrifuging the solution in a tabletop centrifuge for 10 min at 800 × g at room temperature. The upper aqueous phase contains soluble proteins, and the lower, detergent-enriched phase contains proteins bearing hydrophobic domains.

Western Blots

After transfer to polyvinylidene difluoride membrane, the membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.05% Tween 20 for 1 h and then incubated with primary antibody for 3 h and secondary antibody for 1 h at room temperature. Blots were detected with enhanced chemiluminescence (ECL, Amersham Corp.) and quantitated by densitometry using a Bio-Rad GS-670 imaging densitometer. Optimal primary antibody concentration was determined by antibody titration (1:100, 1:500, 1:1000, and 1:5000) using a Mini-protein II multiscreen apparatus (Bio-Rad). Preliminary experiments established that the amount of protein loaded was within the range of linearity of the assays. The percent of particulate p21rhoA was calculated according to particulate p21rhoA/(particulate + cytosolic p21rhoA).

Monoclonal anti-p21rhoA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) generated against amino acids 120-150 of human p21rhoA was used at a 1:2500 dilution. Polyclonal anti-Galpha q/11, anti-Rac1, and anti-Cdc42 antibodies (Santa Cruz Biotechnology, Inc.) generated against amino acids 341-359 common to Galpha q and Galpha 11, amino acids 178-191 of Rac1, and amino acids 166-182 of Cdc42 were used at 1:5000, 1:500 and 1:1000 dilutions, respectively.

ADP-ribosylation of Cytosolic and Particulate p21rhoA

The detergent concentration and the volumes of cytosolic and particulate fractions were adjusted to identical values, and the following reagents were added: 200 µM GTP, 10 mM dithiothreitol, 2 mM thymidine, and 1 µg/ml Clostridium botulinum exoenzyme C3. After initiation of ADP-ribosylation by addition of [32P]NAD (final concentration of 50 µCi/ml), the mixture (total volume of 100 µl) was incubated for 30 min at 30 °C. The reaction was stopped by trichloroacetic acid (24%, 250 µl) and deoxycholate (2%, 6 µl), and the final volume was adjusted to 1 ml with water. After centrifugation (5000 × g, 10 min), the supernatant was carefully removed, and the pellet was resuspended in 20 µl of 2 × sample buffer. 10 µl of 1 M Tris base was added to neutralize the pH. Samples were heated at 85 °C for 5 min, and the proteins were separated by SDS-polyacrylamide gel electrophoresis. ADP-ribosylation of p21rhoA in beta -escin-permeabilized strips was carried out as described previously (11).

Details of the solutions used for study of permeabilized strips were described previously (15-17). The pretreatment with A23187 and the presence of 10 mM EGTA assured (15-17) that the changes in force and MLC20 phosphorylation observed under these conditions were not due to changes in [Ca2+]. alpha -Toxin was purchased from List Biological Laboratories Inc. (Campbell, CA). GTPgamma S was from Boehringer (Mannheim, Germany). ADP-ribosyltransferase C3, tautomycin, and A23187 were from Calbiochem. [32P]NAD (30 Ci/mmol) was from DuPont NEN. Statistical comparisons were made using Student's t test; all values are given as mean ± S.E.


RESULTS

p21rhoA Is Translocated by Agonists, GTPgamma S, and Aluminum Fluoride from the Cytosol to the Particulate Fraction Concomitantly with Ca2+ Sensitization of Rabbit Portal Vein Smooth Muscle

Phenylephrine (PE; 100 µM) plus GTP (10 µM) increased force from 13 ± 1.1% (n = 18) to 41 ± 5.4% (n = 6, p < 0.001) of the maximal Ca2+-induced contraction in alpha -toxin-permeabilized rabbit portal vein strips at constant free Ca2+ (pCa 6.5). Such contractions are the result of increased MLC20 phosphorylation (2, 4). Force development was accompanied by translocation of p21rhoA from the cytosol to the particulate fraction. The particulate fraction contained 10 ± 1.6% (n = 23) of the total p21rhoA in control pCa 6.5 solution, increasing to 18 ± 5.5% (n = 9, p < 0.05) upon stimulation with PE plus GTP (Fig. 1A).


Fig. 1. Translocation of p21rhoA correlated with Ca2+ sensitization of force by PE, AlF4-, and GTPgamma S. alpha -Toxin-permeabilized rabbit portal vein strips were incubated with pCa 6.5 solution (control) or were stimulated with PE (100 µM) plus GTP (10 µM) for 5 min, with AlF4- (10 mM NaF + 30 µM AlCl3) for 20 min, or with various concentrations of GTPgamma S. Stimulated and control tissues were homogenized in ice-cold homogenization buffer and fractionated by centrifugation at 100,000 × g, and p21rhoA in the particulate fraction was quantitated on Western blots (see "Materials and Methods"). Force is normalized to the maximal Ca2+-induced contraction (100%). In A, the solid line represents an exponential fit to the data with the correlation coefficient r = 0.91 (p < 0.05, n = 4~23). bullet , control; black-square, 0.3 µM GTPgamma S; open circle , PE + GTP; square , 1 µM GTPgamma S; ×, AlF4-; black-triangle, 10 µM GTPgamma S; triangle , 50 µM GTPgamma S. In B is shown the dose-dependent p21rhoA translocation induced by GTPgamma S (0.3~50 µM). A linear fit of the data gives the correlation coefficient r = 0.9995 (p < 0.001).
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GTPgamma S translocated p21rhoA from the cytosol to the particulate fraction in a concentration-dependent manner (Fig. 1B; r = 0.9995, p < 0.001) from 10 ± 1.6% (n = 23) to 21 ± 4.2% (n = 8; 0.3 µM), to 31 ± 3.7% (n = 6; 1 µM), to 50 ± 6.9% (n = 2; 10 µM), and 62 ± 9.5% (n = 4; 50 µM). The translocation of p21rhoA induced by GTPgamma S was accompanied by Ca2+ sensitization of force from 13% ± 1.1% (n = 18; pCa 6.5) to 33 ± 4.7% (n = 6; 0.3 µM), to 49 ± 2.8% (n = 10; 1 µM), to 62 ± 2.1% (n = 8; 10 µM), and to 64 ± 1.4% (n = 8; 50 µM), respectively.

If PE-induced activation and translocation of p21rhoA mediates Ca2+ sensitization in portal vein smooth muscle, then ADP-ribosylation of p21rhoA would be expected to inhibit this effect (see the Introduction). ADP-ribosylation of p21rhoA with C3 (see "Materials and Methods") (19, 20) significantly inhibited the Ca2+ sensitization of force in beta -escin-permeabilized rabbit portal vein by PE plus GTP from 38 ± 3.1% (n = 3) to 7 ± 0.7% (n = 3, p < 0.01) and by GTPgamma S (50 µM) from 52 ± 2.8% (n = 3) to 33 ± 2.0% (n = 3, p < 0.05).

Several agonists acting on receptors coupled to heterotrimeric G-protein can both activate the phosphatidylinositol cascade and cause Ca2+ sensitization, as does AlF4-, an agent previously thought to activate heterotrimeric, but not monomeric, G-proteins (21). However, recently, we (11) and others (9) observed that ADP-ribosylation of endogenous p21rhoA also inhibited Ca2+ sensitization of force by AlF4-. Therefore, we determined whether AlF4- also translocated p21rhoA in portal vein. AlF4- (10 mM NaF + 30 µM AlCl3, 20 min) increased force from 13 ± 1.1% (n = 18) to 53 ± 3.1% (n = 4) of the maximal Ca2+-induced contraction and particulate p21rhoA from the control value of 10 ± 1.6% (n = 23) to 26 ± 8.4% (n = 4, p < 0.01) (Fig. 1A).

As shown in Fig. 1A, the translocation of p21rhoA, up to 50%, was quantitatively correlated (r = 0.91, p < 0.05) with Ca2+ sensitization of force induced by agonist (PE plus GTP), AlF4-, and various concentrations of GTPgamma S. Higher concentrations (>= 10 µM) of GTPgamma S caused further translocation of p21rhoA without further increase in force, indicating a "ceiling effect."

To ascertain whether the observed translocation is alpha -adrenergic receptor-specific, we also determined the effect of a muscarinic agonist on the localization of p21rhoA in permeabilized ileum smooth muscles. Surprisingly, a high percentage (61 ± 6.8%, n = 14) of p21rhoA was located in the particulate fraction of unstimulated ileum. This high basal level of particulate p21rhoA was not due to Ca2+ (submaximal, pCa 6.5) because 59 ± 1.4% (n = 2) of p21rhoA was particulate even at cytoplasmic [Ca2+] < pCa 8 (no free Ca2+ added, 10 mM EGTA present). In contrast, inclusion of the muscarinic antagonist atropine (10 µM) during and following dissection decreased particulate p21rhoA to 28 ± 11.6% (n = 6, p < 0.05). This significant decrease in particulate p21rhoA by a highly specific muscarinic antagonist indicates that acetylcholine released from nerve endings in the richly innervated ileum causes translocation of p21rhoA to the particulate fraction and that such translocation is not limited to the action of alpha -adrenergic agonists.

Selective Translocation of p21rhoA, but Not Rac1 and Cdc42, by GTPgamma S

To determine the specificity of translocation of p21rhoA by GTPgamma S, we also determined the distribution of two other Rho family proteins, Rac1 and Cdc42, under conditions identical to those used for determining the partitioning of p21rhoA. GTPgamma S (50 µM, 20 min) (Fig. 2) had no significant effect on the amount of either Rac1 or Cdc42 present in the particulate fraction: 46 ± 2.9% (n = 5) of Rac1 and 14 ± 4.1% (n = 5) of Cdc42 in pCa 6.5 solution and 47 ± 4.2% (n = 6, p > 0.005) of Rac1 and 19 ± 4.8% (n = 6, p > 0.05) of Cdc42 after stimulation with GTPgamma S. Unlike p21rhoA and Cdc42, which were not detected in the detergent-insoluble particulate fraction, 23 ± 7.0% (n = 5) of Rac1 was in the detergent-insoluble particulate fraction, and this fraction was also not changed by GTPgamma S (25 ± 5.7%, n = 6, p > 0.05).


Fig. 2. Selective translocation of p21rhoA, but not Rac1 and Cdc42, induced by GTPgamma S (50 µM, 20 min). Results are representative of five to six experiments showing that p21rhoA, but not Rac1 and Cdc42, is translocated from the cytosol (C) to the particulate (P) fraction; note that only Rac1 is present in the detergent-insoluble (DI) particulate fraction.
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Relaxation of Phenylephrine-induced Ca2+ Sensitization Is Unaccompanied by the Return of p21rhoA from the Particulate to the Cytosolic Fraction

To determine whether the return of p21rhoA from the particulate to the cytosolic fraction is required for the reversal of Ca2+ sensitization, the distribution of p21rhoA after "washout" of the agonist was determined. After increasing the steady-state pCa 6.5-induced contraction by PE plus GTP from 7 ± 2.2% (n = 3) to 32 ± 1.2% (n = 3, p < 0.001), the muscles were transferred into relaxing solution (no added Ca2+, PE, or GTP and containing 1 mM EGTA) and exchanged three times for a total of 25 min. At this time, the pCa 6.5-induced contraction was not significantly different from before exposure to PE plus GTP (9 ± 0.6%, n = 3), indicating that the muscles were no longer Ca2+-sensitized, but 21 ± 4.6% (n = 9) of p21rhoA still remained in the particulate fraction; this was not significantly different from that found in the presence of PE (18 ± 5.5%, n = 9, 5 min, p > 0.05). Even when strips were washed in 10 mM EGTA-containing solution for 60 min, the translocated p21rhoA remained in the particulate fraction (data not shown).

Time Courses of GTPgamma S-induced Contraction and Translocation of p21rhoA and Galpha q/11

The time courses of GTPgamma S-induced potentiation of force and translocation of p21rhoA were determined to evaluate whether they were kinetically consistent with the potential role of p21rhoA as a mediator of agonist-induced Ca2+ sensitization. As shown in Fig. 3 (A and B), within 1 min following addition of GTPgamma S to permeabilized portal vein smooth muscle at pCa 6.5, force reached 21 ± 4.2% (n = 10) of the maximal GTPgamma S-induced contraction; this was accompanied by translocation of p21rhoA to the particulate fraction, increasing from the control value of 10 ± 1.6% (n = 23) to 32 ± 9.7% (n = 6, p < 0.0001). Thus, within the time resolution of this study, the kinetics of translocation of p21rhoA were consistent with its role in GTPgamma S-induced Ca2+ sensitization of force. However, the later time course of GTPgamma S-induced p21rhoA translocation was slower than that of force development: contraction peaked at 5 min, at which time ~51 ± 4% (n = 6) of p21rhoA was in the particulate fraction, whereas p21rhoA continued to translocate, reaching its peak of 62 ± 9.5% (n = 4) at 20 min, consistent with the ceiling effect in the translocation-force relationship (Fig. 1A).


Fig. 3. Time course of translocation of p21rhoA and Galpha q/11 and of Ca2+ sensitization of force induced by GTPgamma S. A, representative Western blots showing that translocation of p21rhoA from the cytosol (C) to the particulate fraction (P) is already detectable at 1 min, the earliest time point checked. Note also that the GTPgamma S (50 µM)-induced translocation of Galpha q/11 to the cytosol is transient, whereas translocation of p21rhoA to the membrane is not reversed during the 1-h period of observation. In control strips incubated in pCa 6.5 solution, both p21rhoA and Galpha q/11 localization remained constant, and Rho-GDI remained in the cytosol at all time points checked. B, summary of results shown in A (n = 3-10 for each point). Force is normalized to maximal contraction (100%) induced by GTPgamma S (n = 12 for each point).
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We also determined the time course of translocation of Galpha q/11, the heterotrimeric G-protein implicated in the activation of phospholipase C, a major contributor to pharmacomechanical coupling in smooth muscle (reviewed in Ref. 4). Under control conditions (pCa 6.5), 86 ± 1.7% (n = 25) of the total Galpha q/11 was in the particulate fraction, and this was reduced by GTPgamma S to 60 ± 12% (n = 6, p < 0.01) at 1 min and 70 ± 8.8% (n = 6, p < 0.05) at 5 min (Fig. 3, A and B). In contrast to p21rhoA, the translocation of Galpha q/11 was transient: by 60 min, the previously translocated protein had returned to the particulate fraction (Fig. 3, A and B).

Particulate p21rhoA Is Hydrophobic

The hydrophobic domain of cytosolic, geranylgeranylated p21rhoA is masked by bound Rho-GDI, and activated p21rhoA is thought to bind to the cell membrane through the unmasked hydrophobic geranylgeranyl group exposed by the release of Rho-GDI (22). Because the particulate fraction obtained through centrifugation may contain both hydrophobic (membrane) and nonhydrophobic (e.g. cytoskeletal) components, we determined by phase separation with Triton X-114 whether the p21rhoA translocated to the particulate fraction by GTPgamma S in vivo was hydrophobic, as indicated by partitioning into Triton X-114. Indeed, GTPgamma S (50 µM, 30 min) increased the fraction of p21rhoA partitioned into the detergent phase when whole homogenates were treated with Triton X-114. p21rhoA in the Triton X-114 phase increased from the control value of 22 ± 6.6% (n = 11) to 81 ± 1.8% (n = 6, p < 0.0001), indicating that most of the particulate p21rhoA was associated with hydrophobic (presumably membrane) components.

To further evaluate whether cytosolic p21rhoA (complexed with Rho-GDI) is hydrophilic, whereas particulate p21rhoA is hydrophobic, the whole homogenate was first separated into cytosolic and particulate fractions, which were subsequently phase-separated with Triton X-114 (see "Materials and Methods"). As shown in Fig. 4, only 5 ± 1.8% (n = 9) of the cytosolic p21rhoA partitioned into the Triton X-114 phase. In contrast, most of the particulate p21rhoA partitioned into the Triton X-114 phase, with only 7 ± 2.5% (n = 8) partitioning into the aqueous phase. Again, there was a dramatic increase from 11 ± 8.6% (n = 3) to 63 ± 13.5% (n = 3, p < 0.05) in the amount of p21rhoA in the Triton X-114-treated particulate fraction of GTPgamma S (50 µM, 20 min)-stimulated muscles. The small quantities of cytosolic p21rhoA partitioning into the Triton X-114 phase and particulate p21rhoA partitioning into the aqueous phase may result from "carryover" during the experimental procedure.


Fig. 4. Triton X-114 partitioning of p21rhoA in the cytosolic and particulate fractions of control and GTPgamma S-stimulated rabbit portal vein. GTPgamma S (50 µM, 20 min)-treated and control (in pCa 6.5 solution) portal vein strips were homogenized and fractionated by centrifugation (see "Materials and Methods"), after which the cytosolic and particulate fractions were further phase-separated with Triton X-114 and blotted with anti-p21rhoA antibody. C, cytosol; P, particulate; Aq, aqueous phase; Tx, Triton X-114 phase.
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Translocated p21rhoA Is Not a Good Substrate for C3-catalyzed ADP-ribosylation

The cytosolic and particulate fractions of control and GTPgamma S-stimulated tissues were incubated with C3 and [32P]NAD (see "Materials and Methods") to determine if the translocated p21rhoA is a good substrate for C3-catalyzed ADP-ribosylation. In unstimulated strips, the largely cytosolic p21rhoA (Figs. 1, 2, and 5) was only minimally ADP-ribosylated; a very faint band was detected in the autoradiograph (Fig. 5, upper left panel), whereas the very small amount of particulate p21rhoA was highly ADP-ribosylated (lower left panel). This is consistent with previous results showing that cytosolic p21rhoA is complexed with Rho-GDI and that the complex is a poor substrate for C3-catalyzed ADP-ribosylation (23, 24), but becomes a better substrate after its dissociation from Rho-GDI. In contrast, the large amount of particulate p21rhoA in GTPgamma S-stimulated tissues (Figs. 1, 2, 3 and 5) was only minimally ADP-ribosylated (Fig. 5, right panels). This lower level of ADP-ribosylation of particulate p21rhoA in GTPgamma S-stimulated strips was not due to the loss of a membrane component to the cytosol because ADP-ribosylation was significantly less even in total tissue homogenates containing both cytosolic and particulate fractions of GTPgamma S-treated tissue compared with controls (data not shown), and cytosolic p21rhoA in GTPgamma S-stimulated tissue was still not a good substrate for ADP-ribosylation (Fig. 5, right panels). This was also not the result of reassociation with Rho-GDI because the latter was not detectable in any of the particulate fractions. These results suggest that membrane-associated p21rhoA exists in at least two states: a resting state that is a good substrate and an activated (and/or inactivated; see "Discussion") state that is a poor substrate for C3-catalyzed ADP-ribosylation.


Fig. 5. Reduced availability of particulate p21rhoA for ADP-ribosylation in GTPgamma S-stimulated, alpha -toxin-permeabilized rabbit portal vein. GTPgamma S (50 µM, 20 min)-treated and control (in pCa 6.5 solution) portal vein strips were homogenized and fractionated by centrifugation (see "Materials and Methods") with the volumes and concentration of the detergent in the two fractions adjusted to the same level. Samples were incubated with [32P]NAD (50 µCi/ml), C3 (1 µg/ml, 30 min, 30 °C), and other reagents (see "Materials and Methods") to ADP-ribosylate p21rhoA. The ADP-ribosylation level was determined by autoradiography (AutoRad). The particulate p21rhoA in the control strips was a good substrate for C3-catalyzed ADP-ribosylation, whereas the particulate p21rhoA in the GTPgamma S-stimulated strips was a poor substrate, although markedly increased (see Western blots). Results are representative of three independent experiments. C, cytosol; P, particulate.
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Effect of Ca2+ and of the Phosphatase Inhibitor Tautomycin on the Localization of p21rhoA

To determine whether Ca2+ alone can induce translocation of p21rhoA, alpha -toxin-permeabilized rabbit portal vein strips were incubated in Ca2+-free solution (no Ca2+ added and containing 10 mM EGTA) for 15 min, homogenized, and fractionated. The particulate fraction under this Ca2+-free condition contained 9 ± 3.0% (n = 6) of the total p21rhoA, which is not significantly different from strips incubated in pCa 6.5 solution: 10 ± 1.6% (n = 23, p > 0.05). However, increasing Ca2+ to very high levels (pCa 4.5, 15 min) increased the p21rhoA content of the particulate fraction to 31 ± 5.8% (n = 10, p < 0.01). This translocation of p21rhoA induced by pCa 4.5 was not due to the release of norepinephrine from nerve endings because inclusion of the alpha -adrenergic blocker prazosin (10 µM) in the solutions during permeabilization and thereafter had no effect on the pCa 4.5-induced translocation of p21rhoA to the particulate fraction (39 ± 6.9%, n = 8, p > 0.05). To exclude the possibility that the translocation of p21rhoA by pCa 4.5 was due to trapping of cytosolic p21rhoA in the cytoskeletal components of the contracted tissue, cytosolic and particulate fractions of pCa 4.5-stimulated tissues were phase-separated by Triton X-114. Similar to the GTPgamma S-stimulated tissues, 89 ± 8.6% (n = 3) of p21rhoA in the particulate fraction (29.0 ± 13.9% (n = 3) of the total p21rhoA) was partitioned into the Triton X-114 phase, suggesting that high [Ca2+] alone can translocate p21rhoA to the membrane.

To determine whether p21rhoA translocated by high [Ca2+] returns to the cytosol after removal of Ca2+, portal vein strips stimulated with pCa 4.5 for 15 min were washed five times in Ca2+-free solution (no Ca2+ added and containing 10 mM EGTA) for a total of 60 min, and the distribution of p21rhoA was determined. Even after this extensive wash, the same amount (38 ± 2%, n = 3) of translocated p21rhoA remained in the particulate fraction.

The Ca2+ dependence of GTPgamma S-induced translocation of p21rhoA was also determined by adding GTPgamma S to alpha -toxin-permeabilized smooth muscle in the presence and absence of Ca2+. GTPgamma S (50 µM, 60 min) caused similar p21rhoA translocation in Ca2+-free, 10 mM EGTA-containing solution as in pCa 6.5 solution: p21rhoA in the particulate fraction was 65 ± 6.9% (n = 6) versus 62 ± 9.5% (n = 4, p > 0.05), respectively.

Tautomycin, a potent inhibitor of protein phosphatases 1 and 2A, (25) causes substantial MLC20 phosphorylation and smooth muscle contraction even in the absence of Ca2+ (26) by inhibiting the catalytic subunit of SMPP-1M. We also wished to localize p21rhoA in tautomycin-stimulated tissues to determine whether phosphorylation of a protein that can be dephosphorylated by phosphatase 1 or 2A is involved in regulating translocation of p21rhoA. No significant translocation of p21rhoA was detected in tautomycin-stimulated tissue at pCa 6.5 (data not shown), although it caused 86 ± 6.3% (n = 3) of the maximal Ca2+-induced contraction.


DISCUSSION

The relationships between the extent and time course of translocation of p21rhoA to the particulate fraction (Figs. 1 and 3) and enhancement of force at constant [Ca2+] are consistent with a causal role of p21rhoA recruitment to the membrane in Ca2+ sensitization. This conclusion is also supported by the hydrophobicity of particulate p21rhoA (partitioning into Triton X-114; this study), the abolition of the Ca2+-sensitizing effect of recombinant p21rhoA by extensive permeabilization of smooth muscle with detergent, and the inactivity of nonprenylated p21rhoA (11). Similarly, GTPgamma S-induced activation of NADPH oxidase in neutrophils involves the dissociation of p21rho from Rho-GDI and its translocation to the membrane by a mechanism that requires a heat- and trypsin-labile membrane component (27). The translocation was limited to p21rhoA in our study: the localization of two other Rho family proteins, Rac1 and Cdc42, was not affected by GTPgamma S under conditions causing extensive translocation of p21rhoA.

The alpha -adrenergic agonist phenylephrine, GTPgamma S, and AlF4- induced translocation of p21rhoA, whereas the muscarinic antagonist atropine inhibited the translocating effect of endogenous acetylcholine. The significant reduction by atropine of the large amount of particulate p21rhoA in "unstimulated" ileum smooth muscle and the translocation of p21rhoA by high [Ca2+] indicate that preparatory conditions alone can affect its localization. Thus, caution is required in interpreting results of fractionation obtained in the presence of high [Ca2+] and/or locally stored and released transmitters.

Because agonists acting on heptameric serpentine receptors coupled to trimeric G-proteins induced Ca2+ sensitization and because AlF4- was not reported to interact with Ras family proteins (21), we had previously thought that the Ca2+ sensitization by AlF4- was mediated by a trimeric G-protein (4). Subsequently, we (11) and others (9) found that ADP-ribosylation of p21rhoA inhibited AlF4--induced Ca2+ sensitization, and we now show (Fig. 1) that AlF4-, like agonists and GTPgamma S, also translocates p21rhoA to the membrane. Furthermore, it has now been shown that although AlF4- cannot interact with another monomeric G-protein (elongation factor G) in solution, such an interaction can occur when elongation factor G is associated with ribosomes (28). On the other hand, other reports show p21rhoA to be a downstream effector of Galpha 12- and Galpha 13-induced stress fiber formation (29) and of Galpha q-initiated hypertrophic signaling to cardiomyocytes (30). Neither these Galpha subunits nor agonist-induced Ca2+ sensitization (11) is sensitive to pertussis toxin, suggesting that they could act as upstream initiators (activated by AlF4-) of a p21rhoA-mediated cascade. At present, we consider it equally likely that a complex of p21rhoA with a cytosolic protein can directly interact with AlF4-.

A larger fraction of cytoplasmic p21rhoA (>60% of the total) could be translocated by GTPgamma S (50 µM) to the membrane than required for maximal Ca2+ sensitization of force. This ceiling effect presumably reflects the fact that the extent (~50%) (2) of inhibition of SMPP-1M attainable by G-protein-coupled mechanisms is achieved by activating only ~50% of endogenous p21rhoA (Fig. 1A). The p21rhoA translocated to smooth muscle membranes did not return to the cytosol during 60 min of incubation without the agonist, although Ca2+ sensitization of force by this time was already reversed. This is consistent with the finding that activated p21rhoA associated with the particulate fraction is transformed, with time, into an inactivated membrane-bound form, and/or its downstream effector(s) is down-regulated (31). According to a recent report, rho translocation in cultured fibroblasts is rapidly reversible (32). The difference between these and our findings may be tissue-dependent or indicate an inhibitory effect of permeabilization (this study) on the reversibility of translocation.

The large cytosolic fraction of p21rhoA present in unstimulated smooth muscle was not ADP-ribosylated, whereas ADP-ribosylation (Fig. 5) of the small amount of membrane-associated p21rhoA was extensive and sufficient to inhibit the Ca2+-sensitizing effect of the alpha -adrenergic agonist PE and of GTPgamma S. In other types of smooth muscle, ADP-ribosylation of endogenous p21rhoA only slowed GTPgamma S-induced Ca2+ sensitization without reducing its amplitude (rabbit mesenteric artery). It also inhibited Ca2+ sensitization by carbachol (ileum smooth muscle) (9, 11) and the increase in MLC20 phosphorylation induced by GTPgamma S in cultured smooth muscle cells (33).

The lack of correlation between p21rhoA detected by Western blotting and ADP-ribosylation, respectively, in several tissues (Ref. 24 and this study) raises the possibility that the variable and often incomplete (agonist- and tissue-dependent) inhibition of Ca2+ sensitization by in situ ADP-ribosylation may reflect slow recruitment of p21rhoA from a cytosolic pool protected from ADP-ribosylation by Rho-GDI. Alternatively, other p21rhoA-independent pathways of Ca2+ sensitization may account for incomplete inhibition of Ca2+ sensitization following ADP-ribosylation of endogenous p21rhoA.

The variable availability of particulate p21rhoA for ADP-ribosylation by C3 (Fig. 5) suggests that membrane-associated p21rhoA can exist in more than one (conformational) state. One state is membrane-associated under resting conditions, available for ADP-ribosylation and presumably ready, "with the trigger cocked," to be activated by an agonist, GTPgamma S, or AlF4-. This would account for the more rapid rate of Ca2+ sensitization achieved by agonists or GTPgamma S than by exogenous p21rhoA (11). Another state (or states) is much less available for ADP-ribosylation, as indicated by the large quantity of p21rhoA that was translocated by GTPgamma S to the membrane, but not ADP-ribosylated (Fig. 5). This may represent a combination of transitional states being activated through membrane association, as well as a third membrane-bound but inactivated state of p21rhoA (31). However, we cannot completely rule out the possibility that a very small fraction of activated p21rhoA that was not translocated is involved in Ca2+ sensitization.

Tautomycin, a potent inhibitor of protein phosphatases 1 and 2, did not translocate p21rhoA, although it had a large "Ca2+-sensitizing" effect on contraction (see also Ref. 26). Therefore, it is unlikely that phosphorylation of a site susceptible to dephosphorylation by these phosphatases plays a role in the translocation of p21rhoA. The possibility of a role of tyrosine phosphorylation in this process is currently under study.

Ca2+-sensitizing mechanisms, whether mediated by protein kinase C (34, 35, 41) or other effectors, appear to converge to inhibit dephosphorylation of a cytosolic SMPP-1M substrate, MLC20 (2). Smooth muscle myosin light chain phosphatase is not known to be associated with the plasma membrane. Therefore, p21rhoA-coupled Ca2+ sensitization is thought to involve the participation of additional downstream messengers/mechanisms, such as inhibitory phosphorylation of the regulatory subunit of SMPP-1M by a p21rhoA-regulated and/or SMPP-1M-associated kinase (36-40) or an atypical protein kinase C (or a related kinase) (41), and/or activation of a phosphatase inhibitor (1). SMPP-1M can also be inhibited directly by arachidonic acid (42). It is likely that multiple mechanisms, including some mediated by p21rhoA, are involved in Ca2+ sensitization.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant P01 HL48807 (to A. V. S and A. P. S.).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.
par    To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, P. O. Box 10011, Charlottesville, VA 22906-0011.
1   The abbreviations used are: MLC, myosin light chain; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); PE, phenylephrine; GDI, guanine nucleotide dissociation inhibitor.

ACKNOWLEDGEMENTS

We thank Barbara Nordin for preparation of the manuscript and Jama Coartney for preparation of the figures.


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