INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE RHO FAMILY OF SMALL GTPases are well-known intracellular signaling proteins that act as molecular switches to control actin cytoskeleton organization in many cell types including smooth muscle (5, 10, 30, 34). Recent evidence suggests that the RhoA-dependent signaling pathway can control many of the functions of vascular smooth muscle cells (VSMCs) such as contraction, migration, and proliferation (13, 25). In VSMCs, the contracting effect of RhoA results from the activation of one of its downstream targets, Rho-dependent kinase (ROK-), which phosphorylates the regulatory subunit of myosin light-chain (MLC) phosphatase [myosin-bound subunit (MBS)], leading to the inhibition of its function by reductions in the phosphatase activity (20, 22) and thus allowing an increase in the level of phosphorylated MLC and contraction at a constant intracellular Ca²⁺ concentration (33). This phenomenon is defined as Ca²⁺ sensitization (23).

Activation of Rho GTPases is regulated by several mechanisms, including the activation of heterotrimeric G protein-coupled receptors (9). After agonist stimulation, Rho is converted from the inactive, GDP-bound form to the active, GTP-bound form in response to stimuli such as serum, lysophosphatidic acid, phenylephrine, and thrombin. Activation of RhoA by these agonists requires translocation of inactive cytosolic RhoA to the membrane fractions. Thus appearance of RhoA in the membrane fraction is indicative of Rho activation (12). Rho activation and/or membrane localization is regulated by posttranslational modification by geranylgeranylation and phosphorylation of Rho (19, 26, 27, 29). Phosphorylation by cAMP-dependent protein kinase as well as by cGMP-dependent protein kinase (cGK1) inhibits Rho signaling by interfering with the membrane anchoring of Rho (6a, 19, 29).

Excessive contractility of VSMCs and abnormal vascular tone due to defective vasorelaxation are the major abnormalities observed in patients with hypertension (16, 35). The Rho signaling pathway has been implicated in the pathogenesis of hypertension as well as of diabetes (28, 37). Although activation of Rho signaling by various stimuli has been described in detail, the agonists and the mechanism that counterregulate Rho signaling in vivo have not been clearly defined. It is important to determine how Rho signaling can be inactivated in vivo to prevent MBS phosphorylation in order to develop therapeutic strategies to overcome abnormal smooth muscle contractility, which is one of the major causes of disease states such as hypertension and diabetes.

Recent studies from this laboratory (1, 28) have shown that insulin rapidly stimulates myosin-bound phosphatase (MBP) activity to cause myosin light-chain dephosphorylation and relaxation of VSMCs isolated from control Wistar-Kyoto rats. MBP activation by insulin was accompanied by a decrease in Rho kinase activity. These effects of insulin were severely impaired in VSMCs isolated from diabetic Goto-Kakizaki rats (28).

Because MBS is one of the downstream targets of RhoA, in this study, we have examined the mechanism by which insulin may decrease MBS site-specific phosphorylation via Rho and the role of the nitric oxide (NO)/cGMP pathway in insulin inactivation of Rho.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell culture reagents, fetal bovine serum, and lipofectamine plus reagent were purchased from Life Technologies (Grand Island, NY). [-³²P]ATP (specific activity of 3,000 Ci/mmol), [³²P]orthophosphoric acid, and [³H]geranylgeranyl pyrophosphate were purchased from DuPont-New England Nuclear (Boston, MA). 8-bromo-cGMP, N^G-monomethyl-L-arginine (L-NMMA), wortmannin, geranylgeranyl pyrophosphate, and (R)-p-8-(4-chlorophenylthio)cGMP (Rp-CPT-cGMP) were purchased from Biomol Research (Plymouth Meeting, PA). Electrophoresis and protein assay reagents were from Bio-Rad (Richmond, CA). Okadaic acid was from Moana Bioproducts (Honolulu, HI). Porcine insulin was a kind gift from Eli Lilly (Indianapolis, IN). Type 1 collagenase was from Worthington Biochemical (Freehold, NJ). SDS-PAGE and Western blot reagents were from Bio-Rad (Hercules, CA). Antibody against the 160-kDa ROK- was purchased from Transduction Laboratories (San Diego, CA). Antibodies against the plekstrin homology domain of insulin receptor substrate-1 (IRS-1) and p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) were obtained from Upstate Biotechnology (Lake Placid, NY). Monoclonal antibody against RhoA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse IgG-agarose, protein A-Sepharose CL-4B, protease inhibitors, calmodulin, sodium orthovanadate, thrombin, phenylephrine, and all other reagents were purchased from Sigma Chemical (St. Louis, MO).

Culture of VSMCs and treatment with insulin. VSMCs in primary culture were obtained by enzymatic digestion of the aortic media of male Wistar-Kyoto rats (200-220 g body wt), as described in recent publications from our laboratory (1-4, 28). Unless otherwise indicated, primary cultures of VSMCs were maintained in -MEM containing 10% FBS and 1% antibiotic-antimycotic mixture. Subcultures of VSMCs at passages 2-5 were used in all experiments. All experiments on Rho translocation, phosphorylation, and Rho kinase activity were performed on highly confluent cells (7-9 days in culture) at identical passages. Before each experiment, cells were serum starved for 24 h in serum-free -MEM containing 5.5 mM glucose and 1% antibiotics. The next day, cells were exposed to insulin (0-100 nM) for 0-30 min. In some experiments, VSMCs were pretreated with various inhibitors for 30 min followed by exposure to insulin, as detailed in Figs. 1-7.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Insulin inhibits Rho translocation. Serum-starved vascular smooth muscle cells (VSMCs) were exposed to thrombin (1 U/ml) for the indicated times or pretreated with insulin (100 nM) for 10 min followed by stimulation with thrombin for 5 and 10 min (InsThr). Equal amounts of membrane proteins were subjected to SDS-PAGE followed by Western blot analysis with anti-RhoA antibody. A: representative autoradiogram. Similar results were obtained in 4 different experiments. B: data from 4 different experiments were quantitated by densitometric scanning. The optical densities obtained in the basal state were assigned a value of 1 arbitrary densitometric unit (ADU), and the effect of thrombin and insulin was expressed relative to basal RhoA levels. *P < 0.05 vs. thrombin.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Nitric oxide (NO) and cGMP inhibitors abolish the inhibitory effect of insulin on RhoA translocation. VSMCs were pretreated with NG-monomethyl-L-arginine (L-NMMA; 1 mM), (R)-p-8-(4-chlorophenylthio)cGMP (RpcGMP; 100 µM), and 8-bromo-cGMP (8-Br-cGMP; 1 mM) for 30 min followed by insulin (100 nM) for 10 min and then stimulated with thrombin (1 U/ml) for 10 min. Equal amounts of membrane proteins were subjected to immunoblot analyses, as detailed in Fig. 1. A: representative autoradiogram. B: quantitation of membrane-associated RhoA content by densitometry. Results from different experiments are expressed relative to control, which was assigned a value of 1.*P < 0.05 vs. control, **P < 0.05 vs. thrombin, ***P < 0.05 vs. insulin + thrombin.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   NO and cGMP inhibitors abolish insulin-mediated inactivation of Rho kinase activity. VSMCs were exposed to inhibitors, insulin, and thrombin as detailed in Fig. 2. Equal amounts of cell lysate proteins (100 µg) were immunoprecipitated with anti-Rho-dependent kinase (ROK)- antibody. Rho kinase activity in the immunoprecipitates was assayed using myosin-bound subunit (MBS) as a substrate along with [-32P]ATP. Results are means ± SE of 5 separate experiments. *P < 0.05 vs. control, **P < 0.05 vs. control/insulin, ***P < 0.05 vs. thrombin, ****P < 0.05 vs. insulin pretreatment followed by thrombin. DPM, disintegration/minute.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Inhibitors of NO synthase (NOS) and cGMP signaling block the insulin-mediated decrease in MBSThr695 site-specific phosphorylation. VSMCs were exposed to inhibitors, as detailed in Fig. 2. Equal amounts of proteins from TCA lysates were subjected to SDS-PAGE followed by Western blot analysis with anti-Thr-695-specific MBS antibody. A: representative autoradiogram. B: quantitation by densitometric analyses. Data from 4 different experiments were quantitated, and results are expressed as means ± SE relative to control, which was assigned a value of 1 ADU. *P < 0.05 vs. control, **P < 0.05 vs. thrombin, *** P < 0.05 vs. insulin pretreatment  thrombin.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Expression of dominant-negative RhoA decreases basal and thrombin-induced MBSThr695 phosphorylation. Serum-starved VSMCs were treated with the inhibitors as detailed in Figs. 2-4. Equal amounts of TCA protein lysates in duplicate were subjected to Western blot analyses with anti-phospho MBSThr695 antibody and anti-MBS antibody. A: representative autoradiogram. B: data from 2 different experiments were quantitated, and results are expressed as mean relative to control, which was assigned a value of 1 ADU.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Expression of dominant-negative RhoA stimulates myosin-bound phosphatase (MBP) activation. Serum-starved VSMCs were exposed to insulin for 10 min before and after treatment with thrombin for 10 min. MBP activity was assayed in myosin-enriched pellets using 32P-labeled myosin light chains (MLC) as substrate. Results are means ± SE of 3 different experiments performed in duplicate. *P < 0.05 vs. wild-type basal, **P < 0.05 vs. thrombin, ***P < 0.05 vs. wild-type basal. Similar results were obtained with phosphorylase-a as a substrate.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Schematic representation of the proposed pathways for insulin activation of MBP. Insulin-stimulated receptor tyrosine kinase phosphorylates insulin receptor substrate (IRS)-1 and IRS-2, promoting association and activation of phosphatidylinositol 3-kinase (PI3-kinase) and the NO-cGMP pathway, which inactivates Rho by phosphorylation and inhibits Rho kinase, thereby preventing MBP inactivation due to Rho kinase-mediated MBS site-specific phosphorylation at Thr-695. Hypertension (HT) and diabetes (DM) are accompanied by upregulation of Rho kinase due to impaired activation of PI3-kinase and NO/cGMP signaling pathways, resulting in impaired MBP activation due to excessive MBS phosphorylation by the activated Rho/Rho kinase.

Immunoprecipitation and in vitro assay of Rho kinase activity in the immunocomplexes. Rho kinase was immunoprecipitated by incubating equal amounts of precleared lysate proteins (100 µg) with anti-ROK- antibody (6 µg/tube) at 4°C with constant shaking. Kinase activity in the immunoprecipitates was assayed using the recombinant MBS as a substrate (1, 28). After incubation at 30°C for 10 min (enzyme concentration was adjusted to ensure first-order kinetics), 25-µl aliquots of the reaction mixture were spotted on phosphocellulose paper; this was followed by extensive washing of the paper and ³²P incorporation, determined by liquid scintillation spectrometry.

Analyses of agonist-induced Rho translocation to the membrane fraction. Cytosolic and membrane fractions were prepared by differential centrifugation according to previously published protocols (1, 28). Equal amounts of membrane proteins were subjected to SDS-PAGE, transferred to polyvinyl difluoride membrane, and probed with mouse anti-RhoA antibody; this was followed by incubation with horseradish peroxidase-labeled secondary antibody and subsequent detection with enhanced chemiluminescence.

Measurement of MBP activity. Phosphatase activity in myosin-enriched fractions was assayed using ³²P-labeled phosphorylase-a as well as ³²P-labeled MLCs as substrates (1, 28). ³²P-labeled phosphorylase-a was prepared by incubating [-³²P]ATP with purified phosphorylase kinase and phosphorylase-b (1, 28). ³²P-labeled MLC was prepared according to the published protocol (1) by incubating MLC (0.8 mg/ml) with purified MLC kinase (50 µg/ml), 0.1 mg/ml calmodulin, and 50 µM [-³²P]ATP.

Retrovirus-mediated expression of dominant-negative RhoA in VSMCs. Plasmid carrying dominant-negative RhoA (Y35A and T37A) was kindly provided by Dr. Van Aelst (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY). The construct was subcloned into BamHI/SalI and EcoRI sites of the retroviral vector, pBabe, carrying a puromycin resistance marker. The resulting constructs were verified by restriction enzyme analyses as well as by automated DNA sequencing. pBabe-RhoA^ve was introduced into a retroviral packaging cell line [LE, a derivative of Bosc23 (kindly given by Dr. Hannon, Cold Spring Harbor Laboratories)] by transfection using lipofectamine plus reagent according to the manufacturer's instructions. The next day, cells were fed with complete growth medium containing 5 mM sodium butyrate and 1 µM dexamethasone and incubated at 32°C. Culture supernatants containing the retroviral recombinant virus particles were collected 48 h after transfection and used for infection of VSMCs. Briefly, overnight cultures of VSMCs at a density of 2 × 10⁴ cells per well at passage 3 were infected with a cocktail containing filtered retroviral supernatant and polybrene (8 µg/ml) in 2 ml of growth medium. The culture plates were centrifuged at 17,000 rpm in a Beckman table-top centrifuge (Allegra 6) for 1 h at room temperature and incubated overnight at 32°C. At the end of 48 h, VSMCs were trypsinized and plated into five 100-mm dishes containing 2 µg/ml puromycin to generate stably expressing clones. A pool of stable clones expressing dominant-negative Rho was amplified and used for functional assays to examine the impact of inactive RhoA on MBS^Thr695 phosphorylation and MBP activation.

Protein assay. Proteins in the cellular extracts and lysates were quantitated by the bicinchoninic acid (32) or Bradford (6) technique.

Statistics. The results are presented as means ± SE of four to six independent experiments, each performed in triplicate at different times. Unpaired Student's t-test was used to compare the basal and thrombin-treated vs. the insulin-treated preparations. One-way ANOVA was used for multiple comparisons. A P value of <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Insulin inhibits RhoA translocation to membrane fractions. In the initial studies, kinetics of thrombin-induced Rho translocation was examined by Western blot analysis of equal amounts of VSMC membrane proteins isolated from control and agonist-treated VSMCs. As seen in Fig. 1, a considerable amount of RhoA was present in the membrane fraction under basal conditions. Incubation with thrombin (1 U/ml) for 5 min caused a threefold increase in RhoA content in the membrane fraction. The increase was sustained for the 30-min incubation period tested (Fig. 1A, lanes 2-4). Pretreatment with 100 nM insulin for 10 min prevented the thrombin-induced increase in membrane RhoA content (Fig. 1A, lane 5), which is indicative of RhoA inactivation. As a control for membrane proteins, the blots were stripped and probed with the anti-₁-subunit of Na⁺-K⁺-ATPase antibody as well as anti-GLUT-1 antibody. The signal intensities in each lane were similar with these two membrane markers, suggesting that the reductions observed in membrane Rho content in insulin-treated preparations were not due to variations in membrane proteins. The agonist-mediated increase in membrane-associated RhoA was accompanied by a concomitant decrease in cytosolic RhoA. However, this observation was not always consistent, probably because a large fraction of the total Rho protein still remained cytosolic (data not shown), as reported earlier (30).

Inhibitors of the NO/cGMP pathway abolish insulin-mediated RhoA inactivation. It is well known that insulin's vasodilatory effects are mediated via NO (36) and that cGMP inhibits MLC phosphorylation (23). These observations, together with the fact that inhibition of the NO/cGMP signaling blocks insulin-mediated MBP activation in VSMCs (1, 28), suggest that insulin may inhibit VSMC contraction by inactivating Rho signaling via the NO/cGMP pathway. To test this hypothesis, VSMCs were pretreated with 1 mM L-NMMA [a NO synthase (NOS) inhibitor] and 100 µM Rp-CPT-cGMP (a cGMP antagonist) for 30 min, incubated with 100 nM insulin for 10 min, and then exposed to thrombin (1 U/ml) for 10 min. VSMCs were examined for translocation of RhoA in the membrane fraction and Rho kinase activity in the anti-ROK- immunoprecipitates. Both L-NMMA and Rp-CPT-cGMP prevented insulin's inhibitory effect on thrombin-mediated RhoA translocation (Fig. 2, compare lanes 5 and 6 with lane 4). Furthermore, exposure of VSMCs to 8-bromo-cGMP, a cGMP agonist, abolished thrombin-mediated RhoA translocation and decreased membrane-associated RhoA content below basal values (Fig. 2, compare lane 7 vs. lane 3), as seen with insulin treatment (lane 4). The concentrations of inhibitors were selected from a preliminary dose-response study that established that 1 mM L-NMMA and 100 µM Rp-CPT-cGMP were most effective in inhibiting insulin's effect on Rho, Rho kinase, and MBP.

NOS and cGMP inhibitors prevent insulin-mediated inactivation of Rho kinase activity. We next examined the impact of NO/cGMP pathway inhibitors on Rho kinase, the downstream target of Rho. Rho kinase activity was assayed in ROK- immunoprecipitates using recombinant MBS protein as a substrate. Mouse IgG was always used as an internal control instead of anti-ROK- antibody. Rho kinase activity, which was negligible in mouse IgG immunoprecipitates, was subtracted from the values obtained with ROK- antibody. As detailed in recent publications (1, 28), insulin treatment for 10 min caused a 35% decrease in basal Rho kinase activity. Treatment with thrombin caused a 25% increase in Rho kinase activity over the basal values, which was prevented by preincubation with insulin. Pretreatment with L-NMMA and Rp-CPT-cGMP before insulin abolished insulin's inhibitory effect on Rho kinase and restored thrombin's effect on Rho kinase activity (Fig. 3). These inhibitors alone had very little effect on basal Rho kinase activity (data not shown). Furthermore, treatment with 8-bromo-cGMP inhibited the thrombin-mediated increase in Rho kinase activity and decreased the enzymatic activity below basal levels (Fig. 3).

NO/cGMP pathway mediates the insulin-induced decrease in MBS^Thr695 phosphorylation. Recent studies have identified two major inhibitory Rho kinase phosphorylation sites on MBS that appear to profoundly influence MBP enzymatic activity (7, 17). For example, in Swiss 3T3 cells, Rho kinase activation by lysophosphatidic acid was accompanied by an increase in Thr-695 phosphorylation on MBS, and this effect was blocked by a Rho kinase inhibitor, Y-27632 (7). Therefore, we next examined whether the previously observed decrease in MBS phosphorylation in insulin-treated VSMCs (1, 28) was due to a reduction in MBS^Thr695 phosphorylation by using site-specific and phosphospecific antibodies. As shown in Fig. 4, MBS is phosphorylated to a considerable extent at Thr-695 in the basal state. Exposure to insulin for 10 min caused a 60% decrease in basal MBS^Thr695 phosphorylation (Fig. 4, lane 2). Furthermore, insulin treatment completely abolished the thrombin-mediated increase in MBS^Thr695 phosphorylation (Fig. 4, compare lane 4 with lane 3). Pretreatment with L-NMMA and Rp-CPT-cGMP prevented the insulin-mediated decrease in MBS^Thr695 phosphorylation and restored the thrombin-mediated increase in MBS phosphorylation (Fig. 4, compare lanes 5 and 6 with lane 4). In contrast, pretreatment with 8-bromo-cGMP, a cGMP agonist, prevented the thrombin-mediated MBS^Thr695 phosphorylation in a manner comparable to insulin (Fig. 4, lane 7).

Dominant-negative RhoA decreases basal MBS^Thr695 phosphorylation and stimulates MBP activation. The above results clearly indicate that inactivation of RhoA/Rho kinase signaling by insulin via NO/cGMP pathway may in part mediate MBP activation by decreasing site-specific MBS^Thr695 phosphorylation. To further confirm these observations, we examined the phosphorylation status of MBS in VSMCs stably expressing dominant-negative RhoA. As shown in Fig. 5, expression of this mutant caused marked reductions in MBS^Thr695 phosphorylation in the basal state. Furthermore, thrombin treatment caused a very small increase in MBS^Thr695 phosphorylation and the NO/cGMP inhibitors were able to still prevent insulin's effect on MBS phosphorylation (Fig. 5). The observed decrease in MBS^Thr695 in these cells was accompanied by a 30% increase in basal MBP activity compared with wild-type VSMCs transfected with vector alone (Fig. 6). Insulin treatment further increased MBP activity. Furthermore, cells expressing dominant Rho exhibit no inhibition of MBP activity on treatment with thrombin in contrast to cells transfected with vector alone (Fig. 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results presented in this study clearly indicate that insulin inhibits Rho signaling by preventing thrombin-induced RhoA translocation to the membrane fraction via the NO/cGMP signaling pathway. Insulin inactivation of RhoA leads to inhibition of Rho kinase activity, thereby decreasing MBS^Thr695 phosphorylation to cause MBP activation. Thus these studies provide a molecular mechanism by which insulin induces smooth muscle relaxation (see Fig. 7).

Several lines of evidence presented in this study suggest that inactivation of RhoA signaling by insulin in VSMCs is mediated by multiple inputs from the NO/cGMP signaling pathway. First, treatment with L-NMMA (a NOS inhibitor) and Rp-CPT-cGMP (a cGMP antagonist) prevented insulin inhibition of Rho translocation to the membrane fraction as well as Rho kinase inactivation, whereas 8-bromo-cGMP (a cGMP agonist) mimicked insulin's inhibitory effect on thrombin-induced Rho translocation and Rho kinase activation. Second, both insulin and 8-bromo-cGMP inhibited the thrombin-induced increase in MBS^Thr695 phosphorylation, which is effectively blocked by L-NMMA and Rp-CPT-cGMP. Third, expression of a dominant-negative RhoA decreased basal and thrombin-induced MBS^Thr695 phosphorylation, resulting in elevated MBP activity. As reported earlier, phosphorylation of MBS on Thr-695 by Rho kinase inhibits MBP. Thus insulin appears to induce dephosphorylation of the inhibitory phosphorylation site on MBS by inactivating Rho kinase. Figure 7 depicts the putative signaling pathways involved in MBP activation by insulin in normal VSMCs and potential defects in hypertension and diabetes.

Posttranslational modification of RhoA is critical to its translocation to the membrane fraction (19, 26, 29). Our preliminary studies indicated that insulin may inhibit Rho translocation by decreasing geranygeranylation via reductions in geranylgeranyl transferase-1 activity via the NO/cGMP signaling pathway (data not shown). These observations, together with recent studies that inhibition of protein geranylgeranylation causes superinduction of inducible NOS (iNOS) by interleukin-I in VSMCs (8), suggest that the NO/cGMP and Rho signaling pathways regulate the activation status of each other via a complex cross-talk. Thus insulin-induced iNOS expression and cGMP generation in VSMCs isolated from control rats (3) may attenuate RhoA signaling by inhibiting geranylgeranylation as well as by altering the phosphorylation status of RhoA, as suggested by a recent study (29). In contrast, chronic activation of RhoA by vasoconstrictors as well as by disease states such as diabetes and hypertension inhibit iNOS and cGMP generation by specifically blocking insulin signaling via IRS-1/PI3-kinase pathway (1-4, 28, 38) leading to hyperphosphorylation of MBS, which results in MBP inactivation. Our preliminary results with the site-specific and phosphospecific MBS^Thr695 antibody indicate that the previously reported increase in MBS phosphorylation status in VSMCs isolated from diabetic Goto-Kakizaki rats (28) was due to a specific increase in MBS^Thr695 phosphorylation. A similar increase in MBS^Thr695 phosphorylation was observed in VSMCs isolated from spontaneously hypertensive rats. Reductions in PI3-kinase-generated signals leading to impaired iNOS induction and cGMP generation have been reported by our laboratory in earlier studies that used VSMCs isolated from spontaneous hypertensive rats and diabetic Goto-Kakizaki rats (3, 28). Furthermore, recent studies from Dr. Quon's laboratory (38) have shown that the PI3-kinase pathway mediates endothelial NOS activation via phosphorylation by its downstream target, the cellular homologue of the viral oncogene V-Akt, also known as protein kinase B, in endothelial cells. Thus defective iNOS protein expression and cGMP generation observed in diabetes and hypertension may contribute to the impairment in MBP activation due to lack of inhibition of Rho kinase. Thus insulin resistance of IRS-1/PI3-kinase signaling seen in diabetic Goto-Kakizaki rats and spontaneously hypertensive rats may lead to hyperphosphorylation of MBS and MBP inactivation due to excessive Rho kinase activation (1). The impaired vasorelaxation observed in patients with diabetes and hypertension may be due to inherent reductions in MBP activity resulting from defective regulation of MBP activation in response to insulin. Given the knowledge that protein kinase C (PKC) levels are elevated in VSMCs isolated from diabetic rat aortas (18) and the fact that PKC can activate Rho kinase and inhibit MBP, it is plausible that the exaggerated Rho kinase signaling and impaired MBP activation may be due to an elevation in PKC activity via excessive release of arachidonic acid by phospholipase A2 (15). Arachidonic acid could increase Rho kinase activity as well as interact directly with MBS, causing dissociation of the holoenzyme and thereby reducing MBP activity. In addition, both PKC and Rho kinase are known to activate the heat-stable inhibitors of MBP via phosphorylation (24).

In summary, we have demonstrated that insulin inhibits thrombin-mediated Rho translocation and Rho kinase activation via the NO/cGMP signaling pathway. Insulin inactivation of Rho kinase results in a decrease in MBS^Thr695 phosphorylation, which leads to MBP activation and vasodilation.