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1 The Diabetes Research Laboratory, Winthrop University Hospital, Mineola 11501; 3 School of Medicine, State University of New York, Stony Brook, New York 11794; and 2 First Department of Internal Medicine, Mie University School of Medicine, Mie 514-8507, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Our laboratory has recently demonstrated that insulin induces relaxation of vascular smooth muscle cells (VSMCs) by activating myosin-bound phosphatase (MBP) and by inhibiting Rho kinase (Begum N, Duddy N, Sandu OA, Reinzie J, and Ragolia L. Mol Endocrinol 14: 1365-1376, 2000). In this study, we tested the hypothesis that insulin via the nitric oxide (NO)/cGMP pathway may inactivate Rho, resulting in a decrease in phosphorylation of the myosin-bound subunit (MBSThr695) of MBP and in its activation. Treatment of confluent serum-starved VSMCs with insulin prevented thrombin-induced increases in membrane-associated RhoA, Rho kinase activation, and site-specific phosphorylation of MBSThr695 of MBP and caused MBP activation. Preexposure to NG-monomethyl-L-arginine, a NO synthase inhibitor, and R-p-8-(4-chlorophenylthio)cGMP, a cGMP antagonist, attenuated insulin's inhibitory effect on Rho translocation and restored thrombin-mediated Rho kinase activation and site-specific MBSThr695 phosphorylation, resulting in MBP inactivation. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked insulin's inhibitory effects by abolishing thrombin-mediated Rho signaling and promoted dephosphorylation of MBSThr695. Furthermore, expression of a dominant-negative RhoA decreased basal as well as thrombin-induced MBSThr695 phosphorylation and caused insulin activation of MBP. Collectively, these results indicate that insulin inhibits Rho signaling by decreasing RhoA translocation via the NO/cGMP signaling pathway to cause MBP activation via site-specific dephosphorylation of its regulatory subunit MBS.
myosin-bound phosphatase site-specific phosphorylation; myosin-bound phosphatase activation; vasodilation; Rho kinase; hypertension
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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,
NG-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.
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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
MBSThr695 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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 MBSThr695 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 MBSThr695 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 MBSThr695 phosphorylation (Fig. 4, lane 2). Furthermore, insulin treatment completely abolished the thrombin-mediated increase in MBSThr695 phosphorylation (Fig. 4, compare lane 4 with lane 3). Pretreatment with L-NMMA and Rp-CPT-cGMP prevented the insulin-mediated decrease in MBSThr695 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 MBSThr695 phosphorylation in a manner comparable to insulin (Fig. 4, lane 7).
Dominant-negative RhoA decreases basal MBSThr695 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 MBSThr695 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 MBSThr695 phosphorylation in the basal state. Furthermore, thrombin treatment caused a very small increase in MBSThr695 phosphorylation and the NO/cGMP inhibitors were able to still prevent insulin's effect on MBS phosphorylation (Fig. 5). The observed decrease in MBSThr695 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).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 MBSThr695 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 MBSThr695 phosphorylation, which is effectively blocked by L-NMMA and Rp-CPT-cGMP. Third, expression of a dominant-negative RhoA decreased basal and thrombin-induced MBSThr695 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 MBSThr695 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 MBSThr695 phosphorylation. A similar
increase in MBSThr695 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 MBSThr695 phosphorylation, which leads to MBP activation and vasodilation.
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ACKNOWLEDGEMENTS |
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This work was supported in part by an Established Investigator Award, a Grant-in-Aid from the American Heart Association, New York State affiliate, and funds from the medical education and research of Winthrop University Hospital.
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. Begum, Diabetes Research Laboratory, 222 Station Plaza North, Suite 511-B, Mineola, NY 11501 (E-mail: nbegum{at}winthrop.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 April 2001; accepted in final form 5 June 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Thr).
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.
