The Diabetes Research Laboratory (N.B., N.D., O.S., J.R., L.R.)
Winthrop University Hospital Mineola, New York 11501
School of Medicine (N.B., L.R.) State University of New York
@ Stony Brook New York, New York 11794
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ABSTRACT |
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INTRODUCTION |
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Smooth muscle contraction and relaxation are largely mediated by the phosphorylation and dephosphorylation of the 20-kDa regulatory myosin light chain (MLC20) at threonine 18 and serine-19 by myosin light chain kinase (MLCK) and a myosin-bound serine/threonine specific protein phosphatase (MBP, Refs. 5, 6). Intracellular calcium levels ([Ca2+]i) are known to modulate the MLCK to MBP activity ratio and ultimately the degree of contractile force because MLCK activity depends on the amount of the Ca2+/calmodulin complex, which itself hinges on [Ca2+]i (7, 8, 9). However, recent studies with intact smooth muscle suggest the existence of other mechanisms that regulate contraction independent of changes in [Ca2+]i. One such mechanism by which this can occur is the inhibition of MBP activity (10, 11, 12). Thus, MLC20 phosphorylation and contractile force can be increased through Ca2+ sensitization, a G-protein-coupled Ca2+ independent process that inhibits MBP (10, 11, 12).
MBP holoenzyme consists of three subunits with molecular masses of 110/130 kDa, 38 kDa, and 20 kDa (13). The 38-kDa subunit is an isoform of the catalytic subunit of protein phosphatase-1 (PP-1C). The two other subunits (110/130 kDa and 20 kDa) are putative regulatory and targeting subunits that bind to myosin and regulate the catalytic activity of the phosphatase (13). Studies on purified preparations of MBP indicate that phosphorylation of the large 130-kDa regulatory myosin-bound subunit (MBS), by an associated kinase, results in an inhibition of phosphatase activity (14). Further studies revealed that an active GTP-bound Rho, a small guanosine triphosphatase, specifically interacts with MBS (15). The Rho-associated kinase phosphorylates MBS and consequently inactivates MBP (14, 15), resulting in an increase in MLC20 phosphorylation and contraction of smooth muscle. Although these results clearly explain how activated Rho increases Ca2+ sensitivity of both MLC20 phosphorylation and contraction, the pathway that leads from receptor, to G proteins, and then to the ultimate inhibition of the phosphatase is not known. Arachidonic acid has been suggested as a possible secondary messenger since it dissociates the trimeric structure of the phosphatase leading to its inhibition (5, 16). Moreover, arachidonic acid is known to be released in smooth muscle at concentrations and times consistent with a physiological role (16). In addition, Ikebe and Brozovich (17) reported that agonist-induced activation of protein kinase C can also increase VSMC contraction via an inhibition of MBP.
If phosphorylation of MBP does indeed have a regulatory function in VSMC contraction, then an effective dephosphorylation mechanism must exist and should occur within the time frame observed for physiological effects. Given that insulins vasodilatory effects are mediated via nitric oxide (NO) (18), and cGMP inhibits MLC20 phosphorylation (19), it can be hypothesized that insulin may inhibit VSMC contraction by activating MBP via the NO/cGMP signaling pathway. Whether or not dephosphorylation of MBS via agonist-induced signal transduction pathways causes activation of MBP remains unknown.
In this study, we examined whether insulin promotes VSMC relaxation via dephosphorylation of MLC20 by activating MBP and studied the mechanism of MBP regulation by insulin. The results of this study indicate that insulin rapidly stimulates MBP by decreasing the phosphorylation of the myosin-bound regulatory subunit, MBS. Furthermore, insulin-mediated MBP activation and decreased MBS phosphorylation are accompanied by Rho kinase inhibition and an increase in NO/cGMP signaling. Blocking signaling through phosphatidylinositol-3 kinase (PI3-kinase), NO, and cGMP inhibits insulins effects on MBP activation. Activation of MBP seems to be coordinated with inhibition of VSMC contraction due to decreased MLC20 phosphorylation.
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RESULTS |
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Treatment with 100 nM insulin for 10 min caused a 80%
increase in MBP activity. The effect of insulin was sustained for the
20-min incubation period studied (Fig. 2A). Dose-response analyses revealed a
maximal increase in MBP activity with 10 nM insulin after
10 min incubation (Fig. 2B
). Similar results were obtained using
[32P]-labeled phosphorylase a substrate (basal
MBP activity = 1.440 ± 0.156 nmol Pi released/mg
protein/min; maximal insulin-stimulated MBP activity = 2.4 ±
0.2 nmol Pi released/mg protein/min). PP-1 activity in myosin-depleted
supernatants was comparable between control and insulin-treated
VSMCs (data not shown). Thus the time and dose-dependent increase
observed in cellular PP-1 activity in response to insulin in VSMCs
shown in Figs. 1
, B and C, was entirely due to an increase in MBP
activity seen in Fig. 2
, A and B.
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Insulin Decreases the MBS Phosphorylation and Prevents
Thrombin-Induced Increase in MBS Phosphorylation
Recent studies suggest that the large 110/130 kDa MBS of
myosin-associated phosphatase (MBP) gets phosphorylated by agonists
that stimulate smooth muscle contraction (14, 15). Furthermore,
phosphorylation of MBS results in a decrease in MBP activity (14, 15).
Our observations that insulin rapidly stimulates MBP suggest that
insulin may be activating the phosphatase either by altering MBS
phosphorylation status and/or activating the catalytic subunit bound to
MBS via another mechanism. Therefore, we examined the effect of insulin
on MBS phosphorylation status. As shown in Fig. 3A, treatment with 100 nM
insulin for 2 min caused a rapid 53% decrease in
[32P] incorporation into MBS (Fig. 3A
, compare
lane 2 vs. lane 1). A 72% decrease in MBS phosphorylation
was observed after 5 min insulin incubation (Fig. 3A
, compare lane 3
vs. lane 1). The observed decrease in MBS phosphorylation
was sustained for 20 min of insulin treatment (Fig. 3A
, compare lane 5
vs. lane 1).
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High Concentrations of Okadaic Acid Inhibit Insulin-Mediated MBS
Phosphorylation
The above results raise the interesting possibility that
insulin may be dephosphorylating MBS via another phosphatase. To test
this possibility, [32P]-labeled VSMCs were
pretreated for 30 min with low (10 nM) and high
concentrations (1 µM) of okadaic acid (OA) to inhibit the
cellular pool of PP-2A and PP-1 activities. These cells were treated
with and without insulin and examined for MBS phosphorylation status.
Insulin caused a 57% decrease in MBS phosphorylation. Low
concentrations of OA that specifically inhibit PP-2A activity did not
prevent insulin-mediated decrease in MBS phosphorylation. In contrast,
OA at a higher concentration of 1 µM completely blocked
insulins inhibitory effect on MBS phosphorylation and increased MBS
phosphorylation by 44% over the basal levels. OA alone did not
appreciably alter basal levels of MBS.
Insulin Inhibits Rho Kinase Activity
Numerous reports (15, 23) suggest that MBS is
phosphorylated by Rho kinase, which is activated upon stimulation with
agonists such as thrombin or angiotensin II (AII). We tested the
possibility that insulin may be inhibiting Rho kinase activity and
thereby decreasing MBS phosphorylation. To test this hypothesis, Rho
kinase activity was assayed in anti-Rok- immunoprecipitates using
MBP as a substrate. As shown in Fig. 4A
, insulin treatment for 10 min caused a 40% decrease in Rho kinase
activity when compared with control lysates. More importantly,
pretreatment with insulin effectively prevented the thrombin-mediated
increase in Rho kinase activity (Fig. 4A
).
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The effect of insulin on Rho kinase inactivation was accompanied
by an inhibition of thrombin-mediated translocation of Rho from cytosol
to the membrane fraction (Fig. 4C). As evident from Fig. 4C
, a
considerable amount of Rho was present in the membrane fraction in
untreated VSMCs (Fig. 4C
, lane 1). Treatment with 100 nM
insulin for 10 min decreased thrombin-mediated translocation of Rho to
the membrane fraction (Fig. 4C
, compare lane 4 vs. lane 3).
Insulin alone caused a very small change in the amount of Rho in the
membrane fraction (Fig. 4C
, lane 2).
Impact of NOS/cGMP Signaling on Insulin-Mediated Rho Kinase
Inactivation and MBP Activation
Given that insulins vasodilatory effects are mediated via
NO (18), we next examined the contribution of NOS and cGMP signaling
pathways in insulin-mediated Rho kinase inactivation as well as MBP
stimulation. VSMCs were pretreated with 1 mM
NGmonoethyl L-arginine acetate
(L-NMMA, a synthetic inhibitor of NOS) and RpcGMP (100
µM, a cGMP antagonist) for 30 min followed by treatment
with and without 100 nM insulin for 10 min. Rho kinase
activity was measured in the immunoprecipitates, and MBP activity was
assayed in myosin-enriched pellets. Both L-NMMA and RpcGMP
prevented insulins inhibitory effect on Rho kinase and restored Rho
kinase activity to control levels [Rho kinase activity (% of control
value): control, 100%; insulin, 60 ± 6%; L-NMMA +
insulin, 98 ± 6%; RpcGMP + insulin, 101 ± 10%] and also
blocked insulins effect on MBP activation (Fig. 5). Furthermore, sodium nitroprusside
(SNP), a NO donor, and 8-bromo cGMP, a cGMP agonist, both mimicked
insulins effects on MBP activation (Fig. 5
) and Rho kinase
inactivation. Combined treatment with insulin and SNP or 8-bromo cGMP
did not further increase MBP activation (data not shown). These results
suggest that insulin-induced NOS/cGMP signaling pathway may participate
in insulin-mediated Rho kinase inactivation and MBP activation. In our
earlier studies, we have demonstrated that insulin rapidly induces iNOS
protein expression and cGMP generation in VSMCs from WKY while VSMCs
isolated from spontaneous hypertensive rats (SHR) exhibit resistance to
insulin in terms of iNOS protein induction and cGMP generation
(24).
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DISCUSSION |
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Several lines of evidence presented in this study suggest that MBP activation by insulin in VSMCs is mediated by multiple inputs from the Rho kinase and PI3-kinase/NO/cGMP signaling pathways. First, insulin rapidly inactivates Rho kinase, and this inactivation is accompanied by a marked time-dependent reduction in MBS phosphorylation and MBP activation. Second, insulin pretreatment prevents thrombin-mediated Rho kinase activation, MBS phosphorylation, and inactivation of MBP and restores MBP enzymatic activity by partially blocking thrombins stimulatory effects on the translocation of Rho to the membrane fraction. Support for our observations comes from a recent study by Feng et al. (26). Using site- and phosphorylation state-specific antibodies, these authors have identified two major inhibitory Rho kinase phosphorylation sites on MBS: Thr695 and Thr850 (26). Direct phosphorylation of recombinant MBS by Rho kinase was accompanied by inhibition of MBP activity. Furthermore, agonists that cause Rho kinase activation in intact Swis 3T3 cells also induced an increase in Thr695 phosphorylation on MBS, and this effect was blocked by a Rho kinase inhibitor, Y-27632 (26). Earlier studies have also shown that treatment of smooth muscle cells with C3 exoenzyme, which ADP ribosylates and inactivates the Rho family members, results in MBP activation because of Rho kinase inhibition (27) supporting the notion that inactivation of RhoA by insulin may cause MBP activation via Rho kinase inhibition.
Unlike the other effects of insulin, which are mediated via the PI3-kinase signaling pathway, insulins effect on Rho kinase inhibition does not appear to involve PI3-kinase as wortmannin, the PI3-kinase inhibitor, did not prevent insulin-mediated Rho kinase inactivation although it completely abolished insulin-mediated PI3-kinase activation. Earlier studies by Kureishi et al (28) have also shown that high concentrations of wortmannin (100 µM), which inhibit myosin light chain kinase, did not abolish contractions mediated by the constitutively active form of Rho kinase nor its catalytic activity in permeabilized smooth muscle of rabbit portal vein. However, in our study, inhibition of PI3-kinase with wortmannin effectively blocks insulin-mediated MBP activation by attenuating insulininduced NO and cGMP generation.
From the inhibitor studies, it appears that the NOS/cGMP signaling pathway may mediate insulins inhibitory effects on Rho kinase inactivation, thereby decreasing MBS phosphorylation as well as MBP activation. For example, blocking NOS activity with L-NMMA attenuates the effect of insulin on Rho kinase inhibition. Furthermore, treatment with RpcGMP, a cGMP antagonist, prevents insulin-mediated Rho kinase inactivation as well as MBP activation. More importantly, treatment with a cGMP agonist, 8-bromo-cGMP, and SNP, a NO donor, mimics the effect of insulin on Rho kinase inactivation, MBS phosphorylation, and MBP activation. It is not clear, at present, why wortmannin failed to block insulins inhibitory effect on Rho kinase. Further studies with transfected VSMCs overexpressing constitutively active and dominant negative mutants of p85PI3-kinase and p110PI3-kinase, NOS, and protein kinase G (PKG) are needed to clearly establish whether insulin inactivates Rho kinase via a signal generated by NOS signaling independent of the PI3-kinase pathway. Nonetheless, our results, though indirect, suggest a complex cross-talk between two major contraction/relaxation signaling pathways, Rho kinase and NO/cGMP, to mediate insulins stimulatory effect on MBP activation.
Regarding the role of the NO/cGMP in the insulin-mediated MBP activation, studies performed by others (29), as well as our laboratory (24), have shown that insulin stimulates the induction of iNOS protein leading to the generation of NO and causes an elevation in the cGMP levels (24). Also, it is well known that cGMP signaling pathway inhibits the contraction of smooth muscle by directly activating MBP (30). It is not exactly clear how cGMP activates MBP. Our preliminary studies suggest that in addition to its directs effects on MBP, cGMP may be inhibiting Rho kinase, thereby decreasing MBS phosphorylation leading to MBP activation. Studies by Wu et al. (30) on smooth muscle from rabbit ileum suggest that cGMP-mediated activation of PKG may phosphorylate MBS on a specific site, resulting in MBP activation. However, recent studies by Nakamura et al. (31) indicated that phosphorylation of MBS by cGMP-dependent PKG did not affect the phosphatase activity toward MLC20 but phosphorylation of the MBP holoenzyme decreased the binding of MBP to phospholipid. Thus, phosphorylation of MBS by PKG is not a direct mechanism in activating MBP.
Our results do not exclude the possibility that decreased MBS phosphorylation and phosphatase activation by insulin may be due to some unknown mechanism other than Rho kinase inhibition. For example, insulin may activate another phosphatase which dephosphorylates MBS causing MBP activation. This possibility was tested with OA. We observed that higher concentrations of OA completely prevented insulin-mediated MBS dephosphorylation. Similar results were obtained in an in vitro study on purified preparations of myosin phosphatase reported by Ichikawa et al. (14). Although these results suggest the involvement of a type 1 phosphatase, we cannot rule out the possibility that MBP holoenzyme itself may be catalyzing the dephosphorylation of its regulatory subunit, MBS. A detailed analysis of the MBS phosphorylation site(s) that are dephosphorylated by insulin is needed. However, at present, these experiments are not feasible due to the nonavailability of a site-specific phosphoantibody that would react with the phosphothreonine 695 on MBS, which is believed to be involved in inhibition of MBP (27).
In addition to these potential mechanisms for the regulation of MBP activity, recent studies have reported the presence of heat-stable inhibitors of MBP that are activated via phosphorylation by protein kinase C (PKC) and presumably by Rho kinase (32). Therefore, it is plausible that inhibition of Rho kinase by insulin will inhibit the heat-stable inhibitors of MBP, resulting in the activation of the phosphatase.
In contrast to WKY, VSMCs isolated from diabetic GK rats and SHR
exhibit marked insulin resistance in terms of MBP activation. In our
recent studies we have demonstrated that VSMCs from SHR exhibit
impaired iNOS induction in response to insulin (24). To our knowledge,
this is the first in vivo study demonstrating MBP activation
by insulin in VSMCs via reductions in the MBS phosphorylation
status and its abnormal regulation in the insulin-resistant states
associated with hypertension and NIDDM. Other studies have demonstrated
an inhibitory regulation of MBP by a G protein-dependent mechanism
(10, 11, 12). Thus, an inhibition of MBP activity by thrombin (22),
arachidonic acid (16), PKC (17), and GTPS (10, 11, 12) have been
reported. This study adds a new dimension to the above observations by
demonstrating that insulins stimulatory effect on MBP activation is
due, in part, to a reduction in the MBS phosphorylation state in
addition to the concomitant activation of the enzyme bound to MBS via
cGMP signaling, thereby causing MLC20
dephosphorylation and insulin-mediated vasorelaxation. Therefore, 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 PKC levels are elevated in VSMCs
isolated from diabetic rat aortas (33) and the fact that PKC can
activate Rho and inhibit MBP, it is tempting to speculate that the
impaired MBP activation by insulin observed in diabetes and SHR may be
due to an elevation in the PKC activity via excessive release of
arachidonic acid by phospholipase A2 (23). Arachidonic acid could
increase Rho kinase activity as well as interact directly with MBS,
causing dissociation of the holoenzyme, thereby reducing MBP activity
(23). Alternatively, arachidonic acid could activate a kinase that
phosphorylates MBS and inhibits MBP. Our results complement the above
observations by documenting that MBS is phosphorylated under basal
conditions and the enzymatic activity of MBP is low. Insulin treatment
relieves the inhibition and restores the enzymatic activity of MBP by
decreasing MBS phosphorylation.
In summary, the results of this study suggest that insulin utilizes the Rho kinase and NO/cGMP signaling pathways to reduce the phosphorylation of MBS and activate MBP which causes vasorelaxation by the dephosphorylation of MLC20.
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MATERIALS AND METHODS |
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Culture of VSMCs and Treatment with Insulin
VSMCs in primary culture were obtained by enzymatic digestion of
the aortic media of male Wistar Kyoto (WKY) rats with body weights of
200220 g, as described in our recent publications (24, 35). Unless
otherwise indicated, primary cultures of VSMCs were maintained in
-MEM containing 10% FBS, and 1% antibiotic/antimycotic mixture.
VSMCs isolated from diabetic Goto-Kakizaki (GK) rats, a model for
NIDDM, were maintained in medium containing 20 mM
glucose to mimic a hyperglycemic condition. Subcultures of VSMCs at
passage 5 were used in all experiments. All experiments on MBP
activation, MLC20 phosphorylation, and Rho kinase
were performed on highly confluent cells (911 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 (0100 nM) for 030 min. In some experiments,
VSMCs were pretreated with various inhibitors for 30 min followed by
exposure to insulin as detailed in the figure legends.
Preparation of Myosin-Enriched Fractions
Myosin-enriched fractions of VSMCs were prepared by extraction
with a high-salt buffer as described previously (20). This fraction
contains only PP-1 activity and essentially no PP-2A activity.
Measurement of Myosin-Bound Phosphatase Activity
Phosphatase activity in myosin-enriched fractions was assayed
using [32P]-labeled phosphorylase a as well as
[32P]-labeled MLC as substrates (21). Briefly,
equal amounts of proteins (1 µg) were diluted with assay buffer
(50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 28
mM ß-mercaptoethanol, and 30 mM KCl). The
reaction was initiated by the addition of
[32P]-labeled substrates and stopped after 5
min incubation at 30 C by the addition of 20% trichloroacetic acid
(TCA). The radioactivity released in the TCA supernatants was counted
as detailed in our recent publications (36, 37).
[32P]-labeled phosphorylase a was prepared
by incubating [-32P]-ATP with purified
phosphorylase kinase and phosphorylase b (38).
[32P]-labeled MLC was prepared according to the
published protocol (39) by incubating MLC with purified MLCK and 50
µM [
-32P]ATP.
Metabolic Labeling of VSMCs and Measurement of MBS
Phosphorylation by Immunoprecipitation and Western Blot Analyses
Serum-starved VSMCs labeled with
[32P]orthophosphoric acid (0.3 mCi/ml for
4 h) were exposed to various agonists as detailed in the figure
legends. The cells were lysed in a buffer containing 50 mM
HEPES, pH 7.5, 2 mM EDTA, 1% Triton X-100, 100
mM NaCl, 50 mM ß-glycerophosphate, 100
mM NaF, 100 mM sodium pyrophosphate, 2
mM sodium orthovanadate, 2 µM microcystin,
and a cocktail of protease inhibitors (36). Precleared lysates with
equal amounts of proteins were immunoprecipitated with anti-MBS
antibody prebound to Protein A Sepharose for 3 h at 4 C
followed by separation of the immunoprecipitates by SDS-PAGE and
autoradiography. The level of MBS phosphorylation was measured by
densitometric scanning of the autoradiograms. To overcome variations in
proteins due to immunoprecipitation, the membranes were probed with
anti-MBS antibody followed by incubation with HRP-conjugated secondary
antibodies and detection by enhanced chemiluminescence (ECL). The
extent of MBS phosphorylation was quantitated by dividing the intensity
of radioactive signal with the protein signal.
Immunoprecipitation and in Vitro Assay of PI3-Kinase
Activity in the IRS-1 Immunoprecipitates
Equal amounts of precleared lysate proteins (100 µg) were
immunoprecipitated with rabbit anti-IRS-1 antibody. PI3kinase
activity was assayed in the IRS-1 immunoprecipitates as detailed in our
recent publication (24).
Immunoprecipitation and in Vitro Assay of Rho Kinase
Activity in the Immune Complexes
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 myosin-enriched fraction as a
substrate (40). 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
followed by extensive washing of the paper and
32P incorporation determined by liquid
scintillation spectroscopy.
Analyses of Agonist-Induced Rho Translocation to the Membrane
Fraction
Cytosolic and membrane fractions were prepared by differential
centrifugation according to previously published protocols (41). Equal
amounts of membrane proteins were subjected to SDS-PAGE, transferred to
polyvinylidene fluoride (PVDF) membrane, and probed with mouse
anti-Rho A antibody followed by incubation with HRP-labeled secondary
antibody and subsequent detection with ECL.
MLC Phosphorylation
MLC20 phosphorylation was analyzed by
urea, glycerol-PAGE separation of the mono and diphosphorylated forms
of MLC20 as detailed earlier (42).
MLC20 was detected by immunoblot analysis with
MLC20 antibody (Sigma) followed by
treatment with HRP-conjugated secondary antibodies and detection
by ECL. The autoradiograms were scanned and quantified, and the percent
maximal MLC20 phosphorylation was determined by
dividing the sum of fast migrating diphosphorylated
MLC20 area and the monophosphorylated
MLC20 area by the total of phosphorylated and
nonphosphorylated areas.
Measurement of VSMC Contraction
VSMC contraction was measured by analyzing the insulin-mediated
decrease in thrombin-stimulated transvascular HRP diffusion according
to the published protocol (22). Briefly, VSMCs plated on
collagen-coated polyethylene trephtalate cell culture inserts (3-µm
pore size, Becton Dickinson and Co.) were treated in
quadruplicate with and without insulin (100 nM) for 30 min
followed by the addition of 500 µl serum free medium containing
thrombin (0.5 U/ml). After 15 min, the lower compartment was filled
with 500 µl serum-free medium and the medium in the upper compartment
was replaced with fresh medium containing HRP (0.34 mg/ml). After 1
min, 60 µl of medium from the lower compartment were transferred to a
tube and mixed with 860 µl of reaction buffer containing 50
mM NaH2PO4, 5
mM Guaiacol and freshly made 0.6 mM
H2O2. The absorbance was
measured at 470 nm after 15 min incubation at room temperature.
Protein Assay
Proteins in the cellular extracts and lysates were quantitated
by the bicinchoninic acid (43) or by the Bradford technique (44).
Statistics
The results are presented as means ± SEM of
four to six independent experiments each performed in triplicate at
different times. Paired Students t test was used to
compare the basal vs. insulin-treated preparations.
Unpaired t test or ANOVA was used to compare the mean values
between treatments. A P value of <0.05 was considered
statistically significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by an Established Investigator Award (N.B.), a Grant-in-Aid New York State affiliate (L.R.) from the American Heart Association, a research award from the American Diabetes Association (N.B.) and funds from the medical eduction and research, Winthrop University Hospital.
Received for publication February 18, 2000.
Revision received April 26, 2000.
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REFERENCES |
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