1 Division of Nephrology and Hypertension, Department of Medicine, University of Texas Health Science Center, and 2 Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Insulin acutely stimulates cyclic guanosine monophosphate
(cGMP) production in primary confluent cultured vascular smooth muscle
cells (VSMC) from canine femoral artery, but the mechanism is not
known. These cells contain the inducible isoform of nitric oxide (NO)
synthase (iNOS), and insulin-stimulated cGMP production in confluent
cultured cells is blocked by the NOS inhibitor,
NG-monomethyl-L-arginine
(L-NMMA). In the present study, it is shown that iNOS is
also present in freshly dispersed VSMC from this artery, indicating
that iNOS expression in cultured VSMC is not an artifact of the culture
process. Insulin did not stimulate NOS activity in primary confluent
cultured cells because it did not affect citrulline or combined
NO3/NO
2 production. To see whether insulin required the permissive presence of
NO to stimulate cGMP production, iNOS and basal cGMP production were
inhibited with L-NMMA, and the cells were incubated with or
without 1 nM insulin and/or the NO donor,
S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at a concentration (0.1 µM) that restored cGMP production to
the basal value. In the presence of L-NMMA, insulin no
longer affected cGMP production but when insulin was added to
L-NMMA plus SNAP, cGMP production was increased by 69%
(P < 0.05 vs. L-NMMA plus SNAP). Insulin, which
increases glucose uptake by these cells, increased the cell lactate
content and the lactate-to-pyruvate ratio (LPR) by 81 and 97%,
respectively (both P < 0.05), indicating that the hormone
increased aerobic glycolysis and the redox potential. The effects of
insulin on LPR and cGMP production were blocked by removing glucose or
by adding 2-deoxyglucose to the incubation media and were duplicated by
the reducing substrate,
-hydroxybutyrate. We conclude that insulin
does not acutely affect iNOS activity in these VSMC but it does augment
cGMP production induced by the NO already present in the cell while
increasing aerobic glycolysis and the cell redox potential.
nitric oxide synthase; cyclic guanosine monophosphate; aerobic glycolysis; vascular smooth muscle cells
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INTRODUCTION |
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CYCLIC GUANOSINE MONOPHOSPHATE (cGMP), by activating cGMP-dependent protein kinase, has important effects on several critical activities of vascular smooth muscle cells (VSMC) (5, 14, 22, 31). cGMP attenuates the contractile agonist-induced intracellular Ca2+ (Ca2+i) transient in vascular smooth muscle cells (VSMC), thereby decreasing VSMC contraction. cGMP also inhibits VSMC contraction at sites distal to Ca2+i by affecting contractile protein function (14). In addition, cGMP inhibits VSMC proliferation and migration (5, 22, 31), which are important events contributing to atherosclerosis and restenosis following balloon angioplasty (20).
It has been suggested that insulin stimulates cGMP production in blood vessels. For instance, insulin acutely stimulates nitric oxide (NO) production in cultured human umbilical vein endothelial cells (32), and insulin-induced vasodilation in humans is blocked by an inhibitor of nitric oxide synthase (NOS) (23, 27). It has been proposed that insulin-induced vasodilation is mediated, at least in part, by the hormone's stimulation of NO production by endothelial cells causing inhibition of contraction of the underlying VSMC, in part, by stimulating VSMC guanylate cyclase activity. In addition, it has been reported that insulin directly increases cGMP production in cultured VSMC (29) and inhibits VSMC contraction in the absence of endothelial cells (12). We reported that physiological concentrations of insulin acutely increased cGMP production (9) and lowered serotonin [5-hydroxytryptamine (5-HT)]-induced Ca2+ influx and the Cai2+ transient in primary confluent cultured cells from canine femoral artery in a glucose-dependent manner (11). Immunoblots of these cells identified inducible NOS (iNOS) protein but not the endothelial constitutive NOS protein (10). The cells had NOS activity (9), and insulin's stimulation of cGMP production was blocked by NG-monomethyl-L-arginine (L-NMMA) (9).
A major route of glucose metabolism in vascular smooth muscle (VSM) is to lactate via aerobic glycolysis (19), which increases the redox potential of cells by generating NADH (17). Previous studies with VSMC and other cell types have shown that reducing and oxidizing agents increase and decrease nitrovasodilator-stimulated guanylate cyclase activity, respectively (2, 4, 13, 15).
The purpose of the present study was to determine the mechanism of insulin-induced stimulation of cGMP production in these primary confluent cultured VSMC. We found that insulin did not stimulate iNOS activity but that insulin increased NO-stimulated cGMP production, regardless of whether the NO was derived from VSMC iNOS or an exogenous source. We found that insulin's increase in NO-stimulated cGMP production was associated with an insulin-induced increase in aerobic glycolysis and the cell redox potential.
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METHODS |
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Cell culture. Adult mongrel dogs of both sexes were killed with intravenous pentobarbital sodium, and the femoral arteries were dissected free. Freshly dispersed VSMC and primary confluent VSMC cultures were prepared as previously described (8). Endothelia and adventitia were stripped away, and the media of the arteries were minced and incubated at 37°C in a solution containing elastase (type V, Sigma, St. Louis, MO) and collagenase (type I, Worthington Biochemical, Freehold, NJ). After 2 h, the enzyme solution was discarded and replaced with fresh solution, and the tissue was incubated for an additional 2 h. The dispersed cells were pelleted and washed three times in Hanks' balanced salt solution and used for immunoblot studies or for cell culture, where they were suspended to a density of 2 × 105 cells/ml in DMEM that contained 1% glutamine, 10% FCS, 1% penicillin-streptomycin solution (10,000 U/ml penicillin, 10 mg/ml streptomycin). One milliliter of this suspension was placed in 35-mm plastic culture dishes. After seeding, cells were incubated in a humidified tissue culture incubator maintained at 37°C and equilibrated with 5% CO2-95% air. After 72 h and every 72 h thereafter, the media were replaced with 1 ml of the same fresh medium. The cells became confluent in 2 wk when they were used.
Gel electrophoresis and immunoblotting. Proteins (10-20 µg) obtained from freshly dispersed VSMC lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose paper by electroblotting as previously described (10). Subsequent probing of the blots was performed with a site-directed rabbit polyclonal anti-inducible NOS (anti-iNOS) antibody prepared as previously described (30), after preincubation in PBS containing 5% (wt/vol) nonfat dried milk and 0.5% (vol/vol) Tween 20. The antibody was generated with a peptide sequence that was unique to iNOS and yielded negative immunoblots with lysates from cultured endothelial or neuronal cells. Antigen-antibody interactions were detected with peroxidase-conjugated anti-rabbit IgA, IgM, and IgG followed by chemiluminescence and visualization on Hyperfilm-enhanced chemiluminescence film (Amersham, Piscataway, NJ). Positive and negative controls for iNOS (20-30 µg protein) were obtained from cell lysates of RAW 264.7 cells (American Type Culture Collection TIB71), a mouse macrophage cell line, pretreated for 24 h with or without 10 µg/ml of lipopolysaccharide (Escherichia coli 026:B6, Sigma), respectively.
NOS activity.
NOS activity in primary confluent cultured cells was measured by
determining the conversion of [14C]arginine to
[14C]citrulline, or by measuring combined
NO2/NO
3 production by the Greiss reagent (25, 26). Dishes of cells were washed
3 times with 1 ml physiological salt solution (PSS), which contained
(in mM) 140 NaCl, 4 KCl, 1.8 CaCl2, 0.8 MgSO4, 5 glucose and 10 HEPES-Tris, pH 7.4, plus 0.1% BSA and were
preincubated with 1 ml of the same medium with the desired agents plus
5 µM L-arginine and 0.2 µCi
[14C]L-arginine (Amersham) (for
[14C]arginine and
[14C]citrulline measurements) or 1 mM
L-arginine (for
NO
2/NO
3 measurements) for 30 min at 37°C. Miconozole (75 µM) was present to prohibit arginine metabolism by the P-450 system. When
[14C]arginine was used, the incubation solution
was discarded, and the cells were homogenized in 0.4 ml deionized water
by vigorous scraping. For
NO
2/NO
3
measurements, the media plus cells were similarly homogenized. The
homogenates were centrifuged at 10,000 g for 10 min, and the
supernatants were evaporated to dryness in a Speed Vac SC100 (Savant,
Holbrooke, NY). For [14C]arginine and
[14C]citrulline measurements, the residue was
resuspended in 25 µl deionized water containing 2 mg/ml
L-arginine and 2 mg/ml L-citrulline and applied
to silica gel-60 plates, 20 × 20 cm, aluminum (Whatman, Clifton,
NJ) as previously described (9). A solvent system containing
CHCl3-CH3OH-NH4OH-H2O,
1:4:2:1 (vol/vol) was used. Relative fronts for arginine and citrulline
were 0.49 and 0.81, respectively, as determined by autoradiography of
authentic L-[14C]arginine and
L-[14C]citrulline standards
(Amersham). The location of arginine and citrulline was confirmed by
staining with ninhydrin spray. The amounts of
[14C]arginine and
[14C]citrulline associated with the incubated
cells were determined by scraping the silica gel at the appropriate
regions of the chromatograph into liquid scintillation vials, vortexing
with scintillation cocktail, and counting. The percentage of
[14C]arginine that was converted to
[14C]citrulline was calculated and corrected
for the percentage of the [14C]arginine stock
contaminated with [14C]citrulline (~1%,
determined in each experiment by TLC as described above). Each
experiment was performed in triplicate. For combined NO
2/NO
3
measurements, the dried residue of the supernatant of centrifuged
homogenized cells plus incubation media was suspended in 80 µl of
phosphate assay buffer (20 mM, pH 7.4), preincubated with nitrate
reductase plus cofactors, and incubated with Greiss reagent, and
absorbance was measured in 80-well microplates at 540 nm as described
in the Cayman's nitrate/nitrite assay kit (Alexis, San Diego, CA).
NO
2 production per milligram of
protein was calculated after subtracting basal values by use of
authentic NO
2 standards. Each
experiment was performed in triplicate.
cGMP assay. Dishes of primary confluent cultured VSMC were incubated for 30 min at 37°C in PSS plus 0.1% BSA and 0.5 mM of isobutylmethylxanthine to inhibit phosphodiesterase in the presence and absence of 0.1 mM L-NMMA, with or without S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and/or 1 nM of insulin. In some cases, 10 µM 5-HT were added to the incubation solution for the final 5 min. The solution was removed, and the reaction was stopped by adding 1 ml of ice-cold ethanol acidified with 0.8% 12 M HCl. The extract was centrifuged at 10,000 g for 15 min, the supernatant was evaporated to dryness in a Speed Vac SC100, and cGMP content of acetylated samples was measured with the cGMP 125I Assay System (Amersham) as previously described (9). Each experiment was performed in triplicate. Protein was measured by the method of Lowry. Although basal cGMP production varied from preparation to preparation, the relative effects of specific perturbations on cGMP production were consistent among different preparations. Thus cGMP production was calculated as a percentage of the amount produced under control conditions.
Lactate and pyruvate measurements. Dishes of primary confluent cultured VSMC were preincubated for 30 min in 1 ml of physiological salt solution (PSS) in room air at 37°C and then incubated in 1 ml of PSS, with or without glucose, plus the desired agents for an additional 3 min. The reaction was stopped with 0.08 ml of 60% perchloric acid, and the cells were scraped and homogenized. An aliquot was saved for protein measurement, and the rest was centrifuged at 10,000 g for 15 min. The supernatant was neutralized with 3 M KOH. Lactate was measured as previously described (6) by adding NAD+ and lactate dehydrogenase (LDH) and measuring the appearance of NADH spectrophotometrically. Pyruvate was measured as previously described (18) by adding NADH and LDH and measuring the disappearance of NADH. The lactate-to-pyruvate ratio (LPR) was taken as an index of the redox potential of the cells (1, 17).
Bovine insulin and 5-HT were obtained from Sigma and L-NMMA and SNAP from Alexis (San Diego, CA). Anti-iNOS antibody was a gift from the Trauma Center, University of Texas Medical School at Houston (30). Statistical analysis was performed on paired data by use of Student's t-test and by ANOVA with multiple comparisons using the Newman-Keuls test. Statistical significance was taken as a value of P < 0.05. ![]() |
RESULTS |
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iNOS in freshly dispersed VSMC.
We reported previously that primary confluent cultured VSMC from canine
femoral artery contain iNOS protein (10) and NOS activity (9), and that
L-NMMA blocks insulin-stimulated cGMP production (9). To
determine whether the freshly dispersed VSMC from this source contain
iNOS, protein extracts from these cells were subjected to
electrophoresis and immunoblotted with rabbit polyclonal anti-iNOS
antibody. Protein extracts from the mouse macrophage cell line, RAW
264.7, in which iNOS had or had not been induced by previous exposure
to lipopolysaccharide, were used as positive and negative controls,
respectively. As shown in Fig. 1,
immunoblots of lysates from freshly dispersed VSMC and induced RAW
cells contained a single band stained positively by iNOS antibody, and
it had the expected molecular mass for iNOS (131 kDa) (24). Noninduced
RAW cells did not stain with iNOS antibody. Thus iNOS protein is
present in freshly dispersed VSMC from canine femoral artery.
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Effect of insulin on NOS activity.
Because insulin inhibits 5-HT-induced contraction of primary
cultured nonproliferated VSMC in an L-NMMA-sensitive manner
(9), we determined whether insulin and/or 5-HT increases NOS activity in primary cultured confluent cells. To measure NOS activity, the
percentage of conversion of [14C]arginine taken
up by the cells to [14C]citrulline was
measured. The 30-min uptakes of [14C]arginine
by L-NMMA-, 5-HT-, insulin-, and 5-HT + insulin-treated cells were 93 ± 9, 104 ± 11, 108 ± 12, and 97 ± 7% of control cells, respectively, which averaged 410 pmol arginine/mg protein [n = 4, P = not significant (NS)]. As shown
in Fig. 2, citrulline production was
inhibited by L-NMMA, an inhibitor of NOS (24), but neither
5-HT nor insulin, nor 5-HT + insulin affected citrulline production.
NOS activity was also determined by measuring combined NO3/NO
2
production. The 30-min production of
NO
3 + NO
2 by cells under control conditions,
incubated with 10 µM 5-HT, 1 nM insulin, or insulin + 5-HT, were 17.5 ± 2.4, 14.4 ± 2.1, 13.7 ± 1.9, and 16.0 ± 2.1 pmol/mg protein,
respectively (n = 3; P = NS). These data demonstrate
that insulin, with or without 5-HT, does not acutely affect NOS
activity in these cells.
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Effects of SNAP and insulin on cGMP.
To test whether the NO produced by iNOS in primary confluent cultured
VSMC acted in a permissive way to allow insulin's stimulation of cGMP
production, cells were preincubated with or without 0.1 mM
L-NMMA and/or 1 nM insulin and/or 0.1 µM SNAP for 30 min.
Figure 3 shows that insulin increased cGMP
production in the absence but not presence of L-NMMA, as we
have previously reported (9). SNAP (0.1 µM) increased cGMP production
relative to control, and SNAP + insulin increased cGMP production in an
additive manner. When endogenous NOS activity was blocked by
L-NMMA, the basal production of cGMP fell, and SNAP
increased it to near its control value without L-NMMA.
However, insulin failed to increase cGMP production when iNOS activity
was blocked by L-NMMA, but the provision of enough SNAP to
raise cGMP production to near the normal basal value enabled insulin to
increase cGMP production further by 69% (P < 0.05).
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Lactate-pyruvate measurements.
Increasing and decreasing the redox potential in various cell types,
including VSM, increases and decreases nitrovasodilator-induced guanylate cyclase activity, respectively (2, 4, 13, 15). To examine the
mechanism of insulin's stimulation of NO-induced cGMP production, we
investigated the effect of insulin on aerobic glycolysis and the
resultant effect on the cell redox potential. Primary confluent
cultured VSMC were incubated in the presence or absence of 5 mM glucose
with or without 1 nM insulin, and lactate levels were measured. As
shown in Fig. 5A, basal lactate
levels were greater in the presence (control) than in the absence of glucose, and insulin increased the lactate level, but only in the
presence of glucose. In the presence of the glycolysis inhibitor, 2-deoxyglucose (2-DOG), the lactate level was decreased, and insulin was unable to increase it. These data indicate that these VSMC perform
aerobic glycolysis, which has been described in VSM from other sources
(19), and that insulin, which increases glucose uptake by these cells
(11), stimulates aerobic glycolysis. As also shown in Fig. 5A,
0 glucose or 2-DOG tended to lower the pyruvate levels, but the results
were not statistically significant. Insulin plus glucose did not affect
the pyruvate levels significantly under these conditions, but
importantly, as shown in Fig. 5B, insulin plus glucose
increased the lactate-to-pyruvate ratio (LPR) compared with glucose
alone (control). In the absence of glucose or in the presence of 2-DOG,
the LPR was less than control and not stimulated by insulin. Taken
together, the data in Fig. 5 indicate that insulin increases aerobic
glycolysis, which increases the redox potential of the cells.
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Redox potential and cGMP production.
SNAP-stimulated cGMP production was measured in VSM cells exposed to
the same conditions as those in Figs. 5 and 6. As shown in Fig.
7, in the presence of L-NMMA
and SNAP, 1 nM of insulin increased cGMP production, in agreement with
the data in Figs. 3 and 4. In the absence of glucose or in the presence
of 2-DOG, basal cGMP production was not different from control, and
insulin did not increase it. As is also shown in Fig. 7,
-hydroxybutyrate, which increased the LPR, increased cGMP production
in the absence of insulin. Thus, in the presence of a permissive amount
of NO, insulin's ability to increase cGMP production is associated
with the hormone's increase in the redox potential and can be
duplicated by increasing the redox potential in the absence of insulin.
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DISCUSSION |
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cGMP inhibits VSMC proliferation, migration, and contraction (5, 14, 22, 31). We have reported previously that insulin acutely increased cGMP production in primary confluent cultured VSMC from canine femoral artery (9). These cells contained iNOS protein (the endothelial constitutive NOS protein was not detected by immunoblots) (10), and had NOS activity (9), and insulin-stimulated cGMP production was blocked by L-NMMA (9). The present study was performed to determine the mechanism of insulin-stimulated cGMP production in these cells. We predicted that insulin would stimulate NOS activity; this was not the case.
In the present study, we showed that insulin did not acutely affect iNOS activity in these cells. We showed that insulin increased guanylate cyclase activity if guanylate cyclase was moderately stimulated by NO, regardless of whether the NO came from VSM iNOS or from an exogenous source (SNAP). We do not know whether insulin would increase guanylate cyclase activity in these cells if initially activated by agents other than NO. We also showed that iNOS in primary cultured VSMC from canine femoral artery was not merely an artifact of the culture process, because immunoblots of lysates of freshly dispersed VSMC from this source also stained positively for iNOS protein. This finding is consistent with previous reports that iNOS protein has been found in unstimulated VSM from kidney (16, 28).
In this study, we also examined how insulin increased NO-stimulated guanylate cyclase activity. We had previously shown that insulin stimulated glucose uptake and that insulin's inhibition of VSM contraction required glucose transport (11). Because VSM performs aerobic glycolysis (19), we reasoned that insulin might stimulate aerobic glycolysis, which could increase the redox potential of the cell. This might enhance NO-stimulated guanylate cyclase activity because previous studies in VSM and other cell types showed that reducing and oxidizing agents increased and decreased nitrovasodilator-stimulated guanylate cyclase activity, respectively (2, 3, 13, 15).
In the present study, we demonstrated that in primary confluent
cultured VSMC from canine femoral artery, insulin increased aerobic
glycolysis and the redox potential of the cell by showing that insulin
increased lactate accumulation and the LPR, respectively. This was
accompanied by increased NO-stimulated cGMP production. When
insulin-induced aerobic glycolysis was blocked by omitting glucose from
the incubation media or by adding 2-DOG, insulin could increase neither
LPR nor NO-stimulated guanylate cyclase activity. In the absence of
insulin, the reducing fuel, -hydroxybutyrate, increased both the
redox state of the cell and NO-stimulated guanylate cyclase activity.
Thus, with or without insulin, an increased redox potential is
associated with increased NO-stimulated guanylate cyclase activity.
It is possible that NO from endothelial cells is normally responsible for sensitizing VSM guanylate cyclase to stimulation by insulin. The present studies suggest that, wherever its source, moderate levels of NO stimulate VSM cGMP more in the presence of insulin. It has been reported that iNOS is present at sites of atherosclerosis or balloon angioplasty and in some sources of unstimulated VSMC (3, 16, 21, 28). We speculate that under these conditions moderate amounts of NO from the VSM cells themselves, without contribution from endothelial cells, may stimulate VSM guanylate cyclase activity, thereby allowing insulin to increase additional cGMP production, possibly resulting in decreased VSMC contraction, proliferation, or migration.
It is possible that insulin's increase of the VSMC redox potential reduces thiol groups on the catalytic region of soluable guanylate cyclase, thereby increasing enzyme activity when the regulatory region has been activated by NO. It is also possible that the insulin-increased redox potential protects a finite amount of NO from being degraded within the cell by endogenous oxidizing agents, thereby increasing cGMP production. The present studies, however, show only an association between the insulin-induced increase in the cell redox potential and insulin's stimulation of NO-induced cGMP production. Future studies are needed to determine whether this association is due to a cause-and-effect relationship.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the excellent secretarial assistance of Gina Henderson.
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FOOTNOTES |
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These studies were supported by grants HL-50660 and HL-24585 from the National Heart, Lung, and Blood Institute and a grant from the Diabetes Action Research and Education Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Kahn, 4.107 MSB, University of Texas Health Science Center, PO Box 20708, Houston, Texas 77225 (E-mail: akahn{at}heart.med.uth.tmc.edu).
Received 10 September 1999; accepted in final form 22 October 1999.
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