Insulin inhibits vascular smooth muscle contraction at a site
distal to intracellular Ca2+
concentration
Andrew M.
Kahn1,
Annat
Husid1,
Timothy
Odebunmi2,
Julius C.
Allen2,
Charles L.
Seidel2, and
Tom
Song1
1 Departments of Medicine, The
University of Texas Health Science Center and
2 Baylor College of Medicine,
Houston, Texas 77030
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ABSTRACT |
Several hypertensive states are associated with
resistance to insulin-induced glucose disposal and insulin-induced
vasodilation. Insulin can inhibit vascular smooth muscle (VSM)
contraction at the level of the VSM cell, and resistance to insulin's
inhibition of VSM cell contraction may be of pathophysiological
importance. To understand the VSM cellular mechanisms by which insulin
resistance leads to increased VSM contraction, we sought to determine
how insulin inhibits contraction of normal VSM. It has been shown that
insulin lowers the contractile agonist-stimulated intracellular Ca2+
(Ca2+i) transient in VSM cells. In this
study, our goal was to see whether insulin inhibits VSM cell
contraction at steps distal to Ca2+i and,
if so, to determine whether the mechanism is dependent on nitric oxide
synthase (NOS) and cGMP. Primary cultured VSM cells from canine femoral
artery were bathed in a physiological concentration of extracellular Ca2+ and permeabilized to
Ca2+ with a
Ca2+ ionophore, either ionomycin
or A-23187. The resultant increase in
Ca2+i contracted individual cells, as
measured by photomicroscopy. Preincubating cells with 1 nM insulin for 30 min did not affect basal Ca2+i or the
ionomycin-induced increase in Ca2+i, as
determined by fura 2 fluorescence measurements, but it did inhibit
ionomycin- and A-23187-induced contractions by 47 and 51%,
respectively (both P < 0.05). In the presence of 1.0 µM ionized Ca2+,
ionomycin-induced contractions were inhibited by insulin in a
dose-dependent manner. In the presence of ionomycin, insulin increased
cGMP production by 43% (P < 0.05).
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 µM), a selective inhibitor of guanylate cyclase that blocked cGMP
production in these cells, completely blocked the inhibition by insulin
of ionomycin-induced contraction. It was found that the cells expressed
the inducible isoform of NOS.
NG-monomethyl-L-arginine
or
NG-nitro-L-arginine
methyl ester (0.1 mM), inhibitors of NOS, did not affect
ionomycin-induced contraction but prevented insulin from inhibiting
contraction. We conclude that insulin stimulates cGMP production and
inhibits VSM contraction in the presence of elevated
Ca2+i. This inhibition by insulin of VSM
contraction at sites where Ca2+i could
not be rate limiting is dependent on NOS and cGMP.
cGMP; guanylate cyclase; nitric oxide synthase
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INTRODUCTION |
INSULIN ACUTELY DILATES blood vessels in vivo and
inhibits vascular smooth muscle (VSM) cell contraction in vitro (4, 18, 29, 47). Several investigators (33, 38) have reported that
insulin-induced vasodilation in vivo is dependent, in part, on
endothelial cell nitric oxide synthase (NOS) activity. Nevertheless, insulin inhibits contraction of vascular tissue that has been stripped
of endothelium (47) and inhibits contraction of individual cultured VSM
cells in the absence of endothelial cells (18, 29). This effect of
insulin on VSM may be related to the pathogenesis of essential
hypertension and the hypertension associated with non-insulin-dependent
diabetes mellitus and obesity. In these conditions, there is resistance
to both insulin-induced glucose disposal and insulin-induced
vasodilation (2, 19, 20, 22). The latter could be implicated in the
pathogenesis of hypertension, since this form of insulin resistance may
be related to elevated blood pressure in certain genetically disposed
individuals (22). If resistance to insulin's inhibition of VSM
contraction is related to the pathogenesis of hypertension, therapies
should be directed at reversing the VSM cellular abnormalities by which
insulin resistance leads to increased VSM contraction. In this regard,
it is important to understand how insulin inhibits the contraction of
normal VSM.
Insulin modulates several Ca2+
transport systems in different cultured VSM cell preparations and
attenuates the contractile agonist-induced intracellular
Ca2+
(Ca2+i) transient (15, 18, 32, 37, 41). This is probably responsible, at least in part, for insulin-inhibited VSM contraction. However, there may be sites distal to the
Ca2+i signal in VSM cells where insulin
inhibits contraction. Knowledge of this is important to devise
strategies to reverse the abnormality at that site in insulin-resistant
states. Thus the first purpose of this study was to see whether insulin
inhibits contraction at steps distal to
Ca2+i. In the present study, we show that
this is the case by demonstrating that insulin inhibits the contraction
of primary cultured VSM cells from canine femoral artery even when
Ca2+i has been raised to such high
concentrations that Ca2+i could not be
rate limiting.
In addition, we have recently reported that
NG-monomethyl-L-arginine
(L-NMMA), an inhibitor of NOS,
blocked insulin's inhibition of serotonin (5-HT)-stimulated
Ca2+ influx and contraction in
these cells (16). Excess
L-arginine blocked the effect of
L-NMMA on insulin's inhibition
of contraction, and nitroprusside or dibutyryl cGMP inhibited
5-HT-induced contraction (16). We showed that the cells had NOS
activity and that insulin stimulated the production of cGMP in
5-HT-stimulated cells by a NOS-dependent mechanism (16). Thus the
second purpose of the present study was to determine whether insulin
also inhibited high- Ca2+i-induced
contraction by a NOS- and cGMP-dependent pathway.
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METHODS |
Cell culture of nonproliferating cells.
Adult mongrel dogs of either sex were killed with intravenous
pentobarbital sodium, and the femoral arteries were dissected free. VSM
cells were cultured under conditions in which they do not proliferate
and were used for Ca2+i and contraction measurements, as previously described (18). 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)
and collagenase (type I, Worthington Biochemical). 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
(GIBCO) and suspended to a density of 2 × 105 cells/ml in DMEM (GIBCO)
containing 0.5% FCS (Cyclone), 1% glutamine, and 1%
penicillin-streptomycin (PS) solution (10,000 U/ml penicillin, 10 mg/ml
streptomycin; Sigma). One milliliter of this suspension was placed in
35-mm culture dishes (Falcon) previously coated with rat tail tendon
collagen gels, prepared as previously described (17). 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 were used 5-8 days after seeding.
Cell culture of confluent cells.
Primary confluent cultures of these cells were prepared as previously
described (15). The dispersed cells were pelleted as described above
and suspended in complete DMEM containing 10% FCS, 1% glutamine, and
1% PS solution. The cell suspension was adjusted to 2 × 105 cells/ml, and 1 ml was put in
35-mm plastic dishes, which were placed 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 fresh complete DMEM. The cells reached confluence between days
10 and 15, when they
were used. The identity of the confluent cultured cells as smooth
muscle cells was confirmed, as previously described, by the
"hill-and-valley" pattern of cell growth and by a ratio of actin
to myosin heavy chain characteristic of intact VSM (36).
Fura 2 fluorescence.
Ca2+i of nonproliferated individual cells
was measured by monitoring the fluorescence emissions of fura 2 using a
custom-built epifluorescence microscope, as previously described (18).
Cells were grown on collagen gels for 5-8 days, as described
above, in a 0.6-ml glass-bottomed chamber. The chamber was placed on
the stage of a Nikon Diaphot inverted phase-contrast microscope and
superfused at 1 ml/min with physiological salt solution (PSS)
containing (in mM) 140 NaCl, 4 KCl, 1.8 CaCl2, 0.8 Mg
SO4, 5 glucose, and 10 HEPES-Tris
(pH 7.4) plus 0.1% BSA at 37°C. One cell was centered in the field
of view, and the autofluorescence of that cell was determined by
exciting the cell with alternating 100-ms pulses of light at 340 and
380 nm and recording the emission intensity at 510 nm with a
photomultiplier tube. The emissions from all other cells had been
excluded by adjusting a diaphragm proximal to the photomultiplier tube.
The cell was loaded for 45 min with 2.4 µM fura 2-AM (Molecular
Probes) that had been sonicated for 20 s in DMEM with 0.1% BSA. The
chamber was superfused again at 1 ml/min with PSS plus 4% BSA with or
without 1 nM insulin. The fluorescent emissions were corrected for
autofluorescence, and the 340- to 380-nm intensity ratios were
recorded. Basal Ca2+i values were
calculated from the fluorescence ratio recordings using the formula:
Ca2+i = Kd · (R
Rmin/Rmax
R) · Sf2/Sb2.
Dissociation constant
(Kd) was taken
as 224 nM, and the symbols in the equation have their usual meaning
(13). Rmax,
Rmin, and
Sf2/Sb2
were determined at the end of each experiment by measuring the
autofluorescence-corrected 510-nm emissions with 340- and 380-nm
excitation while the cells were superfused with PSS containing 2.5 µM
ionomycin, followed by perfusion with nominally Ca2+-free HEPES PSS plus 10 mM
EGTA.
cGMP assay.
Dishes of confluent VSM cells were preincubated with or without 10 µm
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ) in PSS for 15 min at 37°C. IBMX (0.5 mM) and ionomycin (2.5 µM) with or without
S-nitroso-N-acetyl-DL-penicillamine
(SNAP, 10
7 M) were added to
the incubation solution for an additional 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. In other experiments, dishes of
confluent VSM cells were preincubated with or without 1 nM insulin in
PSS plus 0.1% BSA at 37°C for 30 min. The media were changed to
PSS plus BSA with ionomycin and IBMX with and without insulin, and the
reaction was stopped 5 min later with acidified ethanol. The extract
was centrifuged at 10,000 g for 15 min, the supernatant was evaporated to dryness in a Speed Vac SC100
(Savant), and the cGMP content of acetylated samples was measured with
the 125I-labeled cGMP Assay System
(Amersham). Although basal cGMP production varied from preparation to
preparation, the relative effects of specific perturbations on cGMP
production were consistent among different preparations. Thus, in some
experiments, cGMP production was calculated as a percentage of the
amount produced under control (basal) conditions.
Cell contraction.
After 5-8 days of culture, the cells grown on the collagen gels
were used for contraction studies, as previously described (18). The
dishes were placed on the heated (37°C) stage of a Nikon Diaphot
inverted phase-contrast microscope, and the culture media were replaced
with the desired solutions. After a 30-min preincubation period, a
field of at least 6-10 cells was photographed at ×200 to
obtain baseline images. The cells were permeabilized to
Ca2+ with
Ca2+ ionophores (ionomycin or
A-23187) in the presence of 1.8 mM or 1.0 µM extracellular
Ca2+, and after 10 min, another
photograph was taken of the same field. Known concentrations of
Ca2+ and EGTA were selected to
achieve 1.0 µM ionized Ca2+, as
calculated in the manner of Fabiato and Fabiato (10) with association
constants from Martel and Smith (23) and corrections for the effects of
temperature and ionic strength.
Gel electrophoresis and immunoblotting.
Proteins (10-20 µg) obtained from confluent cultured VSM cell
lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose paper by electroblotting (1).
Subsequent probing of the blots was performed with a site-directed rabbit polyclonal anti-inducible NOS (anti-iNOS) antibody, prepared as
previously described (44), 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). A positive control for iNOS (20-30 µg protein) was obtained from cell lysates of RAW 264.7 cells (ATCC TIB71), a mouse monocyte-macrophage cell line, pretreated for 24 h with 10 µg/ml of lipolysaccharide
(Escherichia coli 026:B6, Sigma).
Bovine insulin and ionomycin were obtained from Sigma, A-23187 was from
Calbiochem, ODQ was from Tocris Cookson (St. Louis, MO), and
L-NMMA,
NG-nitro-L-arginine methyl ester
(L-NAME), and SNAP were from
Alexis. Anti-iNOS antibody was a gift from the Trauma Center,
University of Texas Medical School at Houston (44). Statistical
analysis was performed on paired data by use of Student's
t-test and ANOVA with multiple
comparisons using the Newman-Keuls test. Statistical significance was
taken as a value of P < 0.05.
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RESULTS |
High-Ca2+i-induced
contraction.
To determine whether insulin inhibits VSM contraction at steps
distal to Ca2+i, we measured the effects
of insulin on cells that were induced to contract by artificially raising Ca2+i to a level that saturated
the ability of the cell to regulate
Ca2+i. We measured
Ca2+i in individual resting cells bathed
in 1.8 mM extracellular Ca2+ by
monitoring the fluorescence emission of intracellular fura 2. Ca2+i was 83 ± 9 nM
(n = 5), a concentration consistent
with our previous findings in resting cultured canine VSM cells (18).
When 2.5 µM ionomycin was added to the bathing media,
Ca2+i increased to high values. We found that preincubating cells with 1 nM insulin for 30 min did not affect
resting Ca2+i, which was 90 ± 9 nM
(n = 5, P = not significant), and adding
ionomycin to the bathing media raised
Ca2+i in a manner that was
indistinguishable from cells not exposed to insulin. Within 10 min
after addition of ionomycin to the bathing media, the 340- to 380-nm
emission ratio was 3.42 ± 0.45 and 3.44 ± 0.33 for
cells pretreated with and without insulin, respectively
(n = 5 for each condition,
P = not significant). A representative
experiment showing ionomycin increasing
Ca2+i in an insulin-treated VSM cell is
shown in Fig. 1. The precise values for
Ca2+i under these conditions cannot be
determined, since the respective R values are also taken as
Rmax, which would give infinite
values when substituted into the Ca2+i
equation.

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Fig. 1.
Graph showing effects of insulin and ionomycin on intracellular
Ca2+
(Ca2+i) in a vascular smooth muscle (VSM)
cell. A VSM cell grown on a collagen gel was loaded with fura 2, and
fluorescence emissions at 510 nm, with excitation with alternating
pulses at 340 and 380 nm, were recorded, autofluorescence was
subtracted, and ratio
(F340/F380)
was calculated. Cell was superfused with 1 nM insulin for 30 min at
37°C in physiological salt solution
(Ca2+ 1.8 mM). Ionomycin (2.5 µM) was then added to superfusion solution where indicated, followed
by nominally Ca2+-free
physiological salt solution (PSS) containing 10 mM EGTA (pH 7.4).
Horizontal axis is time (min:s).
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We then measured contraction of cells whose
Ca2+i was raised. As shown in Fig.
2, 2.5 µM ionomycin contracted individual
VSM cells by 9.2 ± 1.7%. Under the same experimental conditions, 5 µM A-23187, another Ca2+
ionophore, contracted cells by 12.0 ± 2.2%. However, as also shown
in Fig. 2, when the cells had been preincubated with 1 nM insulin for
30 min, ionomycin- and A-23187-induced contractions were inhibited by
47 and 51%, respectively (both P < 0.05). These data demonstrate that insulin inhibits VSM contraction at
steps distal to Ca2+i, since contraction
was inhibited by insulin under conditions in which
Ca2+i clearly was not rate limiting.

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Fig. 2.
Graphs showing effects of insulin on high intracellular
Ca2+-induced contraction of
individual VSM cells. Dishes of nonproliferated VSM cells were
preincubated in PSS at 37°C with or without 1 nM insulin for 30 min, and baseline photomicrographs were obtained. Ionomycin (2.5 µM;
A) or A-23187 (5 µM;
B) was added to bath media, and
subsequent photomicrographs were obtained 10 min later. Each bar
represents mean ± SE of mean contractions from baseline lengths of
6-10 cells in each of 6 dishes.
* P < 0.01 vs. respective
control.
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Because Ca2+i was brought to levels
higher than those that occur in vivo in the above experiments, our goal was to confirm that insulin could inhibit contraction when
Ca2+i was set at a value at which
Ca2+i could not be rate limiting but was
still in the physiological range. As shown in Fig.
3, when cells were preincubated with media
containing 1.0 µM ionized Ca2+,
the subsequent addition of ionomycin contracted cells by 11.9 ± 1.4%. Preincubating the cells with 0.1, 0.5, and 1 nM insulin for 30 min decreased contraction by 25, 41, and 51%, respectively (all
P < 0.05 vs. 0 insulin). Thus
insulin can inhibit cell contraction stimulated by raising
Ca2+i to the high physiological range in
a dose-dependent manner.

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Fig. 3.
Graph showing dose-response effects of insulin on ionomycin-induced
contraction of individual VSM cells in presence of 1.0 µM ionized
extracellular Ca2+. Dishes of
nonproliferated VSM cells were preincubated for 30 min with PSS at
37°C with or without indicated concentrations of insulin for 30 min. Baths were changed to PSS containing concentrations of
Ca2+ and EGTA to yield 1.0 µM
ionized Ca2+ with or without
insulin, and baseline photomicrographs were obtained. Ionomycin (2.5 µM) was added to bath media, and subsequent photomicrographs were
obtained 10 min later. Each bar represents mean ± SE of mean
contractions from baseline lengths of 6-10 cells in each of 6 dishes. * P < 0.05 vs. 0 insulin.
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cGMP production.
We have previously shown that insulin stimulates cGMP production by
48% in cells exposed to the contractile agent 5-HT (16). In the
present study, we found that a similar result occurred in cells exposed
to ionomycin. cGMP production by cells exposed to 2.5 µM ionomycin
for 5 min averaged 18.0 fmol/mg of protein and was stimulated 49 ± 8% by preincubating them for 30 min with 1 nM insulin
(n = 5, P < 0.05).
Dishes of cells were preincubated for 15 min in PSS with or without 10 µM ODQ, a selective inhibitor of guanylate cyclase, and then treated
with ionomycin, with or without 0.1 µM SNAP, a nitric oxide (NO)
donor, for an additional 5 min. As shown in Fig.
4, SNAP stimulated cGMP production, and
this effect was inhibited by ODQ. ODQ did not affect basal cGMP
production. Thus ODQ effectively inhibits guanylate cyclase activity in
the presence of high Ca2+i in these
cells. As shown in Fig. 5,
ionomycin-induced VSM cell contraction was inhibited by insulin. ODQ
(10 µM) alone had no effect on ionomycin-induced contraction but
completely blocked insulin's inhibition of contraction. Taken
together, these data suggest that the inhibition by insulin of
high-Ca2+i-induced contraction is
dependent on cGMP production.

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Fig. 4.
Graph showing effects of
S-nitroso-N-acetyl-DL-penicillamine
(SNAP) and
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ) on cGMP production. Dishes of confluent VSM cells were
preincubated with PSS at 37°C for 15 min with or without ODQ (10 µM). IBMX (0.5 mM) and ionomycin (2.5 µM) with or without SNAP (0.1 µM) were added to bath media, and 5 min later cells were harvested
and cGMP was measured. Each bar represents mean ± SE of mean of
triplicate measurements performed in 3 separate experiments. * P
<0.05 vs. all other values. ** P <0.05 vs. all other
values.
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Fig. 5.
Graph showing effects of insulin and ODQ on ionomycin-induced
contraction of individual VSM cells. Dishes of nonproliferated VSM
cells were preincubated with PSS at 37°C with or without 1 nM
insulin with or without 10 µM ODQ for 30 min, and baseline
photomicrographs were obtained. Ionomycin (2.5 µM) was added to bath
media, and subsequent photomicrographs were obtained 10 min later. Each
bar represents mean ± SE of mean contractions from baseline lengths
of 6-10 cells in each of 6 dishes. * P <0.05 vs. all other
values.
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Contraction and NOS.
Our goal was to test whether insulin inhibits ionomycin-induced VSM
cell contraction by a NOS-dependent mechanism. In this regard, it is
important to demonstrate whether a NOS protein was present in the
cells. Protein extract from confluent VSM cells was subjected to
electrophoresis and immunoblotted with rabbit polyclonal anti-iNOS
antibody. Protein extract from the mouse monocyte macrophage cell line,
RAW 264.7, in which iNOS had been induced by prior exposure to
lipopolysaccharide, was used as a positive control. As shown in Fig.
6, immunoblots of lysates from both VSM
cells and RAW cells contained single identical bands stained positively
by iNOS antibody, and they had the expected molecular mass for iNOS
(131 kDa) (35). A similar procedure using anti-constitutive NOS
(anti-cNOS) antibody, which yielded positive immunoblots with lysates
from endothelial cells, did not reveal cNOS protein in lysates from the
VSM cells (data not shown).

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Fig. 6.
Immunoblot showing presence of inducible NOS (iNOS) in VSM and RAW
264.7 cells (RAW). Cell lysates (20-30 µg protein) were
electrophoresed on SDS-polyacrylamide gels and immunoblotted with
anti-iNOS antibody.
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VSM cells were then preincubated for 30 min with or without 1 nM
insulin in the presence and absence of
L-NMMA or
L-NAME, two inhibitors of NOS
(35). Cell contraction was then induced by raising
Ca2+i with iononmycin. As shown in Fig.
7, insulin inhibited ionomycin-induced
contraction, and L-NMMA or
L-NAME alone did not
affect contraction. As also shown in Fig. 7, both
L-NMMA and
L-NAME blocked insulin's
inhibition of ionomycin (high
Ca2+i)-induced contraction. These data
suggest that insulin acutely inhibits ionomycin-induced contraction by a NOS-dependent mechanism.

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Fig. 7.
Graph showing effects of insulin,
NG-monomethyl-L-arginine
(L-NMMA), and
NG-nitro-L-arginine methyl ester
(L-NAME) on ionomycin-induced
contraction of individual VSM cells. Dishes of nonproliferated VSM
cells were preincubated for 30 min with PSS at 37°C with or without
1 nM insulin and/or 0.1 mM
L-NMMA or 0.1 mM
L-NAME, and baseline
photomicrographs were obtained. Ionomycin (2.5 µM) was added to bath
media, and subsequent photomicrographs were obtained 10 min later. Each
bar represents mean ± SE of mean contractions from baseline lengths
of 6-10 cells in each of 6 dishes.
* P < 0.05 vs. all other
values.
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DISCUSSION |
Insulin inhibits VSM contraction in vivo and in vitro (4, 18, 29, 47).
Insulin's ability to dilate vascular beds may have pathophysiological
importance, since resistance to insulin-induced vasodilation may be
associated with high blood pressure in essential hypertension,
non-insulin-dependent diabetes, and obesity (2, 19, 20, 22). Resistance
to insulin-induced vasodilation may be responsible, in part, for
increased blood pressure in certain genetically disposed individuals
(22).
The mechanism by which insulin inhibits contraction of normal VSM is
not completely understood. Previous studies (33, 38) have suggested
that insulin causes vasodilation in people by an endothelial
NOS-dependent process. On the other hand, it has been demonstrated that
insulin inhibits contraction of endothelium-denuded vascular strips
(47) and decreases the contractile agonist-induced Ca2+i transient and contraction of
endothelial cell-free cultured VSM cells (18, 29, 32, 37).
It has been reported, in studies using different endothelial cell-free
cultured VSM preparations, that insulin's inhibition of contractile
agonist-induced Ca2+i transients is due
to insulin's inhibition of voltage- and receptor-operated Ca2+ channels (18, 37), inhibition
of inositol trisphosphate-induced Ca2+ release from internal stores
(32), or stimulation of sarcolemmal Ca2+-ATPase-mediated
Ca2+ efflux (46). Regardless of
the mechanism, insulin's inhibition of the contractile agonist-induced
Ca2+i transient is probably responsible,
in part, for the inhibition of VSM contraction by insulin. It has never
been determined, however, whether insulin may also have an effect at
steps distal to Ca2+i. It is important to
determine this because therapies directed against hypertension in
insulin-resistant states should be targeted to all the VSM cell
abnormalities by which insulin resistance could cause increased VSM
contraction. To determine whether insulin can also inhibit VSM
contraction at sites distal to Ca2+i, in
the present studies, we tested whether insulin could inhibit VSM
contraction when Ca2+i is brought to a
concentration at which Ca2+
regulatory systems are saturated.
We have demonstrated that preincubating primary cultured canine VSM
cells with a physiological concentration of insulin for 30 min inhibits
high-Ca2+i-induced contraction. Insulin
inhibited contraction of high-
Ca2+i-induced VSM cell contraction when
Ca2+i was raised by permeabilizing cells
to Ca2+ with ionomycin or A-23187
(Fig. 2).
High Ca2+i was used as a tool to test
whether insulin could inhibit contraction at a site distal to
Ca2+i. Of course, the normal cell never
experiences such high Ca2+i levels.
Nevertheless, it has been reported that cGMP, protein kinase C, and
arachidonic acid affect VSM function appropriately in the presence of
similar high Ca2+i values (7, 12, 27). In
addition, when experiments were designed to clamp Ca2+i in these VSM cells to the high
physiological range (1 µM) with a
Ca2+-EGTA bath containing
ionomycin, the subsequent cell contractions were still inhibited by
insulin (Fig. 3).
We found that insulin did not affect basal
Ca2+i or the ionomycin-induced rise in
Ca2+i. It is possible that the
Ca2+ concentration inside putative
compartmentalized regions of the cytoplasm, accessible to the
contractile proteins, was lowered by insulin. Such an event would not
be detected by measuring Ca2+i in the
whole cell. We think that this possibility is unlikely, since A-23187
is known to permeabilize the plasma membrane and internal membrane
barriers within the cell to
Ca2+ (5, 8). Thus A-23187 would
be expected to raise the Ca2+
concentration throughout the cell. Nevertheless, insulin potently inhibited A-23187-induced contractions (Fig. 2).
It has been shown, in recent years, that VSM contraction is not only
controlled by Ca2+i but that
physiological modulation of contractile and related proteins also
controls tone. For example, myosin light-chain phosphatase (30) and
protein kinase C (3) activities can be regulated and affect VSM
contraction independently of Ca2+i. cGMP
is known to inhibit VSM contraction both by blunting the contractile
agonist-induced Ca2+i transient and at
the level of the contractile proteins per se (21, 24, 27). We have
previously reported that insulin increases cGMP production by
5-HT-treated VSM cells by an
L-NMMA-dependent mechanism and
that L-NMMA and
L-NAME block insulin's
inhibition of 5-HT-induced contraction. The cells also converted
arginine to citrulline in an
L-NMMA-dependent manner (16).
These data suggested that insulin inhibited 5-HT-induced contraction by
stimulating guanylate cyclase activity and cGMP production in these
cells by a NOS-dependent mechanism. The present studies are consistent
with the possibility that insulin inhibits VSM contraction at steps
distal to Ca2+i by increasing cGMP
production. We demonstrated that insulin increased cGMP production in
cells in which Ca2+i was raised by
ionomycin and that ODQ, a selective inhibitor of guanylate cyclase that inhibited cGMP production under these conditions (Fig. 4), blunted insulin's inhibition of ionomycin-induced contraction (Fig. 5). We
also showed that these cells express iNOS protein (Fig. 6) and that two
different inhibitors of NOS blocked insulin's ability to inhibit
high-Ca2+i-induced VSM cell contraction (Fig. 7). Taken together, the present data suggest that insulin inhibits VSM contraction at steps distal to
Ca2+i by a NOS- and cGMP-dependent
pathway.
We show, in the present study, that our cells had expressed iNOS even
though we had not exposed the cells to cytokines or endotoxin. This
result is in keeping with prior studies reporting iNOS protein
and/or message in unstimulated intact VSM preparations (9, 25,
40). This phenomenon is not well appreciated, and several examples
deserve mentioning. Darkow et al. (9) reported the presence of iNOS
protein and NOS activity in endothelial-denuded segments of coronary
arteries from unstimulated pigs. Tojo et al. (40) reported positive
immunostaining for iNOS in the renal afferent and efferent arterioles
from unstimulated rats. Mohaupt et al. (25) identified iNOS message in
renal arcuate and interlobular arteries from unstimulated rats using
microdissection and quantitative PCR techniques. In addition, several
authors have reported functional data consistent with the activity of a
NOS in endothelium-free unstimulated vascular preparations. Wood et al.
(45) identified a VSM relaxing factor obtained from unstimulated
endothelium-denuded bovine pulmonary artery with the pharmacological
properties of NO. Moritoki et al. (26) reported that
L-arginine induced cGMP formation and relaxation of unstimulated endothelium-denuded rat aortic
rings and that inhibitors of NOS blocked these effects. Schini and
Vanhoutte (34) showed that endothelium-denuded aortic rings from
unstimulated rats possess biochemical pathways converting L-arginine to NO. It is likely
that the latter studies also reflect the presence of iNOS in these
unstimulated VSM preparations. Although these results might also be
expained by the presence of endothelial or neuronal NOS, we are not
aware that either of these isoforms have been identified in VSM.
Our unstimulated cultured VSM cells had also expressed iNOS, and this
probably accounts for our present results and our previous findings
that insulin inhibited 5-HT-stimulated
Ca2+ influx and contraction of
these cells by a NOS-dependent mechanism. It should be pointed out that
the cell culture procedure may have induced iNOS in the cultured cells,
whereas iNOS may not have been present in the VSM cells in the intact
artery. Saito et al. (32) reported that insulin inhibited the ANG
II-induced Ca2+i transient in
unstimulated cultured rat VSM cells by an
L-NMMA-sensitive pathway.
Trovati et al. (42) reported that insulin stimulated cGMP
production in unstimulated human cultured VSM cells by an L-NMMA-sensitive mechanism. It
thus appears that a NOS (probably iNOS) is also responsible for some of
insulin's effects in other cultured VSM types. On the other hand, Han
et al. (14) reported that 60 mU/ml of insulin (0.4 µM) inhibited
norepinephrine-induced contraction of endothelium-denuded rat aortic
rings and that this was unaffected by
L-NMMA. The explanation for the
different results of Han et al. from ours and those of others regarding
the sensitivity to L-NMMA of
insulin's attenuation of contractile agonist-induced Ca2+i transport and contraction is
currently not known but may involve differences in experimental models
and designs or concentration of insulin employed.
Because iNOS message and protein and NOS activity have been found in
numerous unstimulated intact vascular smooth muscle preparations (9,
25, 26, 34, 40, 45), the present results may have physiological
relevance. Because VSM cells are stimulated to express iNOS in several
pathological states, such as regions of atherosclerosis (31, 43) or
balloon angioplasty (6), the present results may also have
pathophysiological relevance.
It should be pointed out that the VSM cell contraction experiments and
Ca2+i measurements in the present studies were performed with cells cultured in the presence of 0.5% FCS on rat
tail tendon collagen gels that had not proliferated, whereas the cGMP
and immunoblot experiments were performed with cells cultured to
confluence on a plastic surface in the presence of 10% FCS. It was not
feasible to do the latter measurements in nonproliferated cells because
their yield was too low and contaminating blood cells and/or
rat tail collagen could have caused misleading results. Nevertheless,
we have previously shown that Ca2+i responds to 5-HT and insulin in both VSM cell types in the same manner
(15, 18). Both are dependent on glucose transport for insulin's
effects, which in turn are blocked by
L-NMMA (16, 17). Both cell types
respond to ODQ.
The precise relationship among insulin, NOS, cGMP production, and
inhibition of high-Ca2+i-induced
contraction has not been determined by the present studies. One
possibility is that insulin, in the presence of high
Ca2+i, acutely increases NOS activity and
NO production. The latter could stimulate guanylate cyclase activity
and cGMP production. cGMP is known to inhibit VSM contraction at steps
distal to Ca2+i (21, 24, 27). We are not
aware, however, that any other hormone has been reported to acutely
regulate iNOS activity.
Alternatively, the present data are also consistent with the
possibility that insulin increases cGMP production and inhibits VSM
contraction by a mechanism which requires only a permissive role of NOS
but is not dependent on stimulation of NOS activity by insulin. Indeed,
it has been shown that whole body heating-induced vasodilation of
rabbit ear artery is dependent on the permissive presence of NO
production by NOS, but the actual mechanism for vasodilation is not via
increased NOS activity (11). Further research is necessary to determine
precisely how insulin inhibits VSM contraction at sites distal to
Ca2+i.
 |
ACKNOWLEDGEMENTS |
The authors acknowledge the excellent secretarial assistance of
Dorothy Priddy, Rose Marek, and Gina Henderson.
 |
FOOTNOTES |
These studies were supported by National Heart, Lung, and Blood
Institute Grants HL-50660 and HL-24585 and a grant from the Diabetes
Action Research and Education Foundation.
Address for reprint requests: A. M. Kahn, 4.138 MSB, University of
Texas Health Science Center, PO Box 20708, Houston, TX 77225.
Received 19 September 1997; accepted in final form 4 February
1998.
 |
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