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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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).

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.

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.

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.

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.

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.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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