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(Hypertension. 1997;30:928-933.)
© 1997 American Heart Association, Inc.

Articles

Insulin Acutely Inhibits Cultured Vascular Smooth Muscle Cell Contraction by a Nitric Oxide Synthase–Dependent Pathway

Andrew M. Kahn; Annat Husid; Julius C. Allen; Charles L. Seidel; ; Tom Song

From the Departments of Medicine, The University of Texas Health Science Center, Houston (A.M.K., A.H., T.S.), and Baylor College of Medicine (J.C.A.), Houston, Tex.

Correspondence to Andrew M. Kahn, MD, 4.138 MSB, University of Texas Health Science Center, PO Box 20708, Houston, TX 77225.

	Abstract

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Abstract Insulin acutely decreases contractile agonist-induced Ca²⁺ influx and contraction in endothelium-free cultured vascular smooth muscle (VSM) cells, but the mechanism is not known. Since it has been reported that insulin-induced vasodilation in humans is linked to nitric oxide synthase activity, we wished to determine whether insulin inhibits Ca²⁺ influx and contraction of cultured vascular smooth muscle cells by a nitric oxide synthase–dependent pathway. Primary cultures of endothelial cell–free VSM cells from canine femoral artery were preincubated with and without 1 nmol/L insulin for 30 minutes, and the 5-minute production of cGMP was measured. Insulin alone did not affect cGMP production, but in the presence of 10^-5 mol/L serotonin insulin stimulated cGMP production by 60%. N^G-monomethyl-L-arginine (0.1 mmol/L), an inhibitor of nitric oxide synthase, inhibited the conversion of arginine to citrulline by these cells, blocked insulin-stimulated cGMP production, and blocked the inhibition by insulin of 5-hydroxytryptamine (5-HT)–stimulated Mn⁺² (a Ca²⁺ surrogate) influx and contraction. Insulin did not affect contraction of VSM cells grown under conditions designed to deplete the cells of tetrahydrobiopterin, an essential cofactor of nitric oxide synthase. These studies demonstrate that insulin acutely inhibits 5-HT–stimulated Ca²⁺ influx and contraction of endothelium-free cultured VSM cells by a nitric oxide synthase–dependent mechanism.

Key Words: serotonin • cyclic GMP • fura-2 • Ca²⁺ transport • arginine

	Introduction

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Hypertension is associated with resistance to insulin-induced glucose disposal in non–insulin-dependent diabetes mellitus, obesity, and essential hypertension.^{1 2 3 4} It has been demonstrated that insulin inhibits vascular smooth muscle (VSM) contraction in vivo and in vitro^{5 6 7} and that resistance to insulin-induced vasodilation is associated with resistance to insulin-induced glucose disposal.⁸ It has been proposed that this resistance to insulin-induced vasodilation accounts, in part, for increased vascular tone and blood pressure in the aforementioned insulin-resistant states.⁹ Nevertheless, the mechanisms by which insulin inhibits contraction of normal VSM are not completely understood.

Several investigators have reported that insulin dilates human VSM in vivo by a NOS-dependent mechanism.^{10 11} We and others have reported that insulin acutely (within 30 minutes) decreases the contractile agonist-induced Ca²⁺_i transient and contraction of endothelial cell–free cultured VSM cells.^{7 12 13 14} Since VSM cells contain NOS in certain circumstances,¹⁵ we wished to test the hypothesis that these effects of insulin in cultured VSM cells are dependent on NOS activity. We report here that insulin acutely attenuates 5-HT–induced Ca²⁺ influx and contraction of endothelial cell–free cultured VSM cells by an NOS-dependent pathway.

	Methods

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Cell Culture of VSM Cells
Adult mongrel dogs of either sex were killed with intravenous pentobarbital sodium, and the femoral arteries were dissected free. VSM cells were cultured as previously described.^{16 17} The endothelium was stripped away and the media of the arteries were minced and incubated at 37°C in a solution containing elastase (type V; Sigma Chemical Co) and collagenase (type I; Worthington Biochemical). After 2 hours, the enzyme solution was discarded and replaced with fresh solution, and the tissue was incubated for an additional 2 hours. The dispersed cells were pelleted and washed three times in Hanks' balanced salt solution (GIBCO) and suspended to a density of 2x10⁵ cells/mL in DMEM (GIBCO) that contained 0.5% fetal calf serum (Cyclone), 1% glutamine, and 1% penicillin-streptomycin (PS) solution (10000 U/mL penicillin, 10 mg/mL streptomycin; Sigma). One milliliter of this suspension was placed in 35-mm plastic culture dishes (Falcon) on top of rat-tail tendon collagen gels, which were prepared as follows: Sprague-Dawley rat tail tendons were sterilized in 70% ethanol for 4 hours, minced, and extracted with 0.1% acetic acid for 48 hours at 4°C. The protein concentration of the supernatant was adjusted to 0.15 mg/mL and titrated to pH 8.0 with NaOH at 4°C. One mL was placed in 35-mm culture dishes at room temperature. The gels formed within 20 minutes. The gels were incubated with DMEM overnight before being seeded with dispersed VSM cells. After being seeded with cells, dishes were incubated in a humidified tissue culture incubator maintained at 37°C and equilibrated with 5% CO₂/95% air. After 72 hours and every 72 hours thereafter, the media were replaced with 1 mL of the same fresh medium. The cells were used for contraction studies 5 to 8 days after seeding.

Primary confluent cultures of these cells were also prepared, as previously described.¹⁷ The dispersed cells were pelleted as described above and suspended in complete DMEM, which contained 10% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin solution. The cell suspension was adjusted to 1.7x10⁶ cells/mL, and 0.3 mL was placed on the surface of 10x22-mm glass coverslips. Alternatively, the cell suspension was adjusted to 2x10⁵ cells/mL, and 1 mL was placed in 35-mm plastic culture dishes. The coverslips and dishes were placed in a humidified tissue culture incubator maintained at 37°C and equilibrated with 5% CO₂/95% air. After 72 hours and every 72 hours 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.¹⁶

Fura-2 Fluorescence
To assess the activity of Ca²⁺ influx pathways, we took advantage of the facts that Mn²⁺ can enter cells via Ca²⁺ influx pathways in many cell types, including VSM cells, and that once inside the cells, Mn²⁺ quenches fura-2 fluorescence.¹⁸ When the cells are exposed to extracellular Mn²⁺ and excited at a Ca²⁺-insensitive wavelength, a fall in fluorescence indicates Mn²⁺ influx irrespective of any possible changes in Ca²⁺_i.¹⁸ We previously used this technique to assess the activities of Ca²⁺ influx pathways in these VSM cells.¹⁷ Cells on coverslips were preincubated with 2.4 µmol/L fura-2-AM (Molecular Probes) that had been sonicated for 20 seconds in DMEM with 0.1% bovine serum albumin. A coverslip was placed in a quartz cuvette inside a fluorescent spectrophotometer (Perkin-Elmer LS-3B) such that the coverslip was anchored at its bottom and top and sat at a 45° angle to the excitation beam. The cuvette (2 mL), which was held in a thermostatted holder, was superfused with PSS at 37°C. A peristaltic perfusion pump delivered solution at 3 mL/min into the bottom of the cuvette via a glass capillary tube that ran on the opposite side of the coverslip from the excitation light and pumped solution at 3 mL/min from the top of the cuvette. The half-time for the turnover of solution in the cuvette was 0.46 minute. The storage flasks of solutions were kept at 37°C. The coverslip was superfused for 30 minutes at 37°C at 1 mL/min with PSS that contained (in mmol/L) 140 NaCl, 4 KCl, 1.8 CaCl₂, 0.8 MgSO₄, 5 glucose, and 10 HEPES-Tris, pH 7.4, plus 0.1% bovine serum albumin. The coverslip was then superfused with nominally Ca²⁺-free solution (PSS without CaCl₂) at 3 mL/min and excited through a 10-nm slit width at 362 nm, which is the Ca²⁺-insensitive (isosbestic) wavelength for fura-2.¹⁷ The emission at 510 nm was continuously measured through a 10-nm slit width. When a stable value was obtained, 0.5 mmol/L MnCl₂ was added to the superfusion solution, followed by the same solution with 10^-5 mol/L 5-HT, as previously described.¹⁷ The rate of fluorescence quenching was taken as an estimate of Mn²⁺ influx. To standardize this rate from coverslip to coverslip, the superfusion solution was changed again to nominally Ca²⁺-free PSS containing 5 mmol/L MnCl₂ plus 2.5 µmol/L ionomycin. This ionophore rapidly allowed Mn²⁺ influx and quenched the fluorescence to a stable basal value. The difference between this value and the initial stable value before the cells were exposed to Mn²⁺ was taken as 100 arbitrary fluorescence units, as previously described.¹⁷

cGMP Assay
Dishes of confluent VSM cells were preincubated in PSS at 37°C for 30 minutes. The media were changed to PSS with 0.5 mmol/L IBMX, and the cells were harvested 5 minutes later by adding 1 mL of ice-cold PSS containing 65% ethanol. The extract was centrifuged at 10 000g for 15 minutes at 4°C and the supernatant evaporated to dryness in a Speed Vac SC100 (Savant). cGMP content of acetylated samples was measured with the cGMP [¹²⁵I] Assay System (Amersham).

Thin-Layer Chromatography Analysis of Citrulline Production
Dishes of confluent cells were incubated with 1 mL PSS containing 5 µmol/L L-arginine and 0.2 µCi ¹⁴C-L-arginine (Amersham) for 4 hours at 37°C. Miconozole (75 µmol/L) was present to prohibit citrulline production by the P450 system. The incubation solution was discarded and the cells were homogenized in 0.4 mL deionized water. The homogenate was centrifuged at 10 000g for 10 minutes at 4°C and the supernatant evaporated to dryness in a Speed Vac SC 100. The residue was resuspended in 25 µL deionized water containing 2 mg/mL L-arginine plus 2 mg/mL L-citrulline and applied to silica gel 60 plates, 20x20 cm, aluminum (Whatman). A solvent system containing CHCl₃/CH₃OH/NH₄OH/H₂O, 1:4:2:1 (vol/vol) was used. Relative front for arginine and citrulline was 0.49 and 0.81, respectively, as determined by autoradiography of authentic ¹⁴C-L-arginine and ¹⁴C-L-citrulline standards. 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 liquid scintillation spectroscopy. 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 (about 1%, determined in each experiment by thin-layer chromatography, as described above). Each experiment was performed in triplicate.

Cell Contraction
After 5 to 8 days of culture, the cells grown on the collagen gels were used for contraction studies, as previously described.⁷ The dishes were placed on the heated (37°C) stage of a Nikon Diaphot inverted phase-contrast microscope, and the culture medium was replaced with PSS. After a 30-minute preincubation period, a field of at least 6 to 10 cells was photographed at 200x to obtain baseline images. The medium was replaced with the same experimental solution containing 10^-5 mol/L 5-HT, and after 10 minutes another photograph was taken of the same field.

Bovine insulin and 5-HT were obtained from Sigma. 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 set at P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
cGMP Production
We demonstrated previously that preincubation of primary cultured VSM cells from canine femoral artery for 20 to 30 minutes with physiological concentrations of insulin inhibited 5-HT–stimulated Ca²⁺ influx and contraction.^{7 17} We speculated that these effects of insulin were mediated by a VSM NOS since previous studies indicated that insulin causes acute vasodilation in vivo by an NOS-dependent mechanism.^{10 11} If insulin's effects in VSM were related to NOS, it was possible that insulin might increase cGMP production. To test this possibility, the accumulation of cGMP in confluent VSM cells was measured 5 minutes after adding IBMX to the incubation media (control). As shown in Fig 1, incubation of VSM cells for 5 minutes with 10^-5 mol/L 5-HT plus IBMX or preincubation for 30 minutes with 1 nmol/L insulin followed by incubation with insulin plus IBMX for 5 minutes did not affect the 5-minute accumulation of cGMP. When the cells were preincubated with insulin for 30 minutes and then incubated with IBMX and 5-HT plus insulin for 5 minutes, the accumulation of cGMP was increased over control cells by 63±22% (P<.05 versus control, 5-HT alone, or insulin alone). These data demonstrate that insulin increases the production of cGMP in 5-HT–stimulated cells.

View larger version (33K): [in this window] [in a new window]   Figure 1. Graph showing effects of insulin and 5-HT on cGMP accumulation. Dishes of confluent VSM cells were preincubated with PSS at 37°C with or without 1 nmol/L insulin for 30 minutes. IBMX (0.5 mmol/L) with or without 10-5 mol/L 5-HT was added to the bath media, and 5 minutes later the cells were harvested and cGMP was measured. Each bar represents the mean±SEM of the mean of triplicate measurements performed in six separate experiments. Data are expressed as a percentage of control cGMP accumulation in each experiment, which averaged 34.1 fmol/mg protein. *P<.05 vs control.

If this process depends on VSM NOS activity, L-NMMA, an inhibitor of NOS, should inhibit the stimulation of cGMP production by insulin. This was found to be the case. As shown in Fig 2, when compared with control cells, 0.1 mmol/L L-NMMA significantly decreased cGMP production, and insulin plus 5-HT significantly increased it. When compared with insulin plus 5-HT, the addition of L-NMMA significantly inhibited cGMP production to a level not different from L-NMMA alone. These data suggest that insulin plus 5-HT stimulates cGMP production in an NOS-dependent manner.

View larger version (31K): [in this window] [in a new window]   Figure 2. Graph showing effects of L-NMMA, insulin, and 5-HT on cGMP accumulation. Dishes of confluent VSM cells were preincubated with PSS at 37°C with or without 1 nmol/L insulin and/or 0.1 mmol/L L-NMMA for 30 minutes. IBMX (0.5 mmol/L) with or without 10-5 mol/L 5-HT was added to the bath media, and 5 minutes later the cells were harvested and cGMP was measured. Each bar represents the mean±SEM of the mean of triplicate measurements performed in five separate experiments. Data are expressed as a percentage of control cGMP accumulation in each experiment, which averaged 41.2 fmol/mg protein. *P<.05 vs control. P<.05 vs insulin plus 5-HT.

Citrulline Production
NOS converts arginine to citrulline and nitric oxide. To verify that these cells have functional NOS, dishes of cells were incubated with 5 µmol/L ¹⁴C-arginine, and the amount of ¹⁴C-citrulline produced was measured in both the presence and absence of 0.1 mmol/L L-NMMA. In the absence of L-NMMA (control), the cells took up an average of 620 pmol arginine/mg protein per hour of which 1.70±0.31% was converted to citrulline (n=4). In the presence of L-NMMA, ¹⁴C-arginine uptake was 97±8% of the amount taken up without L-NMMA (n=4, NS). The percentage of ¹⁴C-arginine taken up in the presence of L-NMMA that was converted to citrulline was 0.68±0.21% (n=4, P<.05 versus control). These data suggest that the cells have functional NOS since they convert arginine to citrulline by a L-NMMA-inhibitable mechanism.

Cell Contraction
Since we had previously shown that insulin inhibited 5-HT–stimulated Ca²⁺ influx and contraction of these VSM cells,^{7 17} we wished to determine whether these effects are dependent on VSM NOS by determining whether inhibitors of NOS blunted these actions of insulin. As shown in Fig 3, 1 nmol/L insulin inhibited 5-HT–induced contraction of individual VSM cells. L-NMMA (0.1 mmol/L) alone did not affect 5-HT–induced contraction, but it eliminated insulin's ability to inhibit contraction. As shown in Fig 3, dibutyryl cGMP inhibited contraction in these cells, as is the case for VSM in general.¹⁹ To rule out the possibility that L-NMMA nonspecifically negates the action of all in- hibitors of VSM contraction, we determined whether L-NMMA would blunt the inhibition of VSM contraction by nitroprusside, an agent that donates nitric oxide to the VSM cell independently of NOS activity.²⁰ As also shown in Fig 3, 0.1 mmol/L nitroprusside inhibited VSM contraction, and L-NMMA did not alter this effect. L-NMMA competes with L-arginine as a substrate for NOS and inhibits the enzyme's activity.²¹ In order to demonstrate further the specificity of L-NMMA to block insulin's inhibition of VSM contraction, we determined whether a 10-fold excess of L-arginine would prevent 0.1 mmol/L L-NMMA from blunting insulin's inhibition of 5-HT–induced contraction. As shown in Fig 3, 1 mmol/L L-arginine alone did not affect insulin-inhibited contraction. Fig 3 also shows that 0.1 mmol/L L-NMMA did not blunt the inhibition of contraction by insulin in the presence of this large concentration of L-arginine. Taken together, the data in Fig 3 indicate that insulin inhibits 5-HT–induced contraction of VSM by an NOS-dependent mechanism.

View larger version (32K): [in this window] [in a new window]   Figure 3. Graph showing effects of insulin on 5-HT–induced contraction of individual VSM cells. Dishes of nonproliferated VSM cells were preincubated in PSS at 37°C for 30 minutes with no additions or with (in µmol/L) 0.001 insulin, 5 dibutyryl cGMP, 100 L-NMMA, insulin plus L-NMMA, 100 nitroprusside (NTP), L-NMMA plus NTP, 1000 arginine plus insulin, or insulin plus arginine plus L-NMMA, and baseline photomicrographs were obtained. 5-HT (10-5 mol/L) was added to the bath media and subsequent photomicrographs were obtained 10 minutes later. Each bar represents the mean±SEM of mean contractions from baseline lengths of 6 to 10 cells in each of six dishes. *P<.01 vs control.

To demonstrate further that the inhibition of 5-HT–stimulated contraction by insulin was dependent on NOS, we determined whether another NOS inhibitor, L-NAME, could inhibit this effect of insulin.²¹ As shown in Fig 4, 0.1 mmol/L L-NAME alone did not affect 5-HT–induced VSM contraction, but it prevented insulin's inhibition of 5-HT–induced contraction. The fact that two different inhibitors of NOS (L-NMMA, L-NAME) block insulin's ability to attenuate 5-HT–induced VSM cell contraction supports the view that this effect of insulin is dependent on NOS activity.

View larger version (32K): [in this window] [in a new window]   Figure 4. Graph showing effects of insulin and L-NAME on 5-HT–induced contraction of individual VSM cells. Dishes of nonproliferated VSM cells were preincubated with or without 1 nmol/L insulin and/or 0.1 mmol/L L-NAME for 30 minutes, and the 5-HT–induced contraction of cells was assayed as described in the legend of Fig 3. Each bar represents the mean±SEM of mean contractions from baseline lengths of 6 to 10 cells in each of six dishes. *P<.05 vs control.

We predicted that insulin would have no effect on contraction if the cells had been previously depleted of BH₄, an essential cofactor for NOS activity.^{15 21} BH₄ is produced by the enzyme GTP cyclohydrolase I.¹⁵ It has previously been demonstrated that prolonged treatment of VSM cells with DAHP, an inhibitor of GTP cyclohydrolase I, depletes cells of BH₄.²² As shown in Fig 5, insulin inhibits 5-HT–induced contraction of control cells. When the cells had been preincubated for 30 minutes or 18 hours with DAHP, 5-HT–induced contraction was not affected. However, as also shown in Fig 5, insulin no longer inhibited 5-HT–induced contraction of cells after 18 hours of preincubation with DAHP. This suggests that an adequate level of BH₄ is necessary for insulin to inhibit VSM contraction. As also shown in Fig 5, a relatively brief 30-minute preincubation of cells with DAHP did not blunt insulin's effect. This finding tends to rule out the possibility that the prolonged exposure to DAHP did not merely blunt insulin's action by a nonspecific effect, but that the drug's effect to deplete the cells of BH₄ was required to block insulin's ability to inhibit VSM contraction. These data further support the notion that the acute effect of insulin to inhibit contraction is dependent on NOS activity.

View larger version (35K): [in this window] [in a new window]   Figure 5. Graph showing effect of DAHP and insulin on 5-HT–induced contraction of individual VSM cells. Dishes of nonproliferated cells were preincubated for 18 hours with or without 1 mmol/L DAHP in DMEM plus 0.5% fetal calf serum in a tissue culture incubator at 37°C in a humidified atmosphere of 5% CO2/95% air. Cells were then preincubated in PSS at 37°C for 30 minutes with or without the continued presence of DAHP in the presence and absence of 1 nmol/L insulin, and other cells that had not been exposed to DAHP were preincubated with 1 mmol/L DAHP with and without insulin for 30 minutes. The 5-HT–induced contraction of cells was then measured as described in the legend of Figure 3. Each bar represents the mean±SEM of mean contractions from baseline lengths of 6 to 10 cells in each of six dishes. *P<.05 vs control.

Mn²⁺ Influx Rate
We have previously demonstrated that insulin inhibits both 5-HT–induced Ca²⁺ influx and contraction in these VSM cells.^{7 17} The former effect of insulin resulted in a blunted 5-HT–induced Ca²⁺_i transient that presumably contributed to insulin's inhibition of contraction.¹⁷ The data in Figs 3 through 5 indicate that insulin-inhibited contraction is dependent on VSM NOS activity, and the data in Figs 1 and 2 suggest that insulin stimulates cGMP production by 5-HT–treated cells via an NOS-dependent pathway. Since others have reported that cGMP inhibits Ca²⁺ channels in VSM,^{23 24 25} we predicted that L-NMMA would block insulin's inhibition of 5-HT–stimulated Ca²⁺ influx. We assessed Ca²⁺ influx by measuring the rate of quenching of intracellular fura-2 fluorescence by extracellular Mn²⁺. We determined whether L-NMMA would eliminate insulin's ability to reduce the 5-HT–stimulated rate of quenching of fura-2 fluorescence, which we had previously shown.¹⁷ As shown in Fig 6, the basal rate of fura-2 quenching by Mn²⁺ was not affected by insulin or L-NMMA. When the cells were exposed to 5-HT, the rate of fura-2 fluorescence quenching was increased and insulin inhibited that effect. We have previously reported these findings, which indicated that 5-HT stimulates Ca²⁺ influx by an insulin-inhibited mechanism.¹⁷ As also shown in Fig 6, L-NMMA alone did not affect the 5-HT–stimulated rate of fura-2 quenching, but insulin no longer inhibited this rate in the presence of L-NMMA. These data indicate that insulin-inhibited Ca²⁺ influx was also dependent on VSM NOS activity.

View larger version (34K): [in this window] [in a new window]   Figure 6. Graph showing effects of insulin, L-NMMA, and 5-HT on Mn2+-induced quenching of intracellular fura-2 in VSM cells. Confluent cells on a coverslip were loaded with fura-2 and preincubated at 37°C in PSS with or without 1 nmol/L insulin and/or 0.1 mmol/L L-NMMA for 30 minutes. The cells were excited at 362 nm, and the fluorescence emission at 510 nm was monitored. The cells were superfused with PSS with and without insulin and/or L-NMMA in the nominal absence of Ca2+ plus 0.5 mmol/L Mn2+. The rate of quenching of fura-2 fluorescence was measured (Basal). After 3 minutes, 10-5 mol/L 5-HT was added to the superfusion solution and the rate of quenching measured again. Each bar represents the mean±SEM from six separate experiments. *P<.05 vs control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous studies have indicated that insulin induces vasodilation in people by an NOS-dependent mechanism.^{10 11} It is not known whether this in vivo NOS-dependent effect of insulin is mediated by endothelial cell NOS, VSM cell NOS, or both. Since we and others have shown that insulin acutely lowers Ca²⁺_i^{7 12 13 14} and contraction^{6 7} in endothelial cell–free VSM cells, we sought in the present studies to determine whether the acute inhibition of contraction of cultured VSM cells by insulin is dependent on NOS activity in the VSM cell. The present data show that insulin plus 5-HT increases the production of cGMP in these cells in a L-NMMA–inhibitable manner. This indicates that the insulin-stimulated component of cGMP was dependent on NOS activity. A functional NOS is present in the cells since they converted arginine to citrulline by a L-NMMA–inhibitable mechanism.

The present data also show that insulin inhibits 5-HT–stimulated Mn²⁺ (a surrogate for Ca²⁺) influx and inhibits contraction of these cells in a L-NMMA–inhibitable and BH₄-dependent manner. L-NAME, another inhibitor of NOS, also blocked insulin's inhibition of 5-HT–induced contraction. L-NMMA did not affect the inhibition of contraction by nitroprusside, a direct NO donor, and it did not block insulin's inhibition of contraction in the presence of 10-fold excess L-arginine. Taken together, the present data demonstrate that the inhibition of 5-HT–induced VSM contraction by insulin is dependent on NOS in the VSM cell itself.

This study does not determine which isoform or isoforms of NOS are responsible for the present findings. Since 5-HT alone increases Ca²⁺_i in these cells but does not by itself increase the production of cGMP, the NOS in these cells must not be a Ca²⁺-sensitive isoform such as the constitutive NOS found in endothelial cells or neurons.^{15 21} The fact that insulin blunts the 5-HT–induced Ca²⁺ transient in these cells⁷ yet increases the production of cGMP by 5-HT–treated cells supports further the notion that a Ca²⁺-sensitive NOS was not responsible for these findings. Indeed, a Ca²⁺-sensitive NOS (cNOS) has not been found in VSM,¹⁵ but an iNOS has been described in this tissue. It seems that a VSM iNOS is likely to be responsible for the present findings. Although VSM iNOS has usually been identified after exposing the tissue to various cytokines for several hours, other investigators have demonstrated the presence of iNOS in VSM cells that had not been treated with such agents.^{26 27 28 29} Since the time course of insulin's effects was acute (30 minutes), it is unlikely that insulin-induced NOS transcription and translation were responsible for the present findings.

The present studies are consistent with previous reports of the relationship between insulin, NOS, and cGMP in VSM. Saito et al reported that insulin inhibited the angiotensin II–induced Ca²⁺_i transient in cultured rat VSM cells by a L-NMMA–sensitive pathway.¹² Trovati et al reported that insulin stimulated cGMP production in human cultured VSM cells by a L-NMMA–dependent mechanism.³⁰ Thus, the present report is consistent with these prior studies and extends the known relationships between insulin and VSM NOS, cGMP, and Ca²⁺_i to the inhibition of VSM contraction.

We demonstrated previously that the inhibition of Ca²⁺ influx and contraction of individual primary cultured VSM cells by insulin is dependent on insulin-induced glucose uptake.³¹ Intracellular glucose is converted in part to diacylglycerol, and high extracellular glucose concentration has been linked to diacyglyerol production and activation of protein kinase C in VSM.^{32 33} Although speculative, it is possible that insulin-induced glucose transport caused a rise in intracellular diacylglycerol concentration, and that when coupled to a 5-HT–induced rise in Ca²⁺_i, activated one or more Ca²⁺-sensitive isoforms of protein kinase C in the VSM cell. Increased protein kinase C activity might acutely stimulate iNOS activity in the VSM cell by posttranslational modification. Preliminary data from our laboratory have shown that raising Ca²⁺_i with ionomycin does not stimulate cGMP production in these VSM cells, but the addition of insulin to ionomycin-treated cells stimulates cGMP production by 44%.³⁴ Thus, we hypothesize that other contractile agonists that raise Ca²⁺_i in VSM cells, such as angiotensin II or norepinephrine, would also cause an insulin-induced increase in guanylate cyclase activity. Additional research is needed to test this hypothesis.

Alternatively, the present data are also consistent with the possibility that insulin increases cGMP production and inhibits VSM contraction by a mechanism that requires only a permissive role of NOS but that is not dependent on stimulation of NOS activity by insulin. Indeed, others have shown that ₂-adrenoceptor agonist–induced vasodilation of rat middle cerebral artery,³⁵ and whole body heating-induced vasodilation of rabbit ear artery³⁶ are dependent on the permissive presence of nitric oxide production by NOS, but their mechanisms for vasodilation are not by increasing NOS activity. Further research is necessary to determine the precise mechanisms by which insulin-inhibited VSM contraction is dependent on VSM NOS activity.

	Selected Abbreviations and Acronyms

 

BH4 = tetrahydrobiopterin Ca2+i = intracellular Ca2+ cNOS = constitutive nitric oxide synthase DHAP = 2, 4-diamino-6-hydroxypyrimidine DMEM = Dulbecco's modified Eagle's medium 5-HT = 5-hydroxytryptamine IBMX = isobutylmethylxanthine iNOS = inducible nitric oxide synthase L-NAME = NG-nitro-L-arginine-methyl ester L-NMMA = NG-monomethyl-L-arginine NOS = nitric oxide synthase PSS = physiological salt solution VSM = vascular smooth muscle

	Acknowledgments

 
These studies were supported by grants HL-4080 and HL-24585 from the National Heart, Lung, and Blood Institute and a grant from the Diabetes Action Research and Education Foundation.

Received February 12, 1997; first decision February 26, 1997; accepted February 26, 1997.

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