INTRODUCTION

Top 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 N^G-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

Top 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 = K_d · (R  R_min/R_max  R) · Sf₂/Sb₂. Dissociation constant (K_d) was taken as 224 nM, and the symbols in the equation have their usual meaning (13). R_max, R_min, 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, 10⁷ 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).

	RESULTS

Top 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 R_max, which would give infinite values when substituted into the Ca²⁺_i equation.

View larger version (16K): [in this window] [in a new window]   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).

View larger version (34K): [in this window] [in a new window]   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.

View larger version (33K): [in this window] [in a new window]   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).

View larger version (19K): [in this window] [in a new window]   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.

View larger version (32K): [in this window] [in a new window]   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).

View larger version (66K): [in this window] [in a new window]   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.

View larger version (32K): [in this window] [in a new window]   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

Top 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.