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(Circulation. 1995;92:1597-1603.)
© 1995 American Heart Association, Inc.

Articles

Insulin-Stimulated Glucose Transport Inhibits Ca²⁺ Influx and Contraction in Vascular Smooth Muscle

Andrew M. Kahn, MD; Richard A. Lichtenberg, MD; Julius C. Allen, PhD; Charles L. Seidel, PhD; Tom Song, BS

From the Departments of Medicine, University of Texas Medical School, Houston (A.M.K., T.S.); Baylor College of Medicine, Houston (J.C.A., C.L.S.); and Mercy Haverford Hospital, Havertown, Pa (R.A.L.).

Correspondence to Andrew M. Kahn, MD, University of Texas Medical School, PO Box 20708, MSB 4.138, Houston, TX 77025.

	Abstract

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Background Insulin attenuates serotonin-induced Ca²⁺ influx, the intracellular Ca²⁺ transient, and contraction of cultured vascular smooth muscle cells from dog femoral artery. These studies were designed to test whether insulin-induced glucose transport was an early event leading to the inhibitory effects of insulin on Ca²⁺ influx, intracellular Ca²⁺ concentration, and contraction in these cells.

Methods and Results Insulin 1 nmol/L stimulated the 30-minute uptake of [³H]2-deoxyglucose in these cells via a phloridzin-inhibitable mechanism. Contraction of individual cells was measured by photomicroscopy, intracellular Ca²⁺ concentration was monitored by measuring fura 2 fluorescence by use of Ca²⁺-sensitive excitation wavelengths, and Ca²⁺ influx was estimated by the rate of Mn²⁺ quenching of intracellular fura 2 fluorescence when excited at a Ca²⁺-insensitive wavelength. In the presence of 5 mmol/L glucose, preincubation of cells for 30 minutes with 1 nmol/L insulin inhibited 10^-5 mol/L serotonin-induced contraction of individual cells by 62% (P<.01) and decreased the serotonin-stimulated component of Mn²⁺ influx by 78% (P<.05). Removing glucose from the preincubation medium or adding 1 mmol/L phloridzin completely eliminated these effects of insulin. Insulin lowered the serotonin-induced intracellular Ca²⁺ peak by 37% (P<.05), and phloridzin blocked this effect of insulin. When glucose uptake was increased to the insulin-stimulated level by preincubation of the cells for 30 minutes with 25 mmol/L glucose in the absence of insulin, serotonin failed to stimulate Mn²⁺ influx, the serotonin-induced Ca²⁺ peak was decreased by 46% (P<.05), serotonin-induced contraction was inhibited by 60% (P<.01), and addition of insulin did not further inhibit contraction.

Conclusions Since the effects of insulin on serotonin-stimulated Ca²⁺ transport, intracellular Ca²⁺ concentration, and contraction were dependent on glucose transport and were duplicated when glucose transport was stimulated by high extracellular glucose concentration rather than insulin per se, it is concluded that insulin-stimulated glucose transport is an early event that leads to decreased Ca²⁺ influx and contraction in vascular smooth muscle.

Key Words: manganese • hypertension • insulin • fura 2

	Introduction

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The effects of insulin on vascular smooth muscle (VSM) function have recently received a great deal of attention.¹ With some exceptions,^{2 3} most investigators report that insulin inhibits VSM contraction,^{4 5 6 7 8 9 10} but the mechanism is unclear. Several laboratories have shown that insulin attenuates the agonist-induced intracellular Ca²⁺ (Ca²⁺_i) transient in VSM cells (VSMCs), which may be responsible, at least in part, for insulin-induced inhibition of contraction.^{7 11 12 13} We recently reported that physiological concentrations of insulin inhibit serotonin (5-HT), angiotensin II, and high extracellular K⁺–induced contractions of isolated, cultured, nonproliferated VSMCs from canine femoral artery. The insulin-induced inhibition of 5-HT–stimulated contraction was associated with a 50% reduction of the 5-HT–stimulated Ca²⁺_i transient.⁷ We also reported that this attenuation of the Ca²⁺_i transient in confluent primary cultures of these cells was caused by inhibition of 5-HT–induced Ca²⁺ influx but not by stimulation of Ca²⁺ efflux or inhibition of Ca²⁺ release from intracellular stores.¹⁴

Despite the general acceptance that insulin attenuates the agonist-induced Ca²⁺_i transient in VSM, the mechanism of action of insulin is unclear. It has recently been reported that high extracellular glucose concentration inhibits agonist-induced Ca²⁺ influx¹⁵ and contraction¹⁶ in cultured rat VSMCs and the Ca²⁺_i transient in cultured rat mesangial cells, a VSMC-like cell.¹⁷ Since we found that insulin causes the same effects in primary cultures of dog femoral artery VSMCs^{7 14} and since insulin stimulates glucose uptake in cultured VSMCs,^{18 19 20} it seemed possible that insulin and high extracellular glucose both lead to their effects via a common pathway, namely, by increasing glucose influx.

In the present study, we present data that demonstrate that insulin-induced glucose influx is an early event leading to the inhibition by insulin of 5-HT–induced Ca²⁺ influx, the Ca²⁺_i transient, and contraction of these cells. We show that removing glucose from the preincubation and incubation media prevents the effects of insulin and that in the presence of glucose, inhibition of insulin-induced glucose transport also prevents the effects of insulin. Finally, we show that in the absence of insulin, stimulating glucose uptake by increasing the extracellular glucose concentration mimics the effects of a physiological concentration of glucose plus insulin on 5-HT–induced Ca²⁺ influx and the Ca²⁺_i transient. High extracellular glucose per se also inhibits 5-HT–induced contraction, and insulin had no additional effect.

	Methods

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Cell Culture of Nonproliferating Cells
Adult mongrel dogs of either sex were killed with intravenous pentobarbital sodium, and the femoral arteries were dissected free. VSMCs were cultured as previously described.²¹ 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 HBSS (Gibco) and suspended to a density of 2x10⁵ cells/mL in DMEM (Gibco) that contained 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) on top of rat-tail tendon collagen gels, which were prepared as follows²¹ : 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 VSMCs. 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.

Cell Culture of Confluent Cells
Since isotope uptake and fluorescence measurements are more easily performed with confluent than individual cells, primary confluent cultures of these cells were prepared as previously described.²¹ The dispersed cells were pelleted as described above and suspended in complete DMEM, which contained 10% FCS, 1% glutamine, and 1% PS 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 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.²¹

[³H]2-Deoxyglucose Uptake
Confluent cells grown in dishes were preincubated at 37°C for 30 minutes in 1 mL of physiological salt solution (PSS) that contained (in mmol/L) NaCl 140, KCl 4, CaCl₂ 1.8, MgSO₄ 0.8, glucose 5, and HEPES-Tris 10, pH 7.4. The cells were then incubated at 37°C in 0.7 mL of PSS that contained 0.7 µCi [³H]2-deoxyglucose ([³H]2-DOG) (8.1 Ci/mmol, New England Nuclear) with or without 1 nmol/L insulin (bovine pancreas, Sigma) and with or without 1 mmol/L phloridzin (Sigma). Isotope uptake was measured for 30 minutes, since we wished to correlate the effects of insulin on glucose uptake with those on Ca²⁺ transport. We have found that incubating cells with insulin for 30 minutes causes dependable changes in Ca²⁺ transport and contraction.^{7 14} The cells were washed by rapid rinsing of the dishes six times with 5.5 mL ice-cold 100 mmol/L MgCl₂. The cells were dissolved in 1 mL of 1 mol/L NaOH. [³H]2-DOG content was measured by liquid scintillation spectroscopy, and protein was measured by the method of Lowry. In each experiment, the isotopic uptake solution was applied to cells and then removed 1 to 2 seconds later, and the cells were washed, dissolved in NaOH, and counted to estimate binding and/or trapping of isotope by the cells and plastic dishes. This value, which was always <10% of total isotope taken up by a dish in 30 minutes, was subtracted from all uptake values. Isotope uptake experiments were performed in triplicate. Absolute [³H]2-DOG uptake under a given set of experimental conditions varied between experiments, but the relative effect of an experimental perturbation in a given experiment was highly reproducible from experiment to experiment. For this reason, in each experiment, data were expressed as a percent of the value obtained under defined control conditions (5 mmol/L glucose, no insulin). The 30-minute uptake of 0.1 µmol/L [³H]2-DOG under these conditions averaged 0.64 pmol/mg protein.

Fluorescence Measurements of Ca²⁺ Transport
A coverslip with cells 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. Before the cells were exposed to Ca²⁺-sensitive dye, they were excited at 340 nm through a 10-nm slit width, the emitted light at 510 nm was monitored through a 10-nm slit width, and the intensity was blanked to zero units. The excitation beam was changed to 380 nm, and the emission signal at 510 nm was arbitrarily set to 150 units. This value was subtracted from all subsequent values obtained after cells had been loaded with dye to obtain fluorescence data from the dye itself without the contribution of autofluorescence from the cells or other components in the cuvette. The cells were loaded in the cuvette for 45 minutes with 2.4 µmol/L fura 2-AM (Molecular Probes) that had been sonicated for 20 seconds in DMEM with 0.1% BSA. The cuvette was perfused again for 30 minutes at 1 mL/min with PSS with and without 1 µmol/L insulin, 1 mmol/L phloridzin, 20 mmol/L extra glucose, 20 mmol/L mannitol, or zero glucose, according to the goals of each experiment. The cuvette was then perfused at 3 mL/min with these solutions, and baseline fluorescence emissions were obtained by rapidly alternating the excitation wavelength between 340 and 380 nm and recording the 510-nm emission intensity. 5-HT 10^-5 mol/L was added to the perfusion solution, and fluorescence measurements continued for 20 minutes. [Ca²⁺]_i values were calculated from the fluorescence ratio recordings by the following formula: [Ca²⁺]_i=K_d[(R-R_min)/(R_max-R)](Sf₂/Sb₂). K_d was taken as 224 nmol/L, and the symbols in the equation have their usual meanings.²² R_max, R_min, and Sf₂/Sb₂ were determined at the end of each experiment by measuring the 510-nm emissions at 340- and 380-nm excitation while the cells were superfused with PSS containing 4 mmol/L Ca²⁺ plus 2.5 µmol/L ionomycin, followed by superfusion with nominally Ca²⁺-free PSS plus 10 mmol/L EGTA.

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 VSMCs, and that once inside the cells, Mn²⁺ quenches fura 2 fluorescence.²³ 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. We previously used this technique to assess the activities of Ca²⁺ influx pathways in these VSMCs.¹⁴ Cells on coverslips were loaded with fura 2 and preincubated for 30 minutes in the desired solutions as described above. The coverslips were superfused with nominally Ca²⁺-free solution (PSS without CaCl₂) at 3 mL/min and were excited through a 10-nm slit width at 362 nm, which is the Ca²⁺-insensitive (isosbestic) wavelength for fura 2. 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.¹⁴ 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 (AFUs), as previously described.¹⁴

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 21} 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 the desired solutions as described above. After a 30-minute preincubation period, a field of at least 6 to 10 cells was photographed at x200 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. The lengths of the longest axes of 6 to 10 arbitrarily chosen cells were measured in the first photograph, and the lengths of the same cells were measured in the subsequent photograph. For each cell, the percent contraction from the baseline length was calculated, and these values were averaged for all cells.

Statistical Analysis
Statistical analysis was performed on paired and unpaired data with Student's t test and ANOVA with multiple comparisons with the Newman-Keuls test. Statistical significance was taken as a value of P<.05.

	Results

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[³H]2-DOG Uptake
A physiological concentration of insulin stimulates the uptake of glucose in cultured VSMCs from several sources.^{18 19 20} To determine whether this applied to cultured VSMCs from canine femoral artery, we measured the 30-minute uptake of [³H]2-DOG with and without 1 nmol/L insulin and in the presence and absence of 1 mmol/L phloridzin, a glucose transport inhibitor.^{24 25} As shown in Fig 1, insulin significantly stimulated [³H]2-DOG uptake by 22±9% (P<.05) in the absence of phloridzin. In the presence of phloridzin, [³H]2-DOG uptake was inhibited by 50% and was not stimulated by insulin. Thus, a physiological concentration of insulin stimulates glucose uptake by a phloridzin-sensitive mechanism in these VSMCs.

View larger version (14K): [in this window] [in a new window]   Figure 1. Bar graph showing effects of insulin and phloridzin on [3H]2-deoxyglucose ([3H]2-DOG) uptake in vascular smooth muscle cells. Confluent cells in dishes were preincubated in physiological salt solution at 37°C for 30 minutes. The 30-minute uptake of 0.1 µmol/L [3H]2-DOG was then measured in the presence or absence of 1 nmol/L insulin and/or 1 mmol/L phloridzin. Values are expressed as a percent of [3H]2-DOG uptake in the absence of insulin and phloridzin, which averaged 0.64 pmol/mg protein. Each bar represents the mean±SEM of four experiments. *P<.05 vs control.

Fluorescence Quenching by Mn²⁺
We sought to determine whether insulin-stimulated glucose uptake was an early event leading to the inhibition by insulin of Ca²⁺ influx and contraction in these cells. We recently showed that in the presence of 5 mmol/L glucose, insulin inhibited the 5-HT–stimulated component of Mn²⁺ influx (a marker for Ca²⁺ influx).¹⁴ If the stimulation of glucose uptake by insulin was responsible for insulin's inhibition of Mn²⁺ uptake, insulin should not affect Mn²⁺ uptake in the absence of glucose.

As shown in representative tracings in Fig 2, 1 nmol/L insulin in the presence of 5 mmol/L glucose inhibited 5-HT–stimulated Mn²⁺ influx. Fig 3 shows the rates of Mn²⁺ influx in the presence of 5 mmol/L glucose before and after 5-HT and the 5-HT–stimulated component of Mn²⁺ influx for seven coverslips each in the presence and absence of insulin. Fig 4 shows these rates in cells preincubated and incubated in the absence of glucose. Note that insulin significantly inhibited the 5-HT–stimulated component of Mn²⁺ influx in the presence of glucose (Fig 3). These data are similar to those we reported previously.¹⁴ Also note that in the absence of glucose (Fig 4), the 5-HT–stimulated component of Mn²⁺ influx was greater than in the presence of glucose (Fig 3) (12.2±2.3 versus 3.6±0.4 AFU/min; P<.01), and insulin did not decrease it.

View larger version (17K): [in this window] [in a new window]   Figure 2. Graph showing effects of insulin and serotonin (5-HT) on Mn2+-induced quenching of intracellular fura 2 in vascular smooth muscle cells. Confluent cells on a coverslip were loaded with fura 2 and incubated for 30 minutes in physiological salt solution (PSS) with or without 1 nmol/L insulin. The cells were excited at 362 nm, and the fluorescence emission at 510 nm was monitored while they were superfused with PSS with or without insulin in the nominal absence of Ca2+ plus 0.5 mmol/L Mn2+. At the arrow, 10-5 mol/L 5-HT was added to the superfusion solution. Tracings are from a representative experiment.

View larger version (18K): [in this window] [in a new window]   Figure 3. Bar graph showing effects of insulin and serotonin (5-HT) in the presence of 5 mmol/L glucose on Mn2+ influx in vascular smooth muscle cells. Experiments were performed with 5 mmol/L glucose in all solutions, as in Fig 1, and the rates of fluorescence quenching before and after addition of 5-HT to the superfusion solution were taken as estimates of Mn2+ influx under basal and 5-HT–stimulated conditions, respectively. For each coverslip, the difference between the latter values was taken as the 5-HT–stimulated component of Mn2+ influx. Each bar represents the mean±SEM from six coverslips, each preincubated with or without 1 nmol/L insulin. +P<.05 vs basal; *P<.05 vs control.

View larger version (19K): [in this window] [in a new window]   Figure 4. Graph showing effects of insulin and serotonin (5-HT) in the absence of glucose on Mn2+ influx in vascular smooth muscle cells. Experiments were performed as in Figs 2 and 3 except that after the cells were loaded with fura 2, no glucose was present in the preincubation or incubation solutions. Each bar represents the mean±SEM from six coverslips, each preincubated with or without 1 nmol/L insulin. *P<.01 vs basal.

Cell Contraction
Since we had previously shown that the inhibition by insulin of 5-HT–stimulated Ca²⁺ influx was responsible for the attenuation by insulin of the Ca²⁺_i transient¹⁴ and that the latter was associated with the inhibition of contraction by insulin in cultured VSMCs from canine femoral artery,⁷ it was expected that the effect of insulin to inhibit VSM contraction would be impaired in the absence of glucose.

To test this, VSMCs were grown in the presence of 0.5% FCS on collagen gels, and 5-HT–stimulated contraction was measured in cells preincubated in the presence and absence of glucose with and without insulin. As shown in Fig 5, insulin inhibited contraction in glucose-containing media, in agreement with our previous findings.⁷ As also shown, contraction was not significantly affected by preincubation of the cells for 30 minutes in the absence of glucose, but insulin no longer inhibited contraction under these conditions.

View larger version (11K): [in this window] [in a new window]   Figure 5. Bar graph showing effects of glucose and insulin on serotonin (5-HT)–induced contraction in vascular smooth muscle (VSM) cells. Nonproliferated VSM cells were grown on collagen gels preincubated in physiological salt solution (PSS) or glucose-free PSS plus 4% BSA, with or without 1 nmol/L insulin for 30 minutes, and a baseline photomicrograph was obtained. 5-HT 10-5 mol/L was added to the bath media, and a subsequent photomicrograph was 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.

Effects of Phloridzin and 25 mmol/L Glucose
The above data suggest that the effect of insulin on Ca²⁺ transport and contraction was dependent on glucose transport. To further test this hypothesis, the influx of Mn²⁺ and contraction were measured in cells preincubated in the presence and absence of insulin in glucose-containing media with or without 1 mmol/L phloridzin. This compound inhibits basal and insulin-stimulated glucose uptake in these cultured VSMCs (Fig 1). As shown in Fig 6, in the absence of insulin, the Mn²⁺ influx rate in the presence of phloridzin was stimulated by 5-HT. As also shown, insulin did not inhibit the 5-HT–stimulated component of Mn²⁺ influx in the presence of phloridzin. Furthermore, the 5-HT–stimulated component of Mn²⁺ influx was significantly greater than in the absence of phloridzin, as shown in Fig 3 (14.7±3.2 versus 3.6±0.5 AFU/min; P<.01), and was of the same magnitude as in the absence of glucose (Fig 4). These data further support the notion that insulin-induced glucose transport is responsible for the inhibition by insulin of 5-HT–stimulated Ca²⁺ influx.

View larger version (19K): [in this window] [in a new window]   Figure 6. Bar graph showing effects of insulin and serotonin (5-HT) in the presence of phloridzin and 5 mmol/L glucose on Mn2+ influx in vascular smooth muscle cells. Experiments were performed as in Figs 2 and 3 except that 1 mmol/L phloridzin was present in all preincubation and incubation solutions. Results are the mean±SEM from six coverslips, each preincubated with or without 1 nmol/L insulin. *P<.01 vs basal.

Since phloridzin blocked the effect of insulin to inhibit 5-HT–stimulated Mn²⁺ (and presumably Ca²⁺) influx (Fig 6), we expected that phloridzin would block the inhibition by insulin of 5-HT–induced contraction. As shown in Fig 7, insulin inhibited 5-HT–induced contraction of these VSMCs in the absence but not the presence of phloridzin. Phloridzin alone did not affect 5-HT–induced contraction.

View larger version (27K): [in this window] [in a new window]   Figure 7. Bar graph showing effects of insulin and phloridzin on serotonin (5-HT)-induced contraction in vascular smooth muscle cells. Experiments were performed as in Fig 5 except that 5 mmol/L glucose was present in all preincubation and incubation solutions in the presence and absence of 1 mmol/L phloridzin. 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 reasoned that if insulin inhibited 5-HT–induced Ca²⁺ (or Mn²⁺) influx and contraction by stimulating glucose uptake, another maneuver that stimulates glucose uptake in the absence of insulin should cause the same effects. We tested this possibility by increasing glucose uptake via raising extracellular glucose concentration from 5 to 25 mmol/L. To control for increased extracellular osmolarity, parallel dishes of cells were exposed to the same osmolarity by addition of 20 mmol/L mannitol to the preincubation and incubation media. When [³H]2-DOG was used as a tracer to estimate glucose uptake, raising extracellular glucose from 5 to 25 mmol/L and adding 1 nmol/L insulin to medium containing 5 mmol/L glucose increased the appropriate control 30-minute uptake of 5 mmol/L glucose by 32±12% and 22±9%, respectively (n=3, P=NS). As shown in Fig 8, insulin's inhibition of 5-HT–induced contraction of mannitol-exposed cells was similar to that which occurs in the absence of mannitol (compare with Fig 5). When cells were preincubated with 25 mmol/L glucose for 30 minutes in the absence of insulin, cell contraction was inhibited, and it was not inhibited further by addition of insulin to the preincubation and incubation media. In separate experiments, we measured the effect of 5-HT on Mn²⁺ influx into cells preincubated with 25 mmol/L glucose for 30 minutes. The Mn²⁺ influx rates were 6.9±0.6 and 7.2±0.8 AFU/min before and after the cells were exposed to 10^-5 mol/L 5-HT, respectively (n=7; P=NS). For an osmotic control, cells were preincubated for 30 minutes with 5 mmol/L glucose plus 20 mmol/L mannitol. Mn²⁺ influx was increased by 42±3% upon exposure of the cells to 10^-5 mol/L 5-HT (n=7, P<.05). This effect of 5-HT was similar to that in cells preincubated with 5 mmol/L glucose in the absence of mannitol (Fig 3). Thus, a maneuver that increases glucose uptake (25 mmol/L extracellular glucose) without exposing the cells to insulin mimics the inhibitory effects of insulin (with 5 mmol/L extracellular glucose) on 5-HT–stimulated contraction and Mn²⁺ influx.

View larger version (13K): [in this window] [in a new window]   Figure 8. Bar graph showing effects of high glucose concentration and insulin on serotonin (5-HT)–induced contraction in vascular smooth muscle cells. Experiments were performed as in Fig 5 except that preincubation and incubation solutions consisted of physiological salt solution with either 20 mmol/L mannitol plus 5 mmol/L glucose or 25 mmol/L glucose. 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 20 mmol/L mannitol plus 5 mmol/L glucose without insulin.

We have previously shown that in the presence of extracellular Ca²⁺, insulin inhibited by 30% the height of the Ca²⁺_i peak that occurs about 30 seconds after these cells are exposed to 10^-5 mol/L 5-HT.^{7 14} We showed that the inhibition by insulin of 5-HT–induced Ca²⁺ influx was responsible for the decrease in peak Ca²⁺_i. In the present studies, we demonstrate that in the presence of 5 mmol/L glucose, 1 mmol/L phloridzin blocks the inhibitory effects of insulin on 5-HT–stimulated Mn²⁺ influx (Fig 6) and contraction (Fig 7). We also show that 25 mmol/L extracellular glucose mimicked the inhibitory effects of insulin on 5-HT–stimulated Mn²⁺ influx and contraction (Fig 8). On the basis of these findings, we predicted that phloridzin would block insulin's reduction of the 5-HT–induced Ca²⁺_i peak and that in the absence of insulin, 25 mmol/L glucose per se would reduce the Ca²⁺_i peak. To test this, cells on coverslips were loaded with fura 2, and Ca²⁺_i was continuously measured. The cells were preincubated for 30 minutes in PSS with or without 1 nmol/L insulin in the presence or absence of 1 mmol/L phloridzin. In addition, coverslips were preincubated in PSS that contained 20 mmol/L extra glucose or mannitol. After 30 minutes of preincubation, 10^-5 mol/L 5-HT was added to the superfusion solutions. Basal Ca²⁺_i was not affected under these different experimental conditions, averaging 127±14 nmol/L (n=5 coverslips under each condition). As shown in the Table, insulin decreased the 5-HT–induced Ca²⁺_i peak by 37%, in agreement with our previous studies.¹⁴ Phloridzin alone increased the Ca²⁺_i peak, but addition of insulin did not decrease it. Thus, insulin does not lower the Ca²⁺_i peak if insulin-induced glucose transport is blocked by phloridzin. Furthermore, the data in the Table show that 20 mmol/L extra glucose (25 mmol/L total) in the absence of insulin inhibited by 40% the 5-HT–induced Ca²⁺_i peak relative to the mannitol osmotic control. Thus, high extracellular glucose, which stimulates glucose influx in the absence of insulin, mimics the inhibitory effect of insulin on the 5-HT–induced Ca²⁺_i peak.

View this table: [in this window] [in a new window]   Table 1. Effect of Insulin, Phloridzin, and Glucose on the Peak of the Serotonin-Induced Ca2+i Transient in Vascular Smooth Muscle Cells

	Discussion

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Insulin inhibits VSM contraction in vivo and in vitro.^{4 5 6 7 8 9 10} This effect of insulin is especially noteworthy because obesity and non–insulin-dependent diabetes are insulin-resistant states associated with elevated blood pressure.²⁶ Essential hypertension is also associated with insulin resistance.^{26 27} It has been reported that the dose-response curves for insulin-induced vasodilation and glucose disposal were similarly shifted to the right in patients with non–insulin-dependent diabetes, obesity, and essential hypertension.^{8 9 28} Recent studies of patients with essential hypertension and/or obesity and studies of genetically obese hypertensive rats have shown that inhibition by insulin of agonist-induced VSM contraction is impaired.^{6 29} Taken together, these data support the hypothesis that individuals with resistance to insulin-induced glucose disposal also have resistance to insulin-induced vasodilation. This should cause elevated peripheral vascular resistance, which could contribute to the hypertensive state. Despite the interest of the inhibitory effect of insulin on vascular tone in normal and insulin-resistant states, the mechanism of the inhibition by insulin of VSM contraction is incompletely understood.¹

We and others have reported that insulin acutely attenuates contractile agonist-induced Ca²⁺ influx and the Ca²⁺_i transient in cultured VSMCs.^{7 11 12 13 14} It is noteworthy that high extracellular glucose causes the same effects in cultured VSMCs or the VSMC-like mesangial cell.^{15 16 17} Insulin stimulates glucose uptake in many cell types, including cultured VSMCs.^{18 19 20} Raising extracellular glucose concentration in the absence of insulin would be expected to increase glucose uptake by increasing the inwardly directed chemical gradient for glucose. Thus, the present study was designed to determine whether insulin-stimulated glucose influx was an early event leading to the inhibition by insulin of agonist-induced Ca²⁺ influx, the Ca²⁺_i peak, and contraction of cultured VSMCs.

The present study shows that 1 nmol/L insulin stimulates [³H]2-DOG uptake in a phloridzin-inhibitable manner in cultured VSMCs from canine femoral artery (Fig 1). The simulation of [³H]2-DOG uptake by this concentration of insulin is similar to what has been described in other VSMCs.^{18 19 20} Also, insulin-induced inhibition of 5-HT–stimulated Mn²⁺ (and presumably Ca²⁺) influx, inhibition of the peak of the Ca²⁺_i transient, and inhibition of contraction of these cells are dependent on extracellular glucose or are inhibited by phloridzin and are mimicked by another maneuver that stimulates glucose uptake in the absence of insulin, namely, high extracellular glucose. Inhibition of contraction by high extracellular glucose in the absence of insulin was not amplified by addition of insulin. Taken together, the present data indicate that insulin acutely (within 30 minutes) inhibits the 5-HT–induced Ca²⁺_i transient and contraction of these VSMCs by a sequence of events beginning with insulin-stimulated glucose uptake.

It is noteworthy that the stimulation of the rate of Mn²⁺ influx by 5-HT was greater in the absence of glucose (Fig 4) or presence of phloridzin (Fig 6) and that the height of the 5-HT–induced Ca²⁺ peak (Table) was higher in the presence of phloridzin than under control conditions (Fig 3). The reason for this is unknown, but possibly even basal glucose influx under control conditions downregulates the stimulation of Ca²⁺ influx by 5-HT. Zero extracellular glucose or phloridzin would prevent this downregulation.

We have previously reported that preincubation with insulin for 1 week attenuates 5-HT and angiotensin II–induced contraction of these cells. It is unknown whether the mechanism of attenuation of contraction by insulin is the same for chronic preincubation as it is for acute (30-minute) preincubations used in the present study. The effects of chronic exposure of these cells to high glucose or phloridzin are also unknown and will require further study. Future studies are also required to determine how glucose transport into the cell inhibits Ca²⁺ influx and contraction.

	Acknowledgments

 
These studies were supported by grants HL-40480 and HL-24585 from the National Heart, Lung, and Blood Institute and by a grant from Astra/Merck Group, Merck and Co, Inc, and from the Diabetes Action Research and Education Foundation. The authors acknowledge the excellent secretarial support of Tanya Handy and the helpful suggestions of Drs Thomas DuBose, Jr and Susan Wall.

	Footnotes

 
Guest editor for this article was Thomas W. Smith, MD, Brigham and Women's Hospital, Boston, Mass.

Received January 23, 1995; accepted March 12, 1995.

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