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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:394-402.)
© 1997 American Heart Association, Inc.

Articles

Reduced Responsiveness of Hypercholesterolemic Rabbit Aortic Smooth Muscle Cells to Nitric Oxide

Robert M. Weisbrod; Mark C. Griswold; Yue Du; Victoria M. Bolotina; Richard A. Cohen

the Vascular Biology Unit, Robert Dawson Evans Department of Clinical Research, Department of Medicine, Boston (Mass) University Medical Center.

Correspondence to Richard A. Cohen, MD, Vascular Biology Unit, R408, Boston University Medical Center, 80 E Concord St, Boston, MA 02118. E-mail racohen@med-med1.bu.edu.

	Abstract

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The response to nitric oxide of intracellular free Ca²⁺ levels, measured by fura 2 fluorimetry, and cyclic GMP, measured by RIA, was evaluated on smooth muscle cells of the thoracic aorta in primary culture from normal and cholesterol-fed rabbits. Relaxation to acetylcholine and nitric oxide was also determined in isolated rings of aorta. After 10 weeks of high-cholesterol diet, the intact aorta relaxed less to both acetylcholine and nitric oxide. In cultured cells from hypercholesterolemic rabbits, intracellular Ca²⁺ oscillated, and the mean Ca²⁺ levels were approximately twofold greater than in normal aortic cells. Nitric oxide failed to affect basal Ca²⁺ in either cell type. The peak and sustained rise in intracellular Ca²⁺ induced by angiotensin II (10^-7 mol/L) were similar in the two cell types. However, nitric oxide (10^-10 to 10^-6 mol/L) decreased the sustained Ca²⁺ levels to a significantly smaller extent in cells from cholesterol-fed rabbits. In addition, in cells from hypercholesterolemic rabbits, nitric oxide added before angiotensin II inhibited to a smaller degree the transient increase in intracellular free Ca²⁺ caused by angiotensin II in the nominal absence of extracellular Ca²⁺, as well as the increase in Ca²⁺ associated with the addition of extracellular Ca²⁺. Measurements of fura 2 quenching caused by Mn²⁺ influx confirmed that nitric oxide inhibited the entry of extracellular divalent cations significantly less in cells from hypercholesterolemic rabbits. Basal levels of cyclic GMP were significantly less than normal, and nitric oxide increased levels of cyclic GMP to a significantly smaller degree in cells from cholesterol-fed rabbits. These data indicate a substantial resistance to nitric oxide action in aortic smooth muscle cells of cholesterol-fed rabbits. This observation is consistent with the notion that resistance of smooth muscle cells to nitric oxide contributes to abnormal endothelium-dependent vasodilatation during hypercholesterolemia and can play a role in the pathogenesis of atherosclerosis.

Key Words: nitric oxide • calcium • cyclic GMP • cholesterol • atherosclerosis • smooth muscle cell

	Introduction

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An impairment in endothelium-dependent relaxation accompanies the development of atherosclerosis in experimental animals and humans.¹ Reduced activity of nitric oxide, which is the primary mediator of endothelium-dependent relaxation, has been implicated not only in the reduced relaxations, but also in the progression of atherosclerosis.¹ Although reduced endothelial release of nitric oxide has been suggested to explain the reduced activity of nitric oxide, several studies have shown that arteries from atherosclerotic rabbits have impaired relaxations to nitric oxide,^{2 3} and at least in the one study in which it was measured, the acetylcholine-induced release of nitric oxide was shown in fact to be increased from atherosclerotic rabbit aorta.⁴ This finding suggests that a characteristic of atherosclerotic arteries is resistance to nitric oxide action.

Nitric oxide released from the endothelium is recognized to inhibit smooth muscle contractility by several means. Stimulation of smooth muscle cell guanylate cyclase increases levels of cyclic GMP. Cyclic GMP, via activation of cyclic GMP–dependent protein kinase(s), mediates decreases in Ca²⁺_i and decreased sensitivity to Ca²⁺ of the contractile elements.⁵ The reduction in Ca²⁺_i caused by cyclic GMP has been attributed to decreased activity of voltage-dependent⁶ and receptor-operated Ca²⁺ channels⁷ and stimulation of the plasmalemmal Na⁺/Ca²⁺ exchanger and sarcolemmal Ca²⁺ reuptake,⁸ as well as to decreased Ca²⁺ release from the sarcoplasmic reticulum in response to inositol triphosphate.⁹ Hyperpolarization due to cyclic GMP–dependent^{10 11} as well as direct¹² activation of Ca²⁺-dependent K⁺ channels by nitric oxide may also contribute to the reduction in Ca²⁺_i and relaxation.

Aortic smooth muscle cells derived from atherosclerotic rabbits¹³ or those from normal rats in which the membrane cholesterol was enriched in culture¹⁴ were demonstrated by radioisotopic techniques to have increased Ca²⁺ uptake, suggesting an important role for membrane cholesterol in modulating Ca²⁺_i. The purpose of the present study was to measure Ca²⁺_i using fura 2 in cells derived from the aortic media of normal and hypercholesterolemic rabbits, to determine the effect of nitric oxide on Ca²⁺_i, and to compare this response with that of intact rabbit aortic rings. Cyclic GMP levels were measured in the cultured cells as another independent indicator of nitric oxide responsiveness. The results indicate increased basal Ca²⁺_i levels, decreased cyclic GMP levels, and reduced responsiveness to nitric oxide in smooth muscle cells and intact aortic rings derived from cholesterol-fed rabbits.

	Methods

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Animals
Male New Zealand White rabbits were assigned to control and cholesterol-fed groups randomly and received diet (Agway Prolab) ad libitum; those receiving cholesterol were fed the same chow supplemented with 0.5% cholesterol and 4% peanut oil for 10 weeks as previously described.² The aorta was aseptically excised and cleaned of adhering connective tissue.

Tension Studies
Rings of proximal descending aorta were removed for study of isometric tension in organ chambers as previously described.² In some rings, before mounting in the organ chambers, the endothelium was removed. After equilibration at the optimal resting tension (6 g), the aortic smooth muscle was contracted to 50% of maximal contraction with phenylephrine hydrochloride, and after achieving a stable contraction, acetylcholine hydrochloride or nitric oxide was added in increasing concentrations to the organ chambers. Phenylephrine was used as the contractile agent because it provided more stable contractions than did either 5-HT or angiotensin II.

Cell Culture
Aortic smooth muscle cells were cultured by enzymatic dispersion.¹⁵ After removing rings for tension studies, the aorta was opened longitudinally and gently scraped to remove endothelium. Plaques were removed by scraping the intimal surface of hypercholesterolemic rabbit aorta before dissociating the medial layer. Narrow strips of media were removed and placed in a solution of collagenase (4 mg/mL) and elastase (1 mg/mL) for 60 to 90 minutes until cells were dispersed. Cells were grown in a 5% CO₂ incubator with medium 199 supplemented with 20% heat-inactivated fetal bovine serum, penicillin G (10 000 U/dL), streptomycin (10 000 µg/mL), and NaHCO₃ (2.2 g/L). For Ca²⁺_i studies, cells were seeded on 9x22-mm glass coverslips in 6-well plates and for determination of cyclic GMP levels, in 24-well plates. The time from seeding cells to reaching confluence in the 6-well plates was similar for cells from normal and cholesterol-fed rabbits (13±1.1 versus 13±1.5 days, n=5 and 6, respectively). At the time of study, cells from both normal and cholesterol-fed rabbits showed similar positive immunohistochemical staining for smooth muscle -actin. After the cells reached confluence, the serum was reduced to 1% and the cells were maintained at quiescence for 3 to 4 days. For all experiments, the culture medium was replaced with a HEPES-buffered (pH 7.4) PSS of the following composition (mmol/L): NaCl 119, NaHEPES 20, KCl 4.6, MgSO₄ 1.0, Na₂HPO₄ 0.15, KH₂PO₄ 0.4, and NaHCO₃ 5.0, supplemented with 0.5% bovine albumin.

Ca²⁺_i Measurement
Ca²⁺_i was determined with the Ca²⁺-sensitive indicator fura 2 with a custom-designed fluorimeter (IonOptix Inc). The cells on a coverslip were loaded with 5 µmol/L of the membrane-permeable methyl ester form of fura 2 for 45 minutes at 37°C in an orbital shaker at 100 rpm. Cells then were washed for 15 minutes to allow intracellular esterases to cleave the ester group so that Ca²⁺-sensitive fura 2 remained in the cell. The coverslip was then mounted on the wall of a cuvette, with the cell layer facing the interior of the cuvette, which contained 3 mL of PSS at 37°C in a light-tight and temperature-controlled holder. The cuvette was continuously perfused with PSS at a flow rate of 1.5 mL/min. Light from a xenon lamp went through a 30-Hz chopper to alternate irradiation of the cells with 340 nm and 380 nm light (10-nm-bandwidth filters) for fura 2 excitation. The light was then conducted through a 0.5-cm liquid light guide apposed to the exterior wall of the cuvette at a distance of 1 mm from the cell layer. Fluorescence emission was collected via the same light guide and passed through a 510-nm filter (10-nm bandwidth) and into a photon-counting photomultiplier. Data were continuously collected with IonOptix software on a computer. Values were digitally averaged over 5-second periods. Intracellular free Ca²⁺ concentrations were estimated from the ratio of the 340/380-nm signals by using the following formula of Grynkiewicz et al¹⁶ :

where K_d for fura 2 is 225 nmol/L at 37°C, R_max and R_min are the ratio values from standards for saturating Ca²⁺ levels (maximum) and in the absence of Ca²⁺ (minimum), respectively, R is the sample ratio of 340/380-nm fluorescence, and S_f2 and S_b2 are the raw 380-nm fluorescence values for minimum and maximum standards, respectively. R_min, R_max, S_f2, and S_b2 values were determined by using external standard solutions consisting of PSS containing 50 nmol/L of the pentapotassium salt of fura 2 with either no Ca²⁺ (EGTA-buffered, minimum) or 1.2 mmol/L Ca²⁺ (maximum). Background autofluorescence was subtracted at the end of each experimental run by adding 10 µmol/L of the Ca²⁺ ionophore ionomycin and 20 mmol/L MnCl₂ to quench all fura 2 fluorescence. Total 340- and 380-nm fluorescence and background autofluorescence were not significantly different in cells cultured from normal and cholesterol-fed rabbits, indicating that fura 2 loading was comparable in the two groups. Ca²⁺_i levels are expressed as nanomolar concentration as calculated from fluorescence signal ratios acquired at 30 Hz, but it should be understood that the values are estimates made with fura 2.¹⁷ Similar differences between cells from normal and cholesterol-fed rabbits were observed in the ratios of 340/380-nm fluorescence signals.

The duration of each experimental run was 20 minutes. Cells were allowed to equilibrate in the cuvette for 6 minutes before 0.1 µmol/L angiotensin II or, where indicated, 5-HT (10 µmol/L) was added to increase Ca²⁺_i. Angiotensin II was used as the principal agonist in the cultured cells because it gave the most reproducible responses. In analyzing the response to angiotensin II, the maximal rise in Ca²⁺_i during the first 10 seconds (peak) and the level achieved 30 or 60 seconds after the addition of angiotensin II (plateau) were quantitated. Nitric oxide (10^-6 mol/L) was added 1 minute before or after angiotensin II to determine its effect on changes in Ca²⁺_i induced by the vasoconstrictor. In some studies, cells were placed in nominally Ca²⁺-free PSS at the beginning of the run and 50 µmol/L EGTA was added 1 minute before angiotensin II. In other experiments, logarithmic increases in nitric oxide concentration (10^-10 to 10^-6 mol/L) were added beginning 30 seconds after the peak increase in Ca²⁺_i caused by angiotensin II, with each subsequent concentration added after the effect of the previous concentration had subsided and Ca²⁺_i had reached a new stable level. To assure that the response was due to nitric oxide rather than to an oxidation product, it was demonstrated in preliminary experiments that the response was eliminated if the nitric oxide in solution was exposed to air for 15 minutes. Data from these experiments were analyzed by (1) subtracting basal Ca²⁺_i levels and calculating the maximal percent decrease in Ca²⁺_i caused by each concentration of nitric oxide and (2) measuring the duration (seconds) that each concentration of nitric oxide reduced the Ca²⁺_i level, starting from the initial decrease in Ca²⁺_i until the Ca²⁺_i level rose to reach a value that was the mean of the trough Ca²⁺_i value and the stable level that followed the response.

Measurement of Divalent Cation Influx Rate
Mn²⁺-induced quenching of fura 2 fluorescence was used to estimate rates of divalent cation influx, which reflects Ca²⁺ influx.¹⁸ The technique takes advantage of the fact that unlike Ca²⁺, when Mn²⁺ binds to fura 2, the fluorescence of the molecule is quenched. Fluorescence of fura 2–loaded cells was measured at 360 nm, the isosbestic wavelength for the indicator. Cells were placed in nominally Ca²⁺-free PSS for 8 minutes before MnCl₂ (10^-4 mol/L) was added for basal measurements. To determine agonist-induced influx, angiotensin II (10^-7 mol/L) was added 2 minutes before MnCl₂. To determine the effect of nitric oxide, 10^-6 mol/L nitric oxide was added 1 minute before angiotensin II. Mn²⁺ influx rates were calculated from the slope of the decline in fura 2 fluorescence during the first minute after addition of Mn²⁺ and was normalized to the level of fluorescence at the time of Mn²⁺ addition. Data are expressed as the initial rate of Mn²⁺ influx. The effect of nitric oxide and angiotensin II on cation influx was also calculated as a percentage decrease after subtracting basal influx rates from those obtained in the presence of angiotensin II and nitric oxide.

Cyclic GMP Measurement
Cyclic GMP levels were quantitated after extraction from the cells by RIA with a modified method.¹⁹ After equilibrating the cells for 30 minutes in PSS, basal cyclic GMP was determined or nitric oxide was applied as indicated to stimulate cyclic GMP production in the cells. In some studies IBMX was present during this 30-minute period. The PSS on the cells was removed and 200 µL 0.1 N HCl was added and allowed to remain for 1 hour to extract cyclic GMP. The extract was transferred to a vial and stored at 4°C until RIA was performed with a commercially available kit (Biomedical Technologies Inc). Protein in the cell layer was solubilized with 1 N NaOH and quantitated with a bicinchoninic acid assay (Pierce). Results are expressed as femtomoles cyclic GMP per microgram cell protein.

Materials
Acetylcholine chloride, angiotensin II, collagenase, elastase, 5-HT, ionomycin, IBMX, phenylephrine hydrochloride, and SOD were purchased from Sigma; bovine serum albumin was from Intergen. Nitric oxide solutions were made by bubbling authentic nitric oxide gas (Matheson Gas) into airtight vials of water purged of oxygen with nitrogen at 4°C.² All drugs were made in water, except for angiotensin II, which was prepared in 0.9% NaCl, and ionomycin and IBMX, which were prepared in DMSO, and were prepared such that a 1:1000 dilution was made into PSS to obtain the final reported concentration. Cell-culture plates were from CoStar and cell-culture media, serum, and antibiotics from GIBCO.

Statistical Analysis
All data are expressed as mean±SE. Statistical analysis of results was performed with Student's t test in an unpaired fashion between normal and hypercholesterolemic rabbit aorta or cultured cells or in a paired fashion within the same group. Two-way ANOVA for repeated measures was performed on results for relaxations and levels of cyclic GMP, in response to increasing concentrations of acetylcholine or nitric oxide. A value of P<.05 was considered significant. For statistical analysis, n indicates the number of rabbits from which arteries and primary cultures were derived.

	Results

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Acetylcholine and Nitric Oxide–Induced Relaxations of Rings of Rabbit Aorta
Phenylephrine caused similar contractions in aortic rings with or without endothelium from normal and cholesterol-fed rabbits (see Fig 1 legend). In rings with intact endothelium, acetylcholine caused relaxations that were significantly less in cholesterol-fed rabbit aorta than in aorta from normal rabbits (P<.025, Fig 1a). In aortic rings with intact endothelium (P<.008, Fig 1b) or in those denuded of endothelium (Fig 1c, 1d, and 1e), nitric oxide caused significantly smaller relaxations in those from cholesterol-fed rabbits than in those from normal rabbits (P<.002).

View larger version (16K): [in this window] [in a new window]   Figure 1. Relaxations of aortic rings to acetylcholine (a) and nitric oxide (b through e). Rings of aorta with intact endothelium (+E) were contracted with phenylephrine hydrochloride (6.8±0.2 g [n=6] and 7.7±0.4 g [n=6] in normal and cholesterol-fed rabbits, respectively). Acetylcholine induced relaxations that were significantly less in aorta from cholesterol-fed than from normal rabbits (a, *P<.025, ANOVA). In endothelium-intact aortic rings (b) contracted to 7.0±0.1 (n=6) and 7.3±0.5 (n=6) g, respectively, to phenylephrine, nitric oxide caused relaxations that were significantly less in rings from cholesterol-fed animals (b, *P<.008). In rings of aorta denuded of endothelium (-E, panels c through e), phenylephrine caused similar contractions in rings from normal and cholesterol-fed rabbits (7.8±0.4 g, n=5, and 8.4±0.1 g, n=5, respectively). Nitric oxide relaxations were significantly less in rings of aorta from cholesterol-fed rabbits (c, *P<.02, ANOVA). Examples are shown of recorded changes in tension of rings from normal (d) and hypercholesterolemic (e) rabbit aorta denuded of endothelium. After phenylephrine (PE) was used to contract both arteries, responses to nitric oxide (10-10 to 10-5 mol/L) were less in the ring from the hypercholesterolemic rabbit. Concentrations are represented as the negative logarithm.

Effect of Nitric Oxide on Ca²⁺_i
Basal Ca²⁺_i levels were noted to oscillate in aortic smooth muscle cells from hypercholesterolemic rabbits (Figs 2b, 3b, 4c, and 4d), whereas they did not do so in normal aortic cells. The mean levels of Ca²⁺_i under basal conditions were significantly higher in cells from hypercholesterolemic rabbits (204±22 versus 110±11 nmol/L, P<.005).

View larger version (29K): [in this window] [in a new window]   Figure 2. Basal and angiotensin II–stimulated Ca2+i levels in cultured normal and cholesterol-fed (chol-fed) rabbit aortic smooth muscle cells. Representative fura 2 Ca2+i recordings in cultured aortic smooth muscle cells from normal (a) and cholesterol-fed (b) rabbits demonstrate the effect of angiotensin II (AII, 0.1 µmol/L), nitric oxide (NO, 1 µmol/L), ionomycin (IM, 10 µmol/L), and MnCl2 (20 mmol/L). Basal Ca2+i levels in cholesterol-fed rabbit aortic cells typically oscillated (b). Summary data (c, n=6 to 7) from these experiments demonstrate that angiotensin II and ionomycin stimulated Ca2+i levels similarly in both groups, while basal Ca2+i was significantly higher (P<.05) and the effect of nitric oxide on Ca2+i was significantly less (P<.05) in cholesterol-fed rabbit cells. Inset: The percent decrease of plateau phase Ca2+i caused by nitric oxide (1 µmol/L) was significantly less in cholesterol-fed rabbit aortic smooth muscle cells. *P<.05.

View larger version (24K): [in this window] [in a new window]   Figure 3. Changes in Ca2+i in response to increasing concentrations of nitric oxide in normal and cholesterol-fed (chol-fed) rabbit aortic smooth muscle cells. Application of nitric oxide (10-10 to 10-6 mol/L) was made to the angiotensin II– (AII, 10-7 mol/L) stimulated Ca2+i plateau in aortic smooth muscle cells from normal (a) and cholesterol-fed (b) rabbits. c, Summary data showing the percent decrease of Ca2+i caused by each nitric oxide concentration. d, Summary data showing the duration (seconds) that each concentration of nitric oxide reduced the Ca2+i level from the time of its first effect until the value reached the mean of the trough value and the plateau level after the response. *P<.05; n=2 to 3.

View larger version (20K): [in this window] [in a new window]   Figure 4. Representative recordings of Ca2+i in aortic smooth muscle cells from normal (a and b) and cholesterol-fed (chol-fed, c and d) rabbits. Angiotensin II (AII, 10-7 mol/L) was applied in all four experiments, whereas nitric oxide (NO, 10-6 mol/L) was applied 1 minute before angiotensin II in b and d. b, Nitric oxide (10-6 mol/L) was applied a second time after the initial effect of nitric oxide disappeared. Summary data (e) demonstrate that nitric oxide (10-6 mol/L) significantly reduced the angiotensin II–induced peak and plateau Ca2+i levels when applied 1 minute before angiotensin II in aortic cells from normal (N) but not cholesterol-fed (CF) rabbits. *P<.05; n=3 to 6.

Angiotensin II (10^-7 mol/L) caused a rapid increase in Ca²⁺_i followed by a plateau and a gradual decline (Figs 2a and 4a). There was no significant difference in the peak (1003±112 versus 1088±92 nmol/L) or plateau level of Ca²⁺_i 60 seconds after the addition of angiotensin II (565±46 versus 532±28 nmol/L) for cells from normal and cholesterol-fed rabbits, n=6 and 7, respectively (Fig 2c). The Ca²⁺ ionophore ionomycin (10^-5 mol/L) also stimulated Ca²⁺_i similarly in normal (986±131 nmol/L) and hypercholesterolemic (930±68 nmol/L) rabbit aortic smooth muscle cells (Fig 2c).

To determine its effect on Ca²⁺_i, nitric oxide (10^-6 mol/L) was applied 60 seconds after the addition of angiotensin II so that it was added after the peak increase in Ca²⁺_i caused by angiotensin II (Fig 2a and 2b). Application of nitric oxide (10^-6 mol/L) reduced Ca²⁺_i in normal cells to a significantly lower level than in hypercholesterolemic rabbit cells (290±36 versus 406±30 nmol/L, respectively, P<.05, Fig 2c). This represents a 51±5% decrease in normal and a 23±5% decrease in hypercholesterolemic rabbit cells, a difference that was also statistically significant (P<.005). To account for the higher basal Ca²⁺_i levels in hypercholesterolemic rabbit cells, the percent decrease in Ca²⁺_i caused by nitric oxide was also calculated after subtraction of basal Ca²⁺_i levels. Nitric oxide (10^-6 mol/L) still caused a significantly greater decrease in Ca²⁺_i in normal than in hypercholesterolemic rabbit aortic cells (62±7% versus 36±7%, P<.05, Fig 2c, inset). In five experiments each, application of 10^-5 mol/L nitric oxide after the 10^-6 mol/L concentration had no further effect on Ca²⁺_i levels in cells from normal or cholesterol-fed animals (data not shown).

To determine whether greater extracellular superoxide radical scavenging of nitric oxide could account for the decreased response in aortic cells from hypercholesterolemic rabbits, cells were incubated with SOD (150 U/mL) for 20 minutes before addition of angiotensin II. After subtracting basal Ca²⁺_i values, nitric oxide (10^-6 mol/L) decreased Ca²⁺_i by 59±3% (n=2) in normal and 28±4% (n=2) in hypercholesterolemic rabbit aortic cells, values very similar to those obtained in the absence of SOD.

In normal rabbit aortic cells, nitric oxide (10^-10 to 10^-6 mol/L) added during the plateau phase of the increase in Ca²⁺_i induced by angiotensin II caused a concentration-dependent decrease in Ca²⁺_i (Fig 3a). The decrease in Ca²⁺_i also increased in duration as the nitric oxide concentration increased (Fig 3a). In hypercholesterolemic rabbit aortic cells, there was significant attenuation of both the magnitude and duration of the decreases in Ca²⁺_i caused by nitric oxide (Fig 3b, 3c, and 3d).

When nitric oxide (10^-6 mol/L) was applied 1 minute before angiotensin II, it had no significant effect on basal Ca²⁺_i in either normal or hypercholesterolemic rabbit aortic cells (Fig 4). Nitric oxide (10^-6 mol/L) added in this manner significantly decreased both peak and plateau levels of Ca²⁺_i achieved by angiotensin II in normal aortic cells (Fig 4b and 4e). The decrease in peak and plateau levels of Ca²⁺_i by nitric oxide represented 40±5% and 44±2%, n=6, respectively. The duration of action of nitric oxide (10^-6 mol/L) typically was 8 to 10 minutes, at which time a rise in Ca²⁺_i was observed, which in normal cells could again be inhibited by addition of nitric oxide (Fig 4b). In smooth muscle cells from hypercholesterolemic rabbits, nitric oxide had no significant effect on the angiotensin II–induced peak or plateau Ca²⁺_i levels (Fig 4d and 4e).

When placed in nominally Ca²⁺-free PSS containing EGTA (50 µmol/L), basal Ca²⁺_i was not significantly affected in either normal or hypercholesterolemic rabbit aortic smooth muscle cells, although basal Ca²⁺_i in the cells from cholesterol-fed rabbits was not observed to oscillate, as was noted in the presence of extracellular Ca²⁺ (Fig 5). Under these conditions, angiotensin II caused a transient rise in Ca²⁺_i (Fig 5). Three minutes after angiotensin II was added, addition of Ca²⁺ (1.2 mmol/L) increased Ca²⁺_i. Neither the transient rise in Ca²⁺_i nor the increase in Ca²⁺_i associated with addition of extracellular Ca²⁺ was significantly different between cells of normal and hypercholesterolemic rabbits (Fig 5c through 5e). In normal aortic cells, nitric oxide (10^-6 mol/L) applied 1 minute before angiotensin II decreased both the transient rise in Ca²⁺_i caused by angiotensin II in the absence of extracellular Ca²⁺_i (59±8% decrease) and the rise in Ca²⁺_i that occurred on addition of extracellular Ca²⁺_i (75±3% decrease, Fig 5b and 5e). In cells from hypercholesterolemic rabbits, nitric oxide decreased the transient rise in Ca²⁺_i by 37±6% and decreased the rise in Ca²⁺_i associated with Ca²⁺ addition by 22±2%. Only the inhibition caused by nitric oxide of the rise in Ca²⁺_i associated with Ca²⁺ addition was significantly smaller in cells from cholesterol-fed rabbits than from normal rabbits (P<.001).

View larger version (23K): [in this window] [in a new window]   Figure 5. Representative recordings of Ca2+i in the absence and after the addition of extracellular Ca2+ to aortic smooth muscle cells from normal (a and b) and cholesterol-fed (chol-fed, c and d) rabbits. The Ca2+ in the PSS was changed to nominally Ca2+-free PSS for 6 minutes, and 50 µmol/L EGTA was added 1 minute before angiotensin II. In all four tracings, angiotensin II (AII, 10-7 mol/L) was applied, and 1.2 mmol/L extracellular Ca2+ was added 3 minutes later. In b and d, nitric oxide (NO, 10-6 mol/L) was applied 1 minute before angiotensin II. Summary data (e) demonstrate that NO significantly reduced (P<.05) both the angiotensin II–induced Ca2+i peak and Ca2+i peak achieved after the addition of Ca2+ in aortic smooth muscle cells from normal (N) but not cholesterol-fed (CF) rabbits. *P<.05; n=2 to 5.

The basal rate of Mn²⁺ influx was significantly greater than normal in aortic smooth muscle cells from hypercholesterolemic rabbits (0.125±0.22 and 0.072±0.012, respectively, n=5 each, P<.025, Fig 6). However, after the addition of angiotensin II, the rate of influx was similar for both groups (0.286±0.041 and 0.273±0.023, respectively, Fig 6). Application of nitric oxide (10^-6 mol/L) before angiotensin II significantly reduced the agonist-stimulated Mn²⁺ influx in normal cells to 0.127±0.031 (P<.0002, n=5), which represents a 79±7% decrease in the agonist-stimulated influx. In contrast, in hypercholesterolemic rabbit aortic cells, nitric oxide reduced angiotensin II–stimulated influx to 0.198±0.028 (n=5), a 29±22% change that was not statistically significant.

View larger version (31K): [in this window] [in a new window]   Figure 6. Effect of nitric oxide on angiotensin-induced Mn2+ influx in aortic smooth muscle cells cultured from normal and cholesterol-fed (chol-fed) rabbits. Mn2+-induced quenching of fura 2 was used as an estimate of Ca2+ influx. Data represent the normalized influx rate of MnCl2 (10-4 mol/L, Basal), application of angiotensin II (10-7 mol/L) 2 minutes before MnCl2 (Control), and application of nitric oxide (10-6 mol/L) 1 minute before angiotensin II (NO, n=5). The basal Mn2+ influx rate was significantly higher in aortic smooth muscle cells from hypercholesterolemic rabbits (*P<.025), whereas the rates were similar after angiotensin II stimulation. Nitric oxide significantly reduced angiotensin II–induced Mn2+ influx in normal cells only (*P<.0002).

Effect of Nitric Oxide on Cyclic GMP Accumulation
After 30 minutes' equilibration in PSS, basal cyclic GMP levels in aortic cells from normal rabbits were 0.97±0.11 fmol/µg protein. The basal cyclic GMP levels in cells from hypercholesterolemic rabbits were significantly less than normal (0.42±0.15 fmol/µg, P<.05, Fig 7a). When stimulated for 1 minute with nitric oxide (10^-7 or 10^-6 mol/L), levels of cyclic GMP were increased to 2.7±0.4 and 6.3±0.6 fmol/µg, respectively (Fig 7a). Nitric oxide (10^-7 and 10^-6 mol/L) stimulated cyclic GMP levels in cells from hypercholesterolemic rabbits to significantly lower levels (1.3±0.4 and 1.5±0.4 fmol/µg, P<.05 and P<.001, Fig 7a).

View larger version (19K): [in this window] [in a new window]   Figure 7. Basal and nitric oxide–stimulated cyclic GMP levels in aortic smooth muscle cells from normal and cholesterol-fed (chol-fed) rabbits. a) Basal- and nitric oxide– (NO, 10-7 and 10-6 mol/L, 1 minute) stimulated cyclic GMP levels were significantly less in cholesterol-fed rabbit aortic cells (*P<.05, n=6). b, Cyclic GMP production was stimulated with nitric oxide (10-10 and 10-9 mol/L for 5 seconds; 10-8 to 10-6 mol/L for 10 seconds) to correlate with the peak effect of NO on Ca2+i in the experiments shown in Fig 3. Nitric oxide–stimulated cyclic GMP levels were significantly less in cholesterol-fed rabbit aortic smooth muscle cells than in normal cells (P<.05 ANOVA, n=3).

The difference in cyclic GMP levels between cells from normal and cholesterol-fed rabbits after stimulation with nitric oxide (10^-6 mol/L) could not be accounted for by phosphodiesterase activity. Under control conditions 1 minute after addition of nitric oxide (10^-6 mol/L), cyclic GMP levels were stimulated 7.0±1.2-fold (n=5) in cells from normal rabbits compared with 3.8±0.8-fold in those from cholesterol-fed rabbits (n=5), representing a 54% decrease. When cells were pretreated for 30 minutes with the cyclic GMP phosphodiesterase inhibitor IBMX (10^-4 mol/L), cyclic GMP stimulation by nitric oxide was 31±5.2-fold (n=5) and 19±7.9-fold (n=5), respectively, in cells from the two groups, representing a 61% decrease in the cells from hypercholesterolemic rabbits.

The effects of nitric oxide (10^-10 to 10^-6 mol/L) were determined on cyclic GMP levels achieved 5 to 10 seconds after adding nitric oxide (Fig 7b), a time which corresponded to the peak decrease in Ca²⁺_i levels caused by each concentration (Fig 3). Nitric oxide (10^-10 to 10^-6 mol/L) produced a concentration-dependent increase in cyclic GMP of 0.38±0.12, 1.5±0.19, 9.1±1.4, 25.2±8.1, and 24.4±4.5 fmol/µg in normal and 0.5±0.22, 0.97±0.55, 2.7±1.6, 7.4±4.5, and 5.9±4.5 fmol/µg in hypercholesterolemic rabbit aortic cells, respectively (Fig 7b). Nitric oxide–stimulated cyclic GMP levels were significantly less in hypercholesterolemic than in normal aortic cells (P<.05, ANOVA).

	Discussion

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The most important finding in this study is that the inhibition of Ca²⁺_i caused by nitric oxide was strikingly less in smooth muscle cells cultured from the aorta of hypercholesterolemic rabbits, reflecting the impaired response observed in the smooth muscle of the intact aorta. This impairment may be due to an alteration in the mechanisms involved in Ca²⁺_i homeostasis or in the responsiveness to nitric oxide itself. In agreement with the work of others,¹³ the present study found that basal Ca²⁺_i was elevated approximately twofold in smooth muscle cells cultured from atherosclerotic rabbit aorta. The influx of ⁴⁵Ca²⁺ and total cellular Ca²⁺ is also elevated in intact arterial rings and freshly isolated smooth muscle cells from cholesterol-fed rabbits.^{20 21 22} The increased basal Mn²⁺ influx demonstrated in this study is consistent with the previous evidence that increased Ca²⁺ influx contributes to higher resting intracellular Ca²⁺ levels. However, the basal levels of Ca²⁺_i remained high during a brief period in the absence of extracellular Ca²⁺, suggesting that the elevated levels may also depend on impaired Ca²⁺-sequestering mechanisms and are not dependent on increased influx, at least in the short term. It is unlikely that the twofold reduction in basal cyclic GMP levels observed in this study can account for the altered higher basal Ca²⁺_i levels, because increasing cyclic GMP with nitric oxide (10^-6 mol/L) did not lower the basal Ca²⁺_i levels. Bialecki et al¹⁴ and Tulenko et al²² showed that cholesterol-donating liposomes increased ⁴⁵Ca²⁺ influx in cultured rat or rabbit aortic smooth muscle cells, suggesting that membrane cholesterol content is important. This effect may be due to the increased plasma membrane bilayer width that occurs when the cholesterol content increases.²³

Apart from the increased basal levels of Ca²⁺_i, the angiotensin II–induced peak and sustained rise in Ca²⁺_i were not different in cells derived from normal and hypercholesterolemic rabbits. This is in contrast to an increased response to 5-HT observed in arterial rings and cultured smooth muscle cells from aorta of atherosclerotic rabbits.¹³ The similarity in Ca²⁺_i after angiotensin II allowed a comparison of the response to nitric oxide in the two cell types. Even if one takes into account the higher basal Ca²⁺_i level, the reduced response to nitric oxide cannot be attributed to an altered response to angiotensin II in the cells from the hypercholesterolemic rabbits. The reduced nitric oxide responsiveness was, however, not due to a specific interaction between angiotensin II and nitric oxide, because similar reduced nitric oxide responses were observed in cells of two hypercholesterolemic rabbits in which Ca²⁺_i was increased with 5-HT (data not shown).

Unlike basal Ca²⁺_i levels, nitric oxide reduced agonist-stimulated levels of Ca²⁺_i in cells from both normal and cholesterol-fed rabbit aorta, although this effect was significantly less in the cells of hypercholesterolemic rabbits. As demonstrated in experiments in which extracellular Ca²⁺ was eliminated and then restored, nitric oxide inhibited both the release of internal stores of Ca²⁺ and Ca²⁺ influx to a reduced extent. The ability of nitric oxide to inhibit the rise in Ca²⁺_i on Ca²⁺ addition was more markedly affected than was the effect on intracellular Ca²⁺ release caused by angiotensin II, suggesting that agonist-induced Ca²⁺ influx in cells from cholesterol-fed rabbits was particularly resistant to nitric oxide. Resistance to nitric oxide of angiotensin II–induced divalent cation influx was also demonstrated in this study by showing that Mn²⁺ influx was inhibited less by nitric oxide in cells from hypercholesterolemic rabbits.

Measurements of cyclic GMP content suggest reduced stimulation of the cyclic nucleotide levels as a mechanism for the reduced response of Ca²⁺_i to nitric oxide in cells derived from atherosclerotic rabbits. In studies conducted with the phosphodiesterase inhibitor IBMX, the difference in cyclic GMP levels could not be accounted for by a difference in the breakdown of the cyclic nucleotide. Reduced levels of guanylate cyclase activity and cyclic GMP levels in response to nitric oxide donors have been described in arteries from atherosclerotic rabbits^{2 24 25} and attributed to an effect of oxidized low-density lipoproteins.^{26 27 28} Superoxide anion has been implicated in reducing the activity of endothelium-derived nitric oxide in atherosclerotic rabbits.^{29 30} The fact that adding SOD did not improve the responsiveness to nitric oxide in cells from hypercholesterolemic rabbits suggests that increased levels of extracellular superoxide anion are not responsible for the impairment, at least acutely. It remains possible that intracellular oxidative mechanisms, which are not reversed by the acute administration of SOD, modify and interfere with guanylate cyclase and ion channels that normally respond to nitric oxide.

Despite the fact that it is commonly observed that relaxations to some nitric oxide donors such as sodium nitroprusside^{26 29} are normal in atherosclerotic arteries in which endothelium-dependent relaxations are impaired, relaxations to nitric oxide of rings of aorta from which the cells in this study were cultured were significantly reduced compared with those of normal rabbits. This was true whether or not the endothelium was removed, indicating that the response of the smooth muscle itself to nitric oxide is reduced. Therefore, the reduced nitric oxide responsiveness observed in cells cultured from hypercholesterolemic rabbit aorta reflects similar mechanisms in the intact artery. It is difficult to compare quantitatively the magnitude of the response to endogenously released nitric oxide caused by acetylcholine with that to exogenous nitric oxide because of the different modes of delivery of nitric oxide to the smooth muscle cells. It is possible that additional endothelial mechanisms explain the reduced endothelium-dependent relaxation³⁰ or that exogenous nitric oxide has different chemical properties or is metabolized differently by the arterial wall than nitric oxide derived from the endothelium. This leaves open the question of the degree to which resistance to nitric oxide of smooth muscle cells accounts for the reduced response to acetylcholine but supports the fact that the intact arterial smooth muscle does in fact resist the action of nitric oxide in a way that is recapitulated in the cells in primary culture.

When placed in primary culture, the smooth muscle cells of the normal aorta dedifferentiate, losing their contractile properties.¹⁵ They also take on antigenic similarities to cells within the aortic media of atherosclerotic rabbits, which are believed to represent the cells that migrate, proliferate, and become part of atherosclerotic plaque.³¹ The reduced responsiveness to nitric oxide of the aortic smooth muscle cells of hypercholesterolemic rabbits is maintained during this process of dedifferentiation. The transient decreases in Ca²⁺_i in response to nitric oxide in cells cultured from normal rabbit aorta have a very similar time course and concentration dependency to the relaxations of intact aortic rings to nitric oxide, suggesting that the mechanisms in smooth muscle cells, which ordinarily respond to nitric oxide by decreasing Ca²⁺_i, are maintained in primary culture. These characteristics of the response to nitric oxide are maintained during the first four passages of smooth muscle cells from normal rabbits (unpublished observations). This suggests that abnormalities in other mechanisms that persist during primary culture of smooth muscle cells of hypercholesterolemic rabbits may explain the reduced responsiveness to nitric oxide. On the basis of the smaller effect of nitric oxide on Mn²⁺ influx, the impaired regulation of plasmalemmal cation channels in cells from hypercholesterolemic rabbits, perhaps due in part to decreased guanylate cyclase activity,²⁵ is a key mechanism responsible for impaired nitric oxide responsiveness. Increased plasma membrane cholesterol content, which may explain the effects noted earlier on basal influx of Ca²⁺ through such cation channels, however, may not account for the decreased nitric oxide responsiveness of cells derived from hypercholesterolemic rabbits. Direct cholesterol enrichment of normal intact rabbit carotid artery rings increased rather than decreased the sensitivity to sodium nitroprusside.³²

In conclusion, this study indicates an abnormal regulation of Ca²⁺_i in smooth muscle cells of hypercholesterolemic rabbits by nitric oxide. This suggests that in addition to any abnormalities in nitric oxide production that may exist in the setting of hypercholesterolemia, the smooth muscle, a major target of nitric oxide action, is less responsive to the vasodilator. Both proliferation^{33 34} and migration³⁵ of cultured vascular smooth muscle are modulated by nitric oxide, as is the growth of the atherosclerotic plaque.³⁶ Thus, the decreased nitric oxide responsiveness of aortic smooth muscle cells from hypercholesterolemic rabbit aorta described in this study may be an important feature of the smooth muscle cells that dedifferentiate, migrate, proliferate, and participate in the development of atherosclerotic plaque.³¹

	Selected Abbreviations and Acronyms

 

5-HT = 5-hydroxytryptamine IBMX = isobutyl methylxanthine PSS = physiological salt solution SOD = superoxide dismutase

	Acknowledgments

 
These studies were supported by National Institutes of Health (Bethesda, Md) grants HL31607, HL47124, HL55993, and HL38731 and a grant-in-aid from the American Heart Association, Massachusetts Affiliate, Inc.

Received December 29, 1995; revision received June 12, 1996;

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