INTRODUCTION

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THE VASCULAR ENDOTHELIUM plays an important role in the regulation of vascular homeostasis. Imbalance in the release of the endothelium-derived factor nitric oxide (NO), a powerful vasodilator, plays a central role in many cardiovascular diseases such as coronary artery disease, atherosclerosis, hypercholesterolemia, and hypertension, as well as acute coronary dysfunction (8, 33). The synthesis of NO from L-arginine is catalyzed by the constitutively expressed endothelial isoform of NO synthase (eNOS). Localization of the majority of eNOS to receptor-rich membrane regions (caveolae) in the endothelium results from posttranslational eNOS modification (40). At these sites, eNOS activity is affected by direct protein-protein interactions, including inhibition of eNOS activity by the membrane protein caveolin-1 (18, 26). Caveolin-1 is displaced in response to elevated intracellular free calcium levels resulting in eNOS binding of the Ca²⁺/calmodulin complex, the main modulator of constitutive NOS isoforms. Through this mechanism and others, expression and activity of eNOS is modulated by an array of substances and conditions (19, 27).

Acute and chronic stresses are conditions implicated in cardiovascular diseases such as atherosclerosis and coronary artery disease (28). The glucocorticoid hormone cortisol is linked to stress responses. Increased cortisol levels mediated by the hypothalamic-pituitary-adrenal axis in response to stress were first described by Selye in 1936 (41). Traditionally, cortisol is thought to provoke genomic responses mediated by the intracellular glucocorticoid receptor (GR), which is capable of interacting with genomic glucocorticoid responsive elements. Both GR protein and message have been identified in endothelial cells (23, 24). Furthermore, the demonstrated ability of the GR antagonist mifepristone to inhibit many glucocorticoid actions in endothelium tissue indicates that the GR is in fact involved in glucocorticoid signaling mechanisms affecting expression of proteins such as cyclooxygenase-1 and eNOS (24, 48). Glucocorticoids act in a myriad of tissues and produce a range of responses. The response to glucocorticoid release may comprise a suppressive mechanism to limit earlier stress activated responses (39, 32). In the vascular endothelium, glucocorticoid has proven to suppress the production of vasodilators such as prostacyclin and NO (24, 37, 48); it has also been linked to the synthesis of the vasoconstrictor thromboxane (16).

Glucocorticoids suppress eNOS activity in human umbilical vein endothelial cells and decrease plasma NO/NO levels in rats (48). However, there is evidence that the coronary vasculature exhibits a site-specific pharmacological profile in response to agonist stimulation (31). Heterogeneous eNOS distribution in the coronary endothelium may extend these variations to the production of NO (1). Therefore, spatial variations in NO production may alter the response to a stimulus throughout the coronary vessels. Coronary artery disease is a major cause of mortality, which is often associated with stress. Despite this fact, the effect of glucocorticoid on the coronary artery endothelium has yet to be established. Because of the heterogeneous nature of the endothelium from different vascular beds, the present study was designed to test the hypothesis that elevated cortisol levels suppress NO release in bovine coronary artery endothelial cells (BCAEC). To understand the cellular mechanisms of NO suppression, we also determined the effect of cortisol on cytosolic Ca²⁺ mobilization and on eNOS protein stability in BCAEC.

	METHODS

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Cell culture. BCAEC were purchased from Cell Applications (San Diego, CA) and cultured at 37°C in a humidified incubator with 5% CO₂-95% air. Cells were plated at an equal density of 5,000 cells/cm² at the fifth and sixth passages, and near-confluent cells were incubated in phenol red-free DMEM cell culture medium with 16% charcoal-stripped FBS for 24 h. The cells were then treated with cortisol or cortisol plus 3 µM carbenoxolone over a range of concentrations and time points, as indicated in Figs. 1-7. Control cells were incubated in the same culture media for the same time periods in the absence of cortisol or carbenoxolone. To examine the effect of cortisol on eNOS degradation, cycloheximide (50 µg/ml) was added to the medium after the cells had been treated with 1 µM cortisol for 24 h. To determine the effect of cortisol on basal nitrite, nitrate, and NO (NO_x) release, NO_x was measured in the culture medium collected over accumulative time periods of 4, 12, and 24 h in the absence or presence of cortisol. Cell density in each well was the same between the control (4.10 × 10⁵ ± 3.89 × 10⁴, n = 6) and cortisol-treated (4.04 × 10⁵ ± 2.18 × 10⁴, n = 5) groups after 24 h. To determine the effect of cortisol on ATP-induced NO_x release, cells were first treated in the culture media in the absence or presence of cortisol for 24 h. The culture medium was then replaced with HBSS with the continuous presence of cortisol in the cortisol-treatment group, and cells were treated for 1 h in HBSS in the absence or presence of ATP. NO_x was then measured in HBSS.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Time-course effect of cortisol on nitrite, nitrate, and nitric oxide (NOx) release from bovine coronary aortic endothelial cells (BCAEC). BCAEC were incubated with control medium or medium with cortisol (83 nM) for the time periods indicated. Media were collected at 4, 12, and 24 h, and NOx was measured by chemiluminescence as described in METHODS. Data are means ± SE of 4 experiments. *P < 0.05 vs. control.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Dose-dependent effect of cortisol on NOx release from BCAEC. BCAEC were pretreated with cortisol (0-10 µM) in the presence or absence of carbenoxolone (3 µM) for 24 h. After pretreatment, BCAEC were incubated in HBSS buffer for 2 h. Buffer NO levels, measured as NOx through chemiluminesence, were reported as the percentage of NO derived from cells unexposed to cortisol (%control). Data are means ± SE of 4-5 experiments. One-way ANOVA analysis indicated that cortisol produced a significant, dose-dependent decrease in NO release in the absence or presence of carbenoxolone (P < 0.05). Two-way ANOVA indicated that carbenoxolone significantly attenuated cortisol-mediated responses (P < 0.05). *P < 0.05 vs. no carbenoxolone.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Immunoblot analysis for glucocorticoid receptor (GR) protein in BCAEC. Western blot analysis of GR was performed using rabbit polyclonal antibodies. Signal for the GR was evident at 97 kDa in duplicate measurements of 3 independent experiments (Ex 1-3).

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Effect of cortisol on endothelial nitric oxide synthase (eNOS) protein levels in BCAEC. BCAEC were incubated with control medium (C) or medium with cortisol (83 nM or 1 µM; T) for the time periods indicated. Cellular protein was collected after 12 and 24 h, and eNOS levels were determined by Western blot with eNOS monoclonal antibody. The Western blot shown is representative of eNOS band intensities from an 83-nM cortisol treatment. Variations between separation gels were normalized by reporting densitometry values as the percentage of total eNOS band intensity per gel. Data are means ± SE of 5-9 experiments. *P < 0.05 vs. controls.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Effect of cortisol on eNOS protein degradation in BCAEC. BCAEC were incubated with control medium or medium with cortisol (1 µM) for 24 h, followed by the treatment of both control and cortisol-treated cells with cycloheximide (50 µg/ml). Cellular protein was collected after 0, 0.5, 1, 2, 4, and 12 h of cycloheximide exposure, and eNOS levels were determined by Western blot with eNOS monoclonal antibody. Protein densitometry values are reported as the percentage of an equal amount of eNOS standard (%STD) loaded on each gel. Data are means ± SE of 5 experiments. Slope analysis indicated a significant difference between the control and cortisol treatment. *P < 0.05 vs. controls.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Effect of cortisol on agonist-stimulated intracellular free Ca2+ concentration ([Ca2+]i) in BCAEC. BCAEC were incubated with control medium or medium with cortisol (1 µM) for 24 h. Cells were then loaded with fura 2, and [Ca2+]i was measured as described in METHODS. Data are expressed as the percentage of cell response to 10 µM ionomycin and 100 µM ATP and are means ± SE of 4-5 experiments. *P < 0.05 vs. basal; +P < 0.05 vs. control.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effect of cortisol on agonist-stimulated NOx release in BCAEC. BCAEC were incubated with control medium or medium with cortisol (1 µM) for 24 h. Cells were then incubated in HBSS in the absence or presence of 30 µM ATP for 1 h. Buffer NOx levels were measured by chemiluminescence as described in METHODS. Data are means ± SE of 4 experiments. *P < 0.05 vs. basal; +P < 0.05 vs. control.

Measurement of NO_x. NO was measured by the chemiluminescence method as described previously (51). Because of the instability of NO in solution, most NO is rapidly converted to nitrite and further to nitrate. Although nitrite and nitrate are relatively stable in physiological solution, they are readily reduced back to NO in vanadium (III)-HCl solution. The samples (100 µl) taken from the medium or HBSS were injected into a gas purge vessel containing 5 ml vanadium (III)-HCl and allowed to react for 1 min and reduce nitrate/nitrite in the sample back to NO. To achieve a high reducing efficiency, the reduction was performed at 90°C. NO in the sample was then "stripped" into the head space of the gas purge vessel by bubbling it with helium (12 ml/min) for 1 min. NO in the head space was drawn into a NO analyzer (model 270B, Sievers Instruments; Boulder, CO) and mixed with ozone (O₃) in front of a cooled Hamamatsu, red-sensitive photomultiplier tube. Signals from the detector were analyzed by an on-line computer as the area under the peak. The measurement reflected the combined concentrations of nitrate, nitrite, and NO (NO_x) in each sample, as calculated from a standard curve of from 20 to 400 pmol of nitrite run in each assay.

Measurement of intracellular free Ca²+ concentration. Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was measured in single cells as previously described (51). Briefly, after cortisol pretreatment, BCAEC were loaded with the fluorescent Ca²⁺ chelator fura 2 (5 µM fura 2-AM) for 45 min at 37°C in loading buffer [125 mM NaCl, 5 mM KCl, 1 mM KH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂, 25 mM HEPES (pH 7.4), 6 mM D-glucose, 10 mM neostigmine, and 0.02% cremophor EL]. The cells were washed three times and incubated for 15 min in 37°C Krebs solution to allow complete hydrolysis of fura 2 ester groups by endogenous esterases. Fura 2 fluorescence was monitored photometrically at an emission wavelength of 510 nm in a single cell mounted on a Nikon Diaphot inverted microscope alternating illumination at 340- and 380-nm wavelengths using an InCyt Im2 Intracellular Imaging system (Intracellular Imaging, Cincinnati, OH). Photon counting was performed with a photomultiplier tube positioned so that thresholding shutters restricted the field of interest. Data acquisition was accomplished with software that controls a light chopper to alternate excitation wavelengths during rationing operations. [Ca²⁺]_i was calculated in real time from a standard curve established for the same settings using buffers of known Ca²⁺ concentration and was expressed as a percentage of the maximum cellular response to 10 µM ionomycin and 100 µM ATP.

Western blot analysis. The endothelial cells were solubilized by sonication in lysis buffer (150 mM NaCl, 50 mM Tris · HCl, 10 mM EDTA, 0.1% Tween 20, 0.1% -mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin; pH 7.4). After centrifugation, protein was quantified in the supernatant by the method of Bradford (5). Samples with equal protein (15 µg for eNOS, 60 µg for GR) were loaded on a 7.0% polyacrylamide gel with 0.1% sodium dodecyl sulfate and were separated by electrophoresis at 100 V for 1 h. Proteins were transferred onto Hybond enhanced chemiluminescence (ECL) nitrocellulose membranes (Amersham; Arlington Heights, IL) at 360 mA for 1 h at room temperature using a semidry blotter (Bio-Rad). The Hybond membrane was probed by mouse monoclonal antiserum for eNOS (1:600) obtained from Transduction Laboratories (Lexington, KY) and rabbit polyclonal antiserum for GR obtained from Affinity Bioreagents (Golden, CO). The secondary antiserums were horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies obtained from Amersham. Proteins were visualized with ECL reagents (Amersham), and the blots were exposed to hyperfilm. Results were quantified by a scanning densitometer (model 670, Bio-Rad) and expressed as a percentage of the control value.

Data analysis. Data were presented as means ± SE. Statistical analyses were performed with ANOVA followed by Newman-Keuls post hoc tests. Values were considered statistically significant at P < 0.05.

	RESULTS

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Effect of cortisol on NO_x release. The time course of the effect of cortisol on basal NO_x release (Fig. 1) demonstrated a general decrease in the NO_x levels in cortisol-pretreated cell media, with the difference between control and treatment becoming more pronounced over time. Although no significant effect was observed under 4 and 12 h when treated with 83 nM cortisol, a significant decrease of 54% was observed in BCAEC media NO_x levels at 24 h (n = 4, P < 0.05). At 24 h after treatment, cortisol (1 nM-10 µM) produced a dose-dependent decrease in NO_x levels (Fig. 2) with a maximum inhibition of 40% (n = 5, P < 0.05). The cortisol-mediated dose-dependent decrease in NO_x levels was also apparent in the presence of 3 µM carbenoxolone. Two-way ANOVA indicated that carbenoxolone significantly (P < 0.05) decreased the cortisol-mediated inhibitory effect of NO_x release in BCAEC.

Effect of cortisol on eNOS levels. Cortisol pretreatment lowered eNOS protein levels in the cell lysates as detected by Western blotting (Fig. 4). This effect became significant at 24 h when 83 nM (n = 5) and 1 µM (n = 9) cortisol-treated cells exhibited ~25% and 40% decreases, respectively, in eNOS protein levels compared with the control cells (P < 0.05). Previous studies suggested that glucocorticoids might decrease eNOS protein levels partly by inhibiting eNOS gene transcription (48). In the present study, the effect of cortisol on eNOS protein stability was determined by Western blot analysis. BCAEC were treated with control medium or medium with 1 µM cortisol for 24 h, followed by the protein synthesis inhibitor cycloheximide (50 µg/ml) for varying durations up to 12 h. As shown in Fig. 5, pretreatment of the cells with cortisol significantly (n = 5, P < 0.05) increased the degradation rate of eNOS in the presence of cycloheximide (slope: from 0.99 ± 0.75 to 2.50 ± 0.51).

Effect of cortisol on [Ca²+]_i. The effect of cortisol treatment on [Ca²⁺]_i was evaluated by fura 2 fluorescence (Fig. 6). BCAEC were treated with control medium or medium with 1 µM cortisol for 24 h before being loaded with fura 2. Cortisol pretreatment did not significantly alter basal levels of [Ca²⁺]_i in BCAEC. Incubation with the G protein-linked P_2Y receptor agonist ATP significantly (P < 0.05) increased [Ca²⁺]_i in both control cells and cells pretreated with cortisol. However, the ATP-induced [Ca²⁺]_i mobilization was significantly (P < 0.05) lower in the cortisol-treated cells than in the control cells.

Effect of cortisol on ATP-mediated NO_x release. Cortisol altered the ATP-induced NO_x release in BCAEC (Fig. 7). The cells were pretreated with either control medium or medium with 1 µM cortisol for 24 h and then incubated with 30 µM ATP for 1 h. Incubation with ATP significantly increased the NO_x release in both control and cortisol-treated cells (n = 5, P < 0.05). However, the ATP-induced NO_x release was significantly (P < 0.05) lower in the cortisol-treated cells than in the control cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study demonstrates for the first time that prolonged cortisol treatment decreases both basal and agonist-stimulated NO_x release in coronary artery endothelial cells and that the response to cortisol is dose dependent. Other novel findings in the present study include that cortisol increases the eNOS degradation rate and that it inhibits intracellular Ca²⁺ mobilization in coronary artery endothelial cells. It is relatively difficult to accurately measure plasma cortisol levels because plasma cortisol concentrations vary widely, and the variation can occur within a very short time. Human plasma cortisol concentrations range from 40 to 180 ng/ml and may increase as much as 10-fold in response to severe stress (49), which represents 1-5 µM. It has been reported that low intensity prolonged exercise increases plasma cortisol concentration to 371 ng/ml (~1 µM) (43). Although bovine cortisol levels have not been studied in similar depth, normal plasma levels on the order of 10¹ nM have been observed and may increase several times when stressors induce cortisol release (9, 45). In vivo, only a small percentage of cortisol circulates in an active free form, whereas the remainder is reversibly bound to circulating corticosteroid-binding globulin and albumin proteins. In the present study, 16% FBS was used in the culture media, which was likely to produce a similar reduction in free cortisol concentrations. Given that stress of any kind will markedly increase cortisol concentrations, the physiological/pathophysiological relevance of this study is fully warranted. In addition, the present findings are also comparable to studies in human umbilical vein endothelial cells and bovine aortic endothelial cells in which dexamethasone (10-1,000 nM) decreased NO_x release and eNOS protein levels (48).

Previous studies demonstrated that dexamethasone decreased NO release and eNOS protein levels at 36 h after treatment in human umbilical vein endothelial cells (48). However, the time course of the effect of the glucocorticoids was not examined. The present study indicates that a significant decrease in NO release and eNOS protein levels was apparent at a much earlier time period in coronary artery endothelial cells. Nevertheless, the cortisol-induced decrease in NO release and eNOS protein levels in the present study was comparable to that seen in human umbilical vein endothelial cells induced by dexamethasone (48). Previous studies have demonstrated that dexamethasone treatment significantly decreases serum NO/NO levels and increases blood pressure in rats (48). When dexamethasone treatment was discontinued, both serum NO/NO and blood pressure returned to normal levels (48), suggesting that the effects of glucocorticoids are reversible. Similar findings were also obtained in fetal pulmonary artery endothelial cells in which dexamethasone exposure for 24 h caused a decline in cyclooxygenase-1 protein expression, and the ensuing withdrawal of dexamethasone for 24 h resulted in a recovery of cyclooxygenase-1 (24). Glucocorticoids have been found to decrease transcription of proteins involved in vasodilator synthesis such as cyclooxygenase-1 and eNOS (24, 48). In the case of eNOS, mRNA levels are further decreased by glucocorticoid through reduced mRNA stability (48). However, the effect of glucocorticoids on eNOS protein stability has not been previously examined. The present study is the first to demonstrate a direct effect of cortisol in decreasing eNOS protein stability in coronary artery endothelial cells. It is not clear at present whether this effect is unique to coronary artery endothelial cells or is common in endothelial cells among different vascular beds. Nevertheless, this finding suggests a new regulatory mechanism by which glucocorticoids regulate eNOS function. Although the mechanism of cortisol-decreased eNOS protein stability is not clear at present, it is speculated that heat shock protein 90 (HSP90) may play an important role given that HSP90 is a key regulator for both GR and eNOS (20, 36). The glucocorticoid-mediated decrease in NOS protein stability is not unprecedented. Inducible NO synthase (iNOS) protein levels have been shown to be modulated in response to various agents including dexamethasone by increased degradation via various proteolytic enzymes (14, 47).

The current understanding of cell response to glucocorticoid stimuli attributes a decrease in eNOS and iNOS activities to the long-term effects of expressional downregulation, mRNA degradation, and, in the case of iNOS, protein degradation (19, 47). However, the present study indicates that the cortisol-mediated decrease in agonist-induced intracellular Ca²⁺ mobilization may also play an important role in the modulation of NO release. Dependence of eNOS on the binding of a calmodulin/Ca²⁺ complex to reach maximal activity has been well documented (18). Therefore, by decreasing intracellular Ca²⁺ mobilization, cortisol provides an additional means of downregulating eNOS activity. The importance of this finding is emphasized by the significant depression of the ATP-mediated NO release. We have demonstrated the ability of cortisol to depress basal NO release in the present study. However, even the complete suppression of basal NO release would account for less than one-half of the observed cortisol suppression of the agonist-stimulated NO release. This suggests that modulation of agonist stimuli may be the primary mechanism through which cortisol regulates NO production in BCAEC. The effect of glucocorticoids on [Ca²⁺]_i may play a major role in regulating rapid vasodilation in responses to stressors. ATP is a physiologically relevant substance with an important regulatory function in coronary vascular tone (10, 46). Release of ATP by several vascular sources including endothelial cells themselves has been observed (4, 53). The present study suggests the potential of cortisol to regulate physiological stimuli that occur in vivo.

Interaction of ATP with G protein-coupled P_2Y receptors results in an increase of intracellular calcium levels (21, 52). We have previously demonstrated that ATP produces a dose-dependent increase in [Ca²⁺]_i in cultured human coronary artery endothelial cells that is not affected by pretreatment of cells with 17-estradiol (51). In agreement with this previous study, ATP significantly increases [Ca²⁺]_i in BCAEC in the present study. However, in the present study, cortisol pretreatment significantly decreased ATP-mediated [Ca²⁺]_i elevation in BCAEC, suggesting a unique effect of glucocorticoids on Ca²⁺ mobilization. This is in agreement with previous findings that dexamethasone inhibited calcium ionophore A-23187-increased [Ca²⁺]_i in bovine aortic endothelial cells (23). Glucocorticoid-mediated modulation of [Ca²⁺]_i was also observed in other cell types including basophilic leukemia (RBL-2H3) and skeletal muscle (C2C12) cells (22, 35). The findings that glucocorticoids suppress intracellular Ca²⁺ mobilization resulting from both G protein-coupled receptor and calcium ionophore stimulation in endothelial cells suggest that glucocorticoids modulate intracellular Ca²⁺ fluxes downstream of receptor stimulation and may act on Ca²⁺ extrusion or reuptake into Ca²⁺ stores and present a new mechanism by which glucocorticoids regulate NO release. Furthermore, the ability of cortisol to modulate intracellular Ca²⁺ mobilization suggests a multiplicity of coronary artery endothelium-based effects. This is illustrated by the fact that intracellular Ca²⁺ elevation is involved in signal transduction leading to the production of several vasodilators including endothelium-derived hyperpolarizing factor, prostacyclin, and NO in endothelial cells (11, 17, 25). Similarly, elevation of intracellular Ca²⁺ decreases endothelial synthesis of the vasoconstrictor endothelin (38). Interestingly, glucocorticoid stimulates an increase in peak [Ca²⁺]_i response to various vasoconstrictors in vascular smooth muscle in vitro and in vivo (2, 29). While this may or may not represent a separate mechanism of Ca²⁺ modulation, it is clear that glucocorticoid acts in multiple tissues in a concerted fashion to decrease vasodilation and increase vasoconstriction.

Dexamethasone has been used as a substitute for endogenous glucocorticoid in previous studies. However, the endogenous glucocorticoid cortisol may be converted to several compounds intracellularly. A predominant reaction is the reversible conversion of cortisol to cortisone catalyzed by 11-HSD. Two forms of 11-HSD have been identified. 11-HSD₁ predominantly catalyzes the reduction of cortisone but also catalyzes the dehydrogenation of cortisol, whereas 11-HSD₂ catalyzes only the dehydrogenase reaction (30, 50). Carbenoxolone is a potent inhibitor of both 11-HSD isozymes (34). The present finding that carbenoxolone attenuates cortisol-mediated inhibition of NO release suggests a predominance of the 11-HSD₁ isoform and its ability to reform the parent steroid cortisol in BCAEC. Although both isoforms have been identified in rat aortic endothelial cells, type 1 mRNA predominates, supporting this conclusion (7). Low levels of 11-HSD₂ activity in endothelial cells would indicate that cortisol may also potentiate mineralocorticoid effects as the mineralocorticoid receptor binds cortisol with a similar affinity as the endogenous mineralocorticoid aldosterone (42).

Previous studies utilizing dexamethasone, while specific for glucocorticoid actions, suffer from the altered pharmacological profile of dexamethasone. Specifically, dexamethasone is not an active substrate for 11-HSD₁ and may be poorly reactive with 11-HSD₂ (3, 12, 15). The fact that, in other tissues, dexamethasone has produced results that differ from endogenous steroid may be due to differences in respect to the 11-HSD activity (13). However, although no demonstrable differences between dexamethasone and cortisol have been observed in the endothelium, the ability of 11-HSD inhibitors to produce significant changes in the effects of endogenous cortisol in vitro in the present study and of endogenous corticosterone in endothelium-intact vascular rings suggests that 11-HSD may play an important role in the overall effect of reactive glucocorticoids in endothelial cells (6). Despite the possibility that 11-HSD expression and activity may be altered in vitro, the use of endogenous glucocorticoid such as cortisol in functional studies may be the simplest means of evaluating the effects of glucocorticoid. Furthermore, the ability of 11-HSD₂ to control glucocorticoid exposure to mineralocorticoid receptors combined with the existence of dual glucocorticoid and mineralocorticoid pathways involved in the generation of cardiovascular disorders such as hypertension demonstrates the need to utilize endogenous glucocorticoid in the analysis of the physiological response (44).

There is a growing body of data indicating that cortisol downregulates NO synthesis/release in endothelial cells through mechanisms of 1) decreased eNOS mRNA expression and 2) increased degradation of eNOS mRNA (48). The decreased eNOS protein levels observed in this study could support such conclusions. Furthermore, the present study demonstrates the novel mechanisms of 3) decreased eNOS protein stability and 4) decreased agonist-mediated intracellular Ca²⁺ mobilization. The ability of cortisol to inhibit the NO release in coronary artery endothelium illustrates a mechanism linking stress and elevated cortisol to coronary artery disease. The inhibition of cortisol on the agonist-mediated NO release is consistent with the theory that one of the main functions of cortisol is the suppression of effects initiated by a stress response. The further characterization of the ability of glucocorticoids to act in a calcium-dependent fashion and to be modified by 11-HSD may provide alternate treatment options targeting glucocorticoid disorders.