Glucocorticoids play a role in the control of vascular smooth muscle tone through the alteration of vasoconstrictor and vasodilator factor production. We studied the effect of dexamethasone on vasoconstriction induced by electrical field stimulation (EFS) in rat mesenteric arteries (MAs) and the role of hypertension in this effect. Endothelium-denuded MAs were obtained from Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs). EFS response was analyzed by isometric tension recordings and cyclooxygenase (COX-1 and COX-2) expression by Western blot. Noradrenaline (NA) release was evaluated in segments incubated with [3H]NA. Dexamethasone (0.1 and 1 μM; 2–8 h) reduced vasoconstriction to EFS (200 mA, 0.3 ms, 1–16 Hz), in a dose- and time-dependent manner only in SHRs. However, the EFS-induced release of [3H]NA was increased in SHR arteries preincubated with dexamethasone (1 μM; 6 h). The thromboxane A2 (TxA2) synthase inhibitor furegrelate (10 μM), the selective COX-2 inhibitor NS-398 (N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide; 10 μM), or the TxA2 receptor antagonist SQ 29548 (1 μM), reduced EFS and NA induced vasoconstrictor responses. However, the effect of these drugs was abolished in arteries preincubated with dexamethasone. Both dexamethasone and phentolamine (1 μM) inhibited the increased thromboxane B2 levels observed after EFS. COX-2 protein expression was reduced by dexamethasone in SHR arteries. Results suggest that dexamethasone reduces vasoconstriction to EFS in MAs from SHRs by decreasing COX-2 expression, thereby decreasing the smooth muscle TXA2 release induced by α-adrenoceptor activation. The undetectable COX-2 expression in MAs from normotensive animals explains the noneffect of dexamethasone in their arteries.
Vascular tone is determined by a equilibrium among several mechanisms, one of which involves perivascular innervation. This regulation involves adrenergic, cholinergic, nitrergic, peptidergic, and/or sensory innervations that are specific to the vascular bed considered (Loesch, 2002). Electrical field stimulation (EFS) is widely used to study the contribution to vasomotor response made by neurotransmitters released from nerve endings. When applied to rat mesenteric artery, EFS produces a vasoconstrictor response that is the integrated result of different released neurotransmitter, mainly noradrenaline from adrenergic nerve terminals. Furthermore, in spontaneously hypertensive rats (SHRs), neuronal nitric oxide (NO) from nitrergic innervation and calcitonin gene-related peptide (CGRP) from sensory nerves also participate in the vasomotor response to EFS (Kawasaki et al., 1988; Ralevic et al., 1995; Wimalawansa, 1996; Marín et al., 2000).
Glucocorticoid hormones play an important role in the control of vascular smooth muscle tone by acting on both endothelial and vascular smooth muscle cells. In endothelial cells, glucocorticoids suppress the production of vasodilators such as prostacyclin and nitric oxide (Zingarelli et al., 1994; Jun et al., 1999). In vascular smooth muscle cells, glucocorticoids enhance agonist-mediated pharmacomechanical coupling at multiple levels and increase responses to vasoconstrictor agents, like noradrenaline (NA) (Yang and Zhang, 2004). Meanwhile, dexamethasone regulates neuronal CGRP expression and release (Supowit et al., 1995), and we have recently demonstrated that glucocorticoid receptor activation increases the vasodilator response to CGRP in mesenteric arteries (MAs) from SHRs (Balfagón et al., 2004; Márquez-Rodas et al., 2006).
Vascular tone is also regulated by COX-derived prostanoids produced in the vascular wall. Several authors have demonstrated the production of contractile prostanoids in response to vasoconstrictor agents, including α-adrenergic agonists (Tabernero et al., 1999; Xavier et al., 2003; Alvarez et al., 2005). These mediators may be synthesized by the constitutive and inducible isoforms of cyclooxygenase (COX-1 and COX-2, respectively). COX-2 is not normally expressed but can be induced in some pathological situations such as hypertension (Alvarez et al., 2005). Some authors have suggested that contractile COX-2 products contribute to the increased vasoconstrictor responses observed in hypertension (Alvarez et al., 2007). Because glucocorticoids inhibit the COX-2 expression in the vascular wall, it seems possible that inhibition of COX-2 derivates would be implicated in the vascular effects of dexamethasone.
The results mentioned above led us to hypothesize that dexamethasone could alter the vasomotor response induced by EFS to different extents and through different mechanisms in normo- and hypertension. Therefore, the goal of this work was to study the effect of dexamethasone on the EFS-induced vasoconstrictor response in MAs from normotensive and hypertensive rats, as well as the role of COX-2 in that dexamethasone effect.
Materials and Methods
Animals and Tissue Preparation. Six-month-old male Wistar-Kyoto (WKY) rats and SHRs were used. After sacrifice by CO2 inhalation, the superior mesenteric artery was carefully dissected out, cleaned of connective tissue, and placed in Krebs-Henseleit solution (KHS) at 4°C. The investigation conforms to the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health (Institute of Laboratory Animal Resources, 1996).
Vascular Reactivity. The method used for isometric tension recording has been described elsewhere (Balfagón et al., 2004). Experiments were performed in endothelium-denuded segments to eliminate the main source of vasoactive substances and so avoid any action by different drugs on the endothelial cells that could lead to misinterpretation of results. The segments were subjected to a tension of 0.5g that was readjusted every 15 min during a 90-min equilibration period. After that, the vessels were exposed to 75 mM K+ to check their functional integrity. The absence of vascular endothelium was confirmed by the inability of acetylcholine (10 μM) to relax segments precontracted with NA (1 μM).
Frequency-response curves to EFS (1, 2, 4, 8, and 16 Hz) were performed in segments from both strains in a consecutive manner. The parameters used for EFS were 200 mA, 0.3 ms, 1 to 16 Hz, for 30 s with an interval of 1 min between each stimulus, the time required to recover basal tone, and a period of 2 h between consecutive curves. Previously, we have demonstrated that the observed responses were abolished by tetrodotoxin (Marín et al., 2000), indicating the neuronal origin of the contraction. Four consecutive frequency-dependent curves performed in the same segment from WKY rats or SHRs were similar, ruling out any change in EFS during the experimental period. EFS responses in the presence of dexamethasone (0.1 and 1 μM) were performed to evaluate the possible effect of this glucocorticoid on the neural control of vasomotor tone. To analyze a possible time-dependent effect, dexamethasone was added to the bath at different incubation periods: 2, 4, 6, and 8 h for dexamethasone (0.1 μM) and 2, 4, and 6 h for dexamethasone (1 μM), before the frequency-response curves.
The possible participation of nitrergic or sensory innervations in the effect of dexamethasone (1 μM) on the responses to EFS was analyzed. Experiments analyzing participation of nitrergic innervation were performed in the presence of NG-nitro-l-arginine methyl ester (l-NAME; 100 μM), an NO synthase inhibitor that was added to the bath 30 min before the first frequency-response curve and maintained until the end of the experiments. The participation of sensory innervation was studied by adding the sensory neurotoxin capsaicin (0.5 μM) to the bath 60 min before the first frequency-response curve and maintaining it until the end of the experiments.
The possible role of adrenergic mechanisms in the effect of dexamethasone on the EFS-induced vasoconstrictor response was studied by evaluating the response to exogenous NA (10 nM–10 μM) and the release of tritiated NA (see tritium release experiments below) from adrenergic endings in MAs from SHRs. Concentration-response curves to NA were performed in the absence and in the presence of dexamethasone (1 μM) for 6 h.
To analyze a possible role of COX derived in vasoconstriction induced by EFS, frequency-response curve to EFS was performed in the presence of the COX-1/2 inhibitor indomethacin. To analyze the role of COX-2-derived TXA2 in vasoconstriction induced by both EFS and exogenous NA, frequency-response curve to EFS and concentration-dependent curves to NA were performed in the presence of the selective COX-2 inhibitor NS-398 (10 μM), the TXA2 synthase inhibitor furegrelate (10 μM), or the TxA2 receptor antagonist SQ 29548 (1 μM). EFS and exogenous NA responses were also performed in the presence of dexamethasone (1 μM) plus NS-398 (10 μM), furegrelate (10 μM), or SQ 29548 (1 μM).
Tritium Release Experiments. Denuded mesenteric segments from SHRs were set up in a nylon net and immersed for 30 min in 10 ml of KHS at 37°C and continuously gassed with a 95% O2-5% CO2 mixture (stabilization period). Thereafter, they were incubated for 60 min in 1 ml of bubbled KHS at 37°C containing (±)[3H]noradrenaline (0.33 μM, 10 μCi/ml, specific activity of 10 Ci/mmol). Afterward, the arteries were transferred to a superfusion chamber with two parallel platinum electrodes, 0.5 cm apart, connected to a stimulator (model S44; Grass Instruments, Quincy, MA) for EFS. The arteries were superfused at a rate of 2 ml/min with oxygenated KHS at 37°C for 100 min, after which time the steady-state level of basal tritium efflux was reached. Two electrical stimulation periods of 60 s (200 mA, 0.3 ms, 4 Hz) were applied to the arteries as described above for vascular reactivity in the absence and in the presence of dexamethasone. The superfusate was collected in vials (10 in all) at 30-s intervals. These vials were collected in the following manner: three before stimulation, to determine the basal level of tritium efflux, two during stimulation, and five after the stimulation; the latter time was enough to recover the basal level of tritium efflux. Afterward, Ready-Protein solution (PerkinElmer, Turku, Finland) was added to the vials, and radioactivity was measured in a scintillation counter (Beckman LS 5000 TD).
The possible role of β-adrenergic receptors in the dexamethasone effect was analyzed in another set of experiments in which β-antagonist propranolol (1 μM) was added to the bath 30 min before the second stimulation period in the presence and absence of dexamethasone. The stimulated tritium release was calculated by subtracting the basal tritium release (B1 and B2) from that evoked by EFS (S1 and S2). Thereafter, the ratios for net tritium release between S2/S1 were calculated to eliminate differences between arteries. The effect of different drugs on the evoked tritium release was expressed as their effect on these ratios. The amount of radioactivity released was expressed in disintegrations per minute per milligram wet tissue.
Western Blot. Superior mesenteric arteries from control WKY rats and SHRs were incubated 6 h in dexamethasone (1 μM) and then homogenized in a boiling buffer composed of sodium vanadate (1 mM), 1% SDS, and Tris-HCl (0.01 M), pH 7.4. Homogenates containing 20 μg of protein were electrophoretically separated on a 10% SDS-polyacrylamide gel and transferred to a polyvinylfluoride membrane. The membrane was blocked for 2 h at room temperature in a Tris-buffered saline solution (100 mM), 0.9% (w/v) NaCl, 0.1% SDS, and 0.01% Tween 20 with 5% nonfat powdered milk before being incubated overnight at 4°C with mouse monoclonal antibody for COX-1 (1:1000; Cayman Chemical, Ann Arbor, MI) and with rabbit polyclonal antibody for COX-2 (1:200; Cayman Chemical). After washing, the membrane was incubated with a 1:1000 dilution of anti-rabbit IgG antibody conjugated to horseradish peroxidase (Amersham International, Little Chalfont, UK). The membrane was thoroughly washed, and the immunocomplexes were detected using an enhanced horseradish peroxidase/luminol chemiluminescence system (ECL plus; Amersham International). The same membrane was used to determine α-actin expression using a monoclonal anti-α-actin antibody (1:3000 dilution; Sigma-Aldrich, Madrid, Spain).
Measurement of Thromboxane A2 Release. To measure the release of the TxA2 metabolite, thromboxane B2, we used the TxB2 immunoassay (Cayman Chemical). Endothelium-denuded segments of mesenteric arteries were preincubated for 30 min in 5 ml of KHS at 37°C and continuously gassed with a 95% O2-5% CO2 mixture (stabilization period). Afterward, the arteries were transferred to a 200-μl chamber containing two parallel platinum electrodes, connected to a stimulator (model S44; Grass Instruments) for EFS. After two washout periods of 4.5 min, the medium was collected to measure basal TxB2 release. Once the chamber was refilled, an EFS period of 30 s at 1, 2, 4, 8, and 16 Hz was applied at 1-min intervals, and then the medium was taken from the chamber to measure the EFS-induced TxB2 release. Another group of experiments was performed in segments treated with dexamethasone (1 μM) for 6 h before the EFS was applied. To analyze the possible role of α-adrenoceptor activation in the EFS-induced TxA2 release, the arteries were incubated with phentolamine (1 μM) for 30 min before applying EFS. The different assays were made following the manufacturer's instructions. Results were expressed as picograms of TxB2 per milliliter per milligram of tissue.
Drugs and Solutions. Drugs used were acetylcholine chloride (2-acetoxyethyltrimethylammonium chloride), capsaicin (8-methyl-N-vanilyl-trans-6-nonenamide), dexamethasone (9α-fluoro-16α-methylprednisolone-21-phosphate disodium salt), furegrelate ([5-(3-pyridinylmethyl) benzoflurancarboxilic acid sodium-potassium salt]), l-noradrenaline (R-4-[2-amino-1-hydroxyethyl]-1,2 benzenediol), l-NAME, phentolamine (2-[N-(3-hydroxyphenyl)-p-toluidinomethyl-2-imidazolidine hydrochloride]), propranolol ([S]-1-isopropylamino-3-(1-naphthyloxy)-2-propranolol hydrochloride]) (Sigma Chemical Co., St. Louis, MO), NS-398 (Calbiochem, San Diego, CA), SQ 29548 (MP Biomedicals, Irvine, CA), and (±)-[3H]noradrenaline hydrochloride (New England Nuclear, Boston, MA). The composition of the KHS was as follows: 115 mM NaCl, 2.5 mM CaCl2, 4.6 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, 25 mM NaHCO3, 11.1 mM glucose, and 0.03 mM Na2 EDTA.
Data Analysis and Statistics. The responses elicited by EFS or NA were expressed as a percentage of the contraction induced by 75 mM K+. All results are expressed as mean ± S.E.M. The number (n) of rats is indicated in the figure legends. Statistical analysis was done by comparing the curve obtained in the presence of the different substances with the previous or control curve by means of repeatedmeasure analysis of variance followed by Bonferroni's test. For Western blot, TxA2 release, and tritium release experiments, statistical analyses were done using Student's t test. Data analysis was carried out with the Prism 3.0 program (GraphPad Software, Inc., San Diego, CA). p Values less than 0.05 were considered significant.
Five consecutive EFS curves were performed at 2-h intervals in the same segments of MAs from WKY rats, and SHRs induced similar contractions (data not shown). In segments from SHRs, incubation (2–6 h) with dexamethasone (1 μM) reduced the contraction induced by EFS in a time-dependent manner (Fig. 1A), whereas this response remained unmodified in WKY rats (data not shown). The low dexamethasone concentration (0.1 μM) required an 8-h incubation to affect the response to EFS in SHRs (Fig. 1B). Because dexamethasone had no observable effect on the response to EFS in WKY rat segments, the following experiments were only performed with segments from SHRs.
Pretreating segments from SHRs with l-NAME 100 μMor capsaicin 0.5 μM had no effect on the reduction in the EFS-induced vasoconstriction produced by the dexamethasone (1 μM, 2–6 h) incubation (Fig. 2, A and B). The vasoconstrictor response induced by EFS was reduced by indomethacin (10 μM, data not shown), furegrelate (10 μM), NS-398 (10 μM), or SQ 29548 (1 μM) (Fig. 3). No additional effect was observed when these drugs were administered simultaneously with dexamethasone (1 μM, 2–6 h) (Fig. 3).
The vasoconstrictor response to exogenous NA was significantly reduced in segments preincubated for 6 h with 1 μM dexamethasone. Preincubation with indomethacin (data not shown), furegrelate, NS-398, or SQ 29548 also reduced the vasoconstrictor response to NA, and no additional effect was observed when these drugs were simultaneously administered with dexamethasone (Fig. 4, A and B).
Tritium Release. Two consecutive EFS in mesenteric segments from SHRs incubated with [3H]NA induced a similar tritium overflow (S1 = 408 ± 69 dpm/mg, S2 = 340 ± 57 dpm/mg; n = 4). The addition of dexamethasone (1 μM) at the end of the first EFS period and maintained until the end of the experiment increased the tritium overflow at 6 h (S1 = 344 ± 45 dpm/mg, S2 = 454 ± 67 dpm/mg; n = 4). The β-antagonist propranolol (1 μM) added to the bath 30 min before S2 did not modify the tritium overflow in the control situation (S1 = 486 ± 33 dpm/mg, S2 = 437 ± 57 dpm/mg; n = 4). In segments pretreated with dexamethasone, adding propranolol to the bath 30 min before S2 abolished the dexamethasone effect (S1 = 476 ± 74 dpm/mg, S2 = 381 ± 57 dpm/mg; n = 4) (Fig. 5).
Thromboxane A2 Release. EFS produced an increment in the TxB2 release in endothelium-denuded mesenteric arteries from SHRs, and this increase was inhibited by pretreatment with the α-adrenergic antagonist phentolamine (1 μM) (Fig. 6). In addition, basal and EFS-stimulated TxB2 levels were decreased by a 6-h preincubation with 1 μM dexamethasone (Fig. 6).
COX Expression. In endothelium-denuded segments from SHRs, 6-h preincubation with dexamethasone (1 μM) did not affect COX-1 protein expression, but it decreased COX-2 protein expression (Fig. 7).
It has been reported that glucocorticoids play an important role in the control of vascular smooth muscle tone by modifying vasoconstrictor responses to different vasoactive agents and by altering vascular prostanoid and/or nitric oxide production (Ullian, 1999; Yang and Zhang, 2004). However, the involvement of glucocorticoids in the neural vasomotor response has not yet been studied. We and others (Kawasaki et al., 1988; Ralevic et al., 1995; Wimalawansa, 1996; Marín et al., 2000) have previously demonstrated that EFS induces contractile responses in endothelium-denuded mesenteric arteries from WKY rats and SHRs, as the integrated result of the release of different neurotransmitters, mainly vasoconstrictor NA from adrenergic nerve terminals. The first objective of the present study was to analyze whether dexamethasone was able to modify the vasoconstrictor response induced by EFS and whether hypertension also the influenced this effect. Since previous reports have shown that actions of dexamethasone are dose- and time-dependent (Laan et al., 1999), two doses (1 and 0.1 mM) of dexamethasone and different incubation periods were studied. These doses were selected because local glucocorticoids formed in the vascular tissue can locally reach a higher concentration than in plasma, playing an autocrine and/or paracrine role within the vascular wall in physiopathology conditions (Walker, 2007). The dexamethasone had no effect on EFS-induced vasoconstriction in segments from WKY rats at any dose or incubation time. However, in segments from SHRs, it decreased the EFS-induced vasoconstriction in a dose- and time-dependent manner. These results indicate that dexamethasone only modulates the EFS-induced vasomotor response in hypertensive conditions, suggesting that it acts on a specific mechanism that is associated to hypertension.
Therefore, the dexamethasone effect observed in SHRs could be due to increased vasodilator neurotransmitter release or decreased vasoconstrictor neurotransmitter release as well as alterations in vasomotor response. We have previously demonstrated in MAs from SHRs that EFS induces NO release from nitrergic nerves, and this release negatively modulates the vasomotor response in the MA (Marín et al., 2000). Because glucocorticoids are able to increase (Limbourg and Liao, 2003) or decrease NO release in different vessels (Yang and Zhang, 2004), we analyzed the possible participation of this innervation in the effect of dexamethasone. In the presence of l-NAME, dexamethasone maintained the inhibitory effect on EFS-induced vasoconstriction, suggesting the participation of neurotransmitters other than NO in the dexamethasone effect. EFS response in SHR vessels also involves sensory innervation (Marín et al., 2000), and glucocorticoids are known to modify the function of the main mediator of this response, CGRP (Watson et al., 2002). Therefore, we also explored the possible participation of sensory innervation in the observed effect of dexamethasone. Preincubation with the neurotoxin capsaicin did not modify the effect of dexamethasone, excluding the participation of sensory innervation in the dexamethasone effect.
Once we had discounted the participation of nitrergic and sensory innervation, we analyzed possible changes in adrenergic innervation induced by dexamethasone. Because NA is the main adrenergic neurotransmitter to be released by EFS in rat MA, the effect of dexamethasone in SHRs could be due to a decrease in NA release and/or alteration in the sensitivity of vascular smooth muscle cells to NA. It is known that during electrical field stimulation, ATP and neuropeptide Y are coreleased with NA, which in rat mesenteric arteries induces vasoconstriction (Donoso et al., 1997), so it is still possible that a slight participation of these neurotransmitters in the EFS-induced vasomotor response could explain the effect of dexamethasone. However, in the frequency range used in the current work, the direct participation of these neurotransmitters is very scarce since the vasoconstrictor response to EFS was practically abolished in the presence of the α-adrenoceptor antagonist phentolamine (Marín et al., 2000). Taking these earlier observations into account, we focused our investigation on NA release and the vasoconstrictor response to exogenous NA. The results obtained in mesenteric arteries from SHRs indicate that dexamethasone had two opposite effects on the vascular adrenergic mechanism; it increased NA release but decreased the vasoconstrictor response to NA. The increase in NA release contrasts with earlier reports that glucocorticoids did not alter sympathetic activity (Sudhir et al., 1989; Whitworth et al., 1995). However, despite the many studies demonstrating the involvement of several complex mechanisms in the enhancement of catecholamine-stimulated vascular contractions by glucocorticoids, other studies have and still others have, like us, found a reduction in vasoconstriction (Ullian, 1999; Yang and Zhang, 2004).
Presynaptic β2-adrenoceptor modulates noradrenaline release from adrenergic endings by increasing NA release. In addition, it has been demonstrated that dexamethasone upregulates the number and function of adrenoceptors (Lenders et al., 1995; Kalavantavanich and Schramm, 2000). The results presented here show that the β-antagonist propranolol abolished the effect of dexamethasone on NA release. This result led us to speculate that dexamethasone increased NA release by increasing presynaptic β2-adrenoceptor function. However, although dexamethasone increases NA release, this effect cannot compensate for the decreased vasoconstrictor response to NA in mesenteric arteries from SHRs. Our results also suggest that other neuronal-independent mechanisms, probably associated with the NA vasoconstrictor response, are involved in the effects of dexamethasone in the EFS-induced vasoconstriction.
Vasoconstrictor response to α-adrenergic agonist is mediated in part by the release of COX derivates (Tabernero et al., 1999; Xavier et al., 2003; Alvarez et al., 2005). Recently, we have reported that vasoconstrictor response to phenylephrine is largely mediated by COX-2-derived vasoconstrictor prostanoids in rat aorta and that this effect is greater in hypertensive animals (Alvarez et al., 2005). It has been reported that COX-derived TxA2 is produced by α-1 adrenoreceptor activation, which plays an important role in vascular response mediated by these receptors (Pipili et al., 1988; Koltai et al., 1990). Vila et al. (2001) reported in human saphenous vein that the thromboxane analog, U-46619, increased vasoconstrictor response to NA through stimulation of TxA2 receptors. In the current work, we observed that the vasoconstrictor response to exogenous NA in endotheliumdenuded segments from SHRs is mediated in part by TxA2 release because the response was diminished by indomethacin, NS-394, furegrelate, or SQ 29548. However, in arterial segments treated with dexamethasone, the participation of TxA2 in the NA response was abolished. These results suggest that decreased TxA2 release could be implicated in the effect of dexamethasone on the EFS-induced vascular response, and this suggestion was reinforced by the fact that dexamethasone treatment decreased COX-2 expression in segments from SHRs; decreased COX-2 expression could also decrease TxA2 release and vasoconstriction to EFS-released NA. This hypothesis is reinforced by the fact that the COX inhibitor indomethacin, the COX-2 inhibitor NS-398, the thromboxane synthase inhibitor furegrelate, or the thromboxane receptor antagonist SQ 29548 all decreased vasoconstrictor response to EFS in control segments to a similar extent, and no additional effects were observed on EFS-induced vasomotion in segments pretreated with dexamethasone when indomethacin, NS-398, furegrelate, or SQ 29548 were administered. To confirm our hypothesis, we analyzed the TxA2 release induced by EFS in control and dexamethasone-treated arteries. The results obtained showed that under control conditions, EFS induced an increment in TxA2 release by endothelium-denuded SHR segments and that this increase was abolished by the α-adrenergic antagonist phentolamine. However, preincubation with phentolamine did not alter basal TxA2 release, in line with reports indicating that α-receptor activation increases TxA2 release (Pipili et al., 1988; Koltai et al., 1990). In segments treated with dexamethasone, both basal and EFS-stimulated TxA2 release were reduced, confirming our hypothesis that the effect of dexamethasone on EFS-induced vasoconstriction is mediated by decreases in COX-2-derived TxA2 release in response to α-adrenergic activation. In line with these data, Adeagbo et al. (2003) reported that COX-2 could be constitutively expressed in endothelial and smooth muscle cells. Furthermore, since the inhibitory effects of indomethacin, NS-398, furegrelate, or SQ 29548 on EFS-mediated contraction were observed in endothelium-denuded arteries, the observations confirm that, in our experimental conditions, TxA2 release occurs in vascular smooth muscle cells as we have reported in arteries from other rat strain (Blanco-Rivero et al., 2006).
Results obtained here demonstrated that in MAs from SHRs, dexamethasone had an opposite effect on the adrenergic response; it increased NA release and decreased COX-2-derived TxA2 release induced by NA. In the presence of dexamethasone, the vasoconstrictor response to EE was diminished, indicating that the decrease in TxA2 release is the predominant responsible mechanism, and it would be able to compensate for and overcome the effects of increased NA release.
In summary, the results presented here demonstrate that dexamethasone decreased the vasoconstrictor response to EFS in mesenteric arteries from SHRs but not from WKY rats. In SHRs, dexamethasone increases NA release, and this effect is mediated by an increased function of β-presynaptic adrenoceptors. Dexamethasone reduces vasoconstrictor response to NA and EFS in MAs from SHRs by decreasing COX-2 expression and consequently TxA2 release in response to α-adrenergic activation. The undetectable COX-2 expression in MAs from normotensive animals explains the noneffect of dexamethasone in these animals. In summary, in MAs from SHRs, a part of increased β-adrenoceptor-mediated NA release, dexamethasone decreases the vasoconstrictor response to EFS by an inhibition of COX-2 expression that decreases smooth muscle-derived TxA2 release by α-adrenoceptor activation.
We thank M. C. Fernández-Criado for the care of the animals.
This study was supported by the Fondo de Investigaciones Sanitarias (Grant PI051767), by the Comisión de Ciencia y Tecnología (Grant SAF-2006-07888), and by Banco de Santander-Universidad Autónoma de Madrid.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: EFS, electrical field stimulation; SHR, spontaneously hypertensive rat; NO, nitric oxide; CGRP, calcitonin gene-related peptide; NA, noradrenaline; MA, mesenteric artery; COX, cyclooxygenase; WKY, Wistar Kyoto; KHS, Krebs-Henseleit solution; l-NAME, NG-nitro-l-arginine methyl ester; NS-398, N-[2-cyclohexyloxy-4-nitrophenyl] methanesulfonamide; TxA2, thromboxane A2; SQ 29548, ([1S-[1α,2β(5Z),3β,4α]-7-[3-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid); TxB2, thromboxane B2; Dex, dexamethasone; U-46619, 9,11-dideoxy-9α,11α-methanoepoxyprostaglandin F2α.
- Received March 28, 2007.
- Accepted June 8, 2007.
- The American Society for Pharmacology and Experimental Therapeutics