Background and purpose: The present study was designed to assess whether cyclooxygenase-2 (COX-2) activation is involved in the effects of chronic aldosterone treatment on endothelial function of mesenteric resistance arteries (MRA) from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Experimental approach: Relaxation to acetylcholine was measured in MRA from both untreated and aldosterone-treated strains. Vasomotor responses to prostacyclin and U46619 were also analysed. Release of 6-oxo-prostaglandin (PG)F 1a and thromboxane B 2 (TxB 2 ) was determined by enzyme immunoassay. COX-2 protein expression was measured by western blot. Key results: Aldosterone reduced acetylcholine relaxation in MRA from both strains. In MRA from both aldosterone-treated strains the COX-1/2 or COX-2 inhibitor (indomethacin and NS-398, respectively), TxA 2 synthesis inhibitor (furegrelate), prostacyclin synthesis inhibitor (tranylcypromine) or TxA 2 / PGH 2 receptor antagonist (SQ 29 548), but not COX-1 inhibitor SC-560, increased acetylcholine relaxation. In untreated rats this response was increased only in SHR. Prostacyclin elicited a biphasic vasomotor response: lower concentrations elicited relaxation, whereas higher concentrations elicited contraction that was reduced by SQ 29 548. Aldosterone increased the acetylcholine-stimulated production of 6-oxo-PGF 1a and TxB 2 in MRA from both strains. COX-2 expression was higher in both strains of rats treated with aldosterone. Conclusions and implications: Chronic treatment with aldosterone impaired endothelial function in MRA under normotensive and hypertensive conditions by increasing COX-2-derived prostacyclin and thromboxane A 2 . As endothelial dysfunction participates in the pathogenesis of many cardiovascular disorders we hypothesize that anti-inflammatory drugs, specifically COX-2 inhibitors, could ameliorate vascular damage in patients with elevated aldosterone production.

Abstract-The aim of the present study was to analyze the possible involvement of vasoconstrictors prostanoids on the reduced endothelium-dependent relaxations produced by chronic administration of aldosterone in Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). For this purpose, acetylcholine (ACh) relaxations in aortic segments from both strains were analyzed in absence and presence of the cyclooxygenase-1 (COX-1) and COX-2 inhibitor indomethacin, the specific COX-2 inhibitor NS-398, the TP receptor antagonist (SQ 29 548), the thromboxane A 2 (TXA 2 ) synthase inhibitor furegrelate, and the prostacyclin (PGI 2 ) synthesis inhibitor tranylcypromine (TCP). In addition, COX-2 protein expression was studied by Western blot analysis. Release of prostaglandin E 2 (PGE 2 ) and the metabolites of PGF 2␣ , TXA 2 , and PGI 2 , 13,14-dihydro-15-keto PGF 2a , TXB 2 , and 6-keto-PGF 1␣ , respectively, were measured. Treatment with aldosterone did not modify blood pressure levels in any strain. However, aldosterone markedly reduced (PϽ0.05) ACh-induced relaxations in segments from both strains in a similar extent. Indomethacin, NS-398, SQ 29 548, and TCP enhanced (PϽ0.05) ACh relaxations in both strains treated with aldosterone. Aortic COX-2 protein expression was higher in both strains of rats treated with aldosterone. In normotensive animals, aldosterone increases the ACh-stimulated aortic production of 13,14-dihydro-15-keto PGF 2a, PGE 2 , and 6-keto-PGF 1␣ (PϽ0.05). In SHR, ACh only increased the 6-keto-PGF 1␣ production (PϽ0.05). It could be concluded that chronic treatment with aldosterone was able to produce endothelial dysfunction through COX-2 activation in normotensive and hypertensive conditions. PGI 2 seems to be the main factor accounting for endothelial dysfunction in hypertensive rats, whereas other prostanoids besides PGI 2 appear to be involved in endothelial dysfunction under normotensive conditions. Key Words: aldosterone Ⅲ endothelium Ⅲ prostacyclin Ⅲ normotension Ⅲ hypertension A ldosterone is a mineralocorticoid that participates in electrolyte balance and plays an important physiological role in the long-term regulation of Na ϩ and K ϩ in the distal tubule and collecting duct. [1][2][3][4] To date, several studies suggested that aldosterone plays a larger role than once appreciated in the regulation of vascular tone as well as in cardiovascular alterations such as endothelial dysfunction, vascular fibrosis, and inflammation, left ventricular hypertrophy, congestive heart failure, and cardiac arrhythmias. [5][6][7][8][9][10][11][12] Furthermore, aldosterone has also been shown to be involved in the pathogenesis of hypertension. [13][14][15] In general terms, endothelial dysfunction is characterized by reduced endothelium-dependent relaxations, suggesting a reduced availability of NO attributable to a reduction of NO production or enhanced NO inactivation. 16,17 In addition, an increased production of vasoconstrictor factors could be also responsible for the reduced endothelium-dependent...

We have previously described that chronic administration of ouabain induces hypertension and functional alterations in mesenteric resistance arteries. The aim of this study was to analyze whether ouabain treatment also alters the structural and mechanical properties of mesenteric resistance arteries. Wistar rats were treated for 5 wk with ouabain (8.0 microg/day sc). The vascular structure and mechanics of the third-order branches of the mesenteric artery were assessed with pressure myography and confocal microscopy. Total collagen content was determined by picrosirius red staining, collagen I/III was analyzed by Western blot, and elastin was studied by confocal microscopy. Vascular reactivity was analyzed by wire myography. Internal and external diameters and cross-sectional area were diminished, whereas the wall-to-lumen ratio was increased in arteries from ouabain-treated rats compared with controls. In addition, arteries from ouabain-treated rats were stiffer. Ouabain treatment decreased smooth muscle cell number and increased total and I/III collagens in the vascular wall. However, this treatment did not modify adventitia and media thickness, nuclei morphology, elastin structure, and vascular reactivity to norepinephrine and acetylcholine. The present work shows hypotrophic inward remodeling of mesenteric resistance arteries from ouabain-treated rats that seems to be the consequence of a combination of decreased cell number and impaired distension of the artery, possibly due to a higher stiffness associated with collagen deposition. The narrowing of resistance arteries could play a role in the pathogenesis of hypertension in this model.

1 This study compares the role of endothelial factors in a-adrenoceptor contractile responses in mesenteric resistance (MRA) and superior (SMA) mesenteric arteries from ouabain-treated (8.0 mg day À1 , 5 weeks) and untreated rats. The role of the renin-angiotensin system was also evaluated. 2 Ouabain treatment increased systolic blood pressure. In addition, ouabain reduced the phenylephrine response in SMA but did not alter noradrenaline responses in MRA. 3 Endothelium removal or the nitric oxide synthase (NOS) inhibitor (L-NAME, 100 mM) increased the responses to a-adrenergic agonists in both vessels. After ouabain treatment, both endothelial modulation and the L-NAME effect were increased in SMA, while only the L-NAME effect was increased in MRA. Endothelial NOS expression remained unaltered after ouabain treatment. 4 Indomethacin (10 mM) similarly reduced the noradrenaline contraction in MRA from both groups; in contrast, in SMA, indomethacin only reduced phenylephrine-induced contractions in segments from untreated rats. Co-incubation of L-NAME and indomethacin leftward shifted the concentration-response curves for noradrenaline more in MRA from ouabain-treated rats; tetraethylammonium (2 mM) shifted the noradrenaline curves further leftward only in MRA from untreated rats. 5 Losartan treatment prevents the development of hypertension but not all vascular changes observed after ouabain treatment. 6 In conclusion, a rise in endothelial NO and impaired prostanoid participation might explain the reduction in phenylephrine-induced contraction in SMA after ouabain treatment. An increase in the modulatory effect of endothelial NO and impairment of endothelium-dependent hyperpolarizing factor effect might explain why the ouabain treatment had no effect on noradrenaline responses in MRA.

Background/Aims: The purpose of this study was to examine the cardiovascular effects of long-term ouabain treatment at different time points. Methods: Systolic blood pressure (SBP) was measured by tail-cuff method in male Wistar rats treated with ouabain (approx. 8.0 µg·day^–1) or vehicle for 5, 10 and 20 weeks. Afterwards, vascular function was assessed in mesenteric resistance arteries (MRA) using a wire myograph. ROS production and COX-1 and COX-2, TNF-α, and IL-6 protein expression were investigated. Results: SBP was increased by ouabain treatment up to the 6th week and remained stable until the 20th week. However, noradrenaline-induced contraction increased only in MRA in rats treated with ouabain for 20 weeks. NOS inhibition and endothelium removal increased the noradrenaline response, but to a smaller magnitude in MRA in the ouabain group. Moreover, inhibition of COX-2 or incubation with superoxide dismutase restores noradrenaline-induced contraction in the 20-week ouabain group to control levels. ROS production as well as COX-2, IL-6 and TNF-α protein expression increased in MRA in this group. Conclusion: Although ouabain treatment induced hypertension in all groups, a larger noradrenaline induced contraction was observed over 20 weeks of treatment. This vascular dysfunction was related to COX-2-derived prostanoids and oxidative stress, increased pro- inflammatory cytokines and reduced NO bioavailability.

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The endothelium plays a vital role in maintaining circulatory homeostasis by the release of relaxing and contracting factors. Any change in this balance may result in a process known as endothelial dysfunction that leads to impaired control of vascular tone and contributes to the pathogenesis of some cardiovascular and endocrine/metabolic diseases. Reduced endotheliumderived nitric oxide (NO) bioavailability and increased production of thromboxane A2, prostaglandin H2 and superoxide anion in conductance and resistance arteries are commonly associated with endothelial dysfunction in hypertensive, diabetic and obese animals, resulting in reduced endothelium-dependent vasodilatation and in increased vasoconstrictor responses. In addition, recent studies have demonstrated the role of enhanced overactivation of β-adrenergic receptors inducing vascular cytokine production and endothelial NO synthase (eNOS) uncoupling that seem to be the mechanisms underlying endothelial dysfunction in hypertension, heart failure and in endocrine-metabolic disorders. However, some adaptive mechanisms can occur in the initial stages of hypertension, such as increased NO production by eNOS. The present review focuses on the role of NO bioavailability, eNOS uncoupling, cyclooxygenase-derived products and pro-inflammatory factors on the endothelial dysfunction that occurs in hypertension, sympathetic hyperactivity, diabetes mellitus, and obesity. These are cardiovascular and endocrine-metabolic diseases of high incidence and mortality around the world, especially in developing countries and endothelial dysfunction contributes to triggering, maintenance and worsening of these pathological situations.

1 Renin-angiotensin and endothelin systems are involved in the cardiovascular effects produced by treatment with ouabain. We recently demonstrated that the contractile response to phenylephrine is decreased in ouabain-treated rats. The present study investigated whether endothelin-1 (ET-1) and angiotensin II (Ang II) contributes to the vascular changes observed in rats chronically treated with ouabain. 2 Wistar rats were treated with ouabain (8.0 mg day À1 , s.c. pellets for 5 weeks) alone or in combination with an endothelin type A receptor (ET A ) antagonist, BMS182874 (40 mg kg À1 day À1 , per gavage) or an angiotensin type 1 (AT 1 ) receptor antagonist, losartan (15 mg kg À1 day À1 , p.o.). 3 Treatment with ouabain increased systolic blood pressure and treatment with either losartan or BMS182874 prevented the development of ouabain-induced hypertension. 4 The sensitivity and maximal response for phenylephrine were reduced in aortic rings from ouabain-treated rats. Removal of the endothelium or in vitro exposure to an inhibitor of nitric oxide synthase (NOS), N-nitro-L-arginine methyl ester (L-NAME, 100 mM) increased the responses to phenylephrine, an effect that was more pronounced in aortas from ouabain-treated rats. Endothelial NOS protein (eNOS) expression was increased after ouabain treatment. Treatment with BMS182874, but not with losartan, prevented the effects of ouabain on the reactivity of phenylephrine and in eNOS protein expression. 5 Gene expression of pre-pro-ET-1 and ET A receptors was increased in aortic rings from ouabaintreated rats. ET B receptor gene expression was not altered by ouabain treatment. 6 In conclusion, our results suggest that endothelin and angiotensin systems play an important role in the development of ouabain-induced hypertension. However, ET-1, by activation of ET A receptors, but not Ang II, contributes to changes in vascular reactivity to phenylephrine induced by chronic treatment with ouabain. British Journal of Pharmacology (2004) 143, 794-802. doi:10.1038/sj.bjp.0705994 Keywords: Ouabain; hypertension; endothelium; nitric oxide; renin-angiotensin system; endothelin Abbreviations: Ang II, angiotensin II; AT 1 , angiotensin type 1 receptor; dAUC, difference of area under the concentrationresponse curves; E max , maximum response; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; ET A , endothelin type A receptor; ET B , endothelin type B receptor; KHB, Krebs-Henseleit bicarbonate buffer; L-NAME, N-nitro-L-arginine methyl ester; NO, nitric oxide; PAGE, polyacrylamide gel; pD 2 , negative logarithm of concentrations producing 50% of maximum response; RT-PCR, reverse transcriptase-polymerase chain reaction; SBP, systolic blood pressure; SDS, sodium lauryl sulphate