Gloria Balfagón

Participation of Prostacyclin in Endothelial Dysfunction Induced by Aldosterone in Normotensive and Hypertensive Rats

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 A2 (TXA2) synthase inhibitor furegrelate, and the prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP). In addition, COX-2 protein expression was studied by Western blot analysis. Release of prostaglandin E2 (PGE2) and the metabolites of PGF2α, TXA2, and PGI2, 13,14-dihydro-15-keto PGF2a, TXB2, and 6-keto-PGF1α, respectively, were measured. Treatment with aldosterone did not modify blood pressure levels in any strain. However, aldosterone markedly reduced (P<0.05) ACh-induced relaxations in segments from both strains in a similar extent. Indomethacin, NS-398, SQ 29 548, and TCP enhanced (P<0.05) 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 PGF2a, PGE2, and 6-keto-PGF1α (P<0.05). In SHR, ACh only increased the 6-keto-PGF1α production (P<0.05). It could be concluded that chronic treatment with aldosterone was able to produce endothelial dysfunction through COX-2 activation in normotensive and hypertensive conditions. PGI2 seems to be the main factor accounting for endothelial dysfunction in hypertensive rats, whereas other prostanoids besides PGI2 appear to be involved in endothelial dysfunction under normotensive conditions.

Hypertension increases the participation of vasoconstrictor prostanoids from cyclooxygenase-2 in phenylephrine responses.

Objective The present study was designed to analyse whether hypertension alters the involvement of cyclooxygenase-2-derived mediators in phenylephrine-induced vasoconstrictor responses. Methods Vascular reactivity experiments were performed in aortic segments from normotensive, Wistar–Kyoto, and spontaneously hypertensive rats (SHR); protein expression was measured by western blot and/or immunohistochemistry, and prostaglandin F2α (PGF2α), 8-isoprostane and prostacyclin release were determined by enzyme immunoassay commercial kits. Results The protein synthesis inhibitor dexamethasone (1 μmol/l), the non-selective cyclooxygenase inhibitor indomethacin (10 μmol/l), the selective cyclooxygenase-2 inhibitor NS 398 (1 μmol/l), and the thromboxane A2/prostaglandin H2 (TP) receptor antagonist SQ 29,548 (1 μmol/l), reduced the concentration–response curves to phenylephrine more in segments from hypertensive than from normotensive rats; however, the thromboxane A2 (TxA2) synthase inhibitors furegrelate (10 μmol/l) and OKY 046 (1 and 10 μmol/l) had no effect in either strain. Removing endothelium or adding dexamethasone almost abolished the NS 398 effect. Cyclooxygenase-2 protein expression, which was reduced by dexamethasone, was higher in aorta from hypertensive animals. In both strains cyclooxygenase-2 was localized mainly in endothelial cells and adventitial fibroblasts. 13,14-Dihydro-15-keto-PGF2α, 6-keto-PGF1α and 8-isoprostane levels were greater in the medium from hypertensive than from normotensive rats; NS 398 decreased levels of the three metabolites studied only in the medium from SHR. Conclusions PGF2α and 8-isoprostane seem to be involved in the response to phenylephrine in rat aorta; this involvement is greater in hypertensive rats, probably due to a higher endothelial induction of cyclooxygenase-2.

Aldosterone induces endothelial dysfunction in resistance arteries from normotensive and hypertensive rats by increasing thromboxane A2 and prostacyclin

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).

Participation of aldosterone in the vascular inflammatory response of spontaneously hypertensive rats: role of the NFkappaB/IkappaB system.

Objective To investigate the participation of aldosterone in the vascular inflammatory process associated with hypertension, as well as the possible involvement of the NFκB/IκB system. Methods Male spontaneously hypertensive rats (SHR; 20–22 weeks old) untreated or treated with either the aldosterone receptor antagonist, eplerenone (100 mg/kg per day) or triple antihypertensive therapy (HHR: hydralazine + hydrochlorothiazide + reserpine; 20 + 7 + 0.15 mg/kg per day) were used in the study. Wistar–Kyoto rats (WKY) were used as a normotensive reference group. Aortic mRNA expression and plasma levels of interleukin (IL)-1β, IL-6 and tumour necrosis factor α (TNFα) were measured. Likewise, the aortic expression of the nuclear factor κB (NFκB) p50 subunit precursor, p105, and its inhibitor (IκB) were measured. Results SHR showed higher aortic expression of IL-1β, IL-6 and TNFα than WKY (P < 0.05) and higher plasma levels of IL-1β and IL-6 than WKY (P < 0.05). Moreover, SHR also presented increased aortic expression of nuclear transcription factor NFκB p50 subunit precursor (p105), and a reduction of its inhibitor IκB. Both eplerenone and HHR decreased blood pressure to a comparable extent (P < 0.05). This effect was accompanied by a reduction in plasma levels of IL-1β and IL-6 and aortic mRNA expression of IL-1β, IL-6 and TNFα. However, the effect of eplerenone was more marked, since eplerenone-treated rats showed significantly lower inflammatory parameters than SHR receiving HHR. In addition, both antihypertensive treatments increased IκB mRNA expression in a similar manner, but only eplerenone reduced NFκB mRNA expression. Conclusions Aldosterone, as well as an increase in haemodynamic forces produced by hypertension, participate in the vascular inflammatory process associated with hypertension in SHR. This effect seems to be mediated by enhanced vascular expression of cytokines through a modification of the NFκB/IκB system.

Influence of hypertension on nitric oxide synthase expression and vascular effects of lipopolysaccharide in rat mesenteric arteries

Experiments were designed to investigate the effects of the inducible nitric oxide synthase (iNOS) stimulator, lipopolysaccharide (LPS), on noradrenaline (NA) responses and on NOS activity and its expression in intact mesenteric resistance arteries (MRAs) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. In MRAs from WKY, LPS (10 μg ml−1; 1–5 h) reduced the vasoconstrictor responses to NA (0.1–30 μM) in the presence, but not in the absence of L‐arginine (L‐Arg, 10 μM). However, in SHR arteries, LPS induced an incubation‐time dependent reduction of NA responses in the absence, as well as the presence, of L‐Arg. The LPS inhibitory effect was reduced by the non‐specific NOS inhibitor L‐NG‐nitroarginine methyl ester (L‐NAME, 100 μM) and the selective iNOS inhibitor, aminoguanidine (100 μM). L‐NAME alone similarly shifted the concentration‐response curve to NA leftward in arteries from both strains, while aminoguanidine had no effect. L‐Arg shifted the curve to NA rightward only in SHR MRAs. Basal activity of both iNOS and constitutive NOS (conversion of [3H]‐L‐Arg to [3H]‐L‐citrulline) was similar in arteries from both strains. After 5 h incubation with LPS, only iNOS activity in arteries from SHR was increased. Basal iNOS protein expression was undetectable; basal endothelial (eNOS) protein expression was similar in arteries from both strains, while neuronal (nNOS) was greater in arteries from SHR. LPS induced iNOS protein expression, that was higher in arteries from SHR than in those from WKY. These results indicate that NO production, via iNOS induction, is greater than in those from MRAs from SHR to WKY.

Effect of clenbuterol on non‐endothelial nitric oxide release in rat mesenteric arteries and the involvement of β‐adrenoceptors

The aim of the present study was to explore the contribution of adrenergic, sensory and nitrergic innervations to the inhibitory effects of the β2‐adrenoceptor agonist clenbuterol on responses to electrical field stimulation (EFS, 200 mA, 0.3 ms, 1–16 Hz, for 30 s, at 1 min interval) in rat mesenteric artery segments without endothelium and the possible involvement of adrenergic, sensory and nitrergic innervations. Clenbuterol (1 μM) reduced EFS‐induced contractile responses, and this effect was reversed by the β‐antagonist propranolol (1 μM) (contraction at 16 Hz expressed as % of 75 mM K+‐induced contraction was: control, 69±9, clenbuterol, 31±6, n=13, P<0.001; control, 83±5, clenbuterol+propranolol 70±7, n=11, P>0.05). In arteries preincubated with [3H]‐noradrenaline (NA), clenbuterol did not modify the tritium overflow evoked by EFS (200 mA, 0.3 ms, 4 Hz, for 60 s; ratio between tritium release in the second and first stimuli was: control, 0.80±0.05 and clenbuterol added before second stimulus, 0.91±0.11, n=5, P>0.05). The nitric oxide (NO) synthase inhibitors NG‐monomethyl‐L‐arginine (L‐NMMA) and NG‐nitro‐L‐arginine methyl ester (L‐NAME) (10 and 100 μM), and the guanylate cyclase inhibitor methylene blue (10 μM) increased the contractions caused by EFS (% contraction at 16 Hz, control, 81±7, n=26; 10 μM L‐NMMA, 109±12, n=8, P<0.05; methylene blue, 119±6, n=6, P<0.05). However, these contractions were decreased by the NO synthase substrate L‐arginine 10 μM (14±6%, n=6, P<0.001), but not modified by either the sensory neurones toxin capsaicin (0.5 μM, 75±6%, n=6, P>0.05) or the protein synthesis inhibitor cycloheximide (10 μM, 83±6%, n=8, P>0.05). None of these drugs altered the concentration‐response curves to exogenous NA (n=7). Pretreatment with capsaicin or cycloheximide did not modify the reduction of the EFS‐evoked contraction provoked by clenbuterol. However the presence of L‐NMMA (or L‐NAME) or methylene blue did decrease the effect of clenbuterol (% contraction at 16 Hz, clenbuterol, 31±6, n=13; clenbuterol+10 μM L‐NMMA, 93±11, n=8, P<0.05; clenbuterol+methylene blue, 90±7, n=6, P<0.05). These results suggest that the reduction caused by clenbuterol in the contraction induced by EFS in rat mesenteric arteries seems to be mediated by NO release, through the activation of β2‐adrenoceptors probably present on nitrergic nerves.

Serotonergic innervation of cat cerebral arteries

5-HT and 5-HIAA were measured in cat cerebral arteries by HPLC. Removal of both superior cervical ganglia or simultaneous lesions of dorsal and central raphe nuclei significantly decreased 5-HT levels but not those of 5-HIAA. This suggests that cat cerebral blood vessels are innervated by serotonergic fibers of different origin.

Ouabain‐induced hypertension alters the participation of endothelial factors in α‐adrenergic responses differently in rat resistance and conductance mesenteric arteries

This study compares the role of endothelial factors in α‐adrenoceptor contractile responses in mesenteric resistance (MRA) and superior (SMA) mesenteric arteries from ouabain‐treated (8.0 μg day−1, 5 weeks) and untreated rats. The role of the renin–angiotensin system was also evaluated. Ouabain treatment increased systolic blood pressure. In addition, ouabain reduced the phenylephrine response in SMA but did not alter noradrenaline responses in MRA. Endothelium removal or the nitric oxide synthase (NOS) inhibitor (L‐NAME, 100 μM) increased the responses to α‐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. Indomethacin (10 μM) 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. Losartan treatment prevents the development of hypertension but not all vascular changes observed after ouabain treatment. 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.

Role of K+ channels and sodium pump in the vasodilation induced by acetylcholine, nitric oxide, and cyclic GMP in the rabbit aorta.

The endothelium-dependent relaxation caused by acetylcholine (ACh) in rabbit aorta segments was reduced by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester and by blockade of: Na+ pump with ouabain, large-conductance Ca2+-activated K+ (BK(Ca)) channels with charybdotoxin (ChTx), or voltage-dependent K+ (Kv) channels with 4-aminopyridine (4-AP). ACh relaxation was unaltered by glibenclamide, apamin, and Ba2+, blockers of ATP-sensitive K+ channels, small-conductance Ca2+-activated K+ channels, and inward rectifier K+ channels, respectively. The relaxation induced by exogenous NO and 8-bromocyclic GMP (8-BrcGMP) was similar in intact and endothelium-denuded segments, and it was reduced or unaltered by the same drugs used in the case of ACh. However, a 4-AP concentration 20-fold higher was necessary to reduce exogenous NO relaxation. These data suggest a resemblance in the mechanisms implicated in the relaxation elicited by ACh, exogenous NO, and 8-BrcGMP. Therefore, the relaxation caused by ACh is mainly mediated by endothelial NO, which in turn, enhances cGMP levels; this messenger appears to be the major one responsible for the smooth muscle cell hyperpolarization in the relaxation elicited by ACh, which is mediated by activation of the Na+ pump and ChTx- and 4-AP-sensitive K+ channels, likely BK(Ca) and Kv channels.

Heterogeneity of endothelium-dependent mechanisms in different rabbit arteries

The possible endothelial factors involved in endothelium-dependent relaxations induced by acetylcholine (ACh) in aorta, mesenteric and femoral arteries of rabbit were analyzed. In thoracic aorta precontracted with noradrenaline, NG-nitro-L-arginine methyl ester (L-NAME) and methylene blue (MB), inhibitors of nitric oxide (NO) synthase and guanylate cyclase, practically abolished ACh relaxation. This relaxation was reduced by the Na+ pump inhibition with ouabain and K(+)-free solution, and by the blockade of Ca(2+)-dependent K+ channels with tetraethylammonium (TEA). Ouabain reduced the relaxation produced by the NO donor, sodium nitroprusside (SNP). In the mesenteric artery, L-NAME and MB produced a small reduction of ACh relaxation. However, ouabain, K(+)-free medium and TEA markedly decreased this relaxation. SNP induced a relaxation which was diminished by ouabain. In segments precontracted with high K+, ACh relaxation was abolished by L-NAME and MB. In femoral arteries, L-NAME and MB reduced ACh relaxation. The stimulated cGMP concentrations caused by ACh or SNP were less in the aorta than in mesenteric and femoral arteries. These results suggest that ACh relaxation is mediated: in aorta by endothelial NO which may hyperpolarize to some extent the smooth muscle cells through the sodium pump activation, in mesenteric artery by endothelium-derived hyperpolarizing factor and NO, the latter being clearly expressed in segments contracted with high K+, and in femoral artery essentially by endothelial NO release.