Rings Of Spontaneously Hypertensive Rats Research Articles

Contribution of L-type Ca2+ channel-sarcoplasmic reticulum coupling to depolarization-induced arterial contraction in spontaneously hypertensive rats

Evidence has shown that vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) are depolarized and that the expression of L-type Ca2+ channels (LTCCs) and the sarcoplasmic reticulum (SR) Ca2+ buffering system are upregulated. Arterial rings exposed to high K+ solutions develop a contraction with two components, namely, an initial or phasic component and a sustained or tonic component. Because LTCCs and SR have different functions in the phasic and tonic components of depolarization-induced contraction, this study investigated the role of LTCC-SR coupling in depolarized arterial rings of SHRs. In the absence of extracellular Ca2+, high external K+ or LTCC agonists elicited a transitory contraction, which was sensitive to nifedipine and was potentiated in SHRs. In the presence of extracellular Ca2+, cyclopiazonic acid (CPA), an SR Ca2+-ATPase (SERCA) inhibitor, evoked a transient contraction that was significantly increased in SHRs. Although the phasic and tonic components were markedly increased in depolarized arterial rings of SHRs, they showed different voltage-dependence and sensitivity to SERCA inhibition. The tonic component was more sensitive to moderate depolarizations, and CPA selectively reduced the tonic component to the level observed in WKY rats. These results suggested that LTCC-SR coupling is potentiated in the sustained contraction of hypertensive VSMCs.

Acute Treatment with Lauric Acid Reduces Blood Pressure and Oxidative Stress in Spontaneously Hypertensive Rats.

The effects of acute administration of lauric acid (LA), the most abundant medium-chain fatty acid of coconut oil, on blood pressure, heart rate and oxidative stress were investigated in spontaneously hypertensive rats (SHR). Intravenous doses of LA reduced blood pressure in a dose-dependent fashion (1, 3, 4, 8 and 10 mg/kg) in both SHR and Wistar Kyoto rats. LA (10-8 to 3 × 10-3 M) induced vasorelaxation in isolated superior mesenteric artery rings of SHR in the presence (n = 7) or absence (n = 8) of functional endothelium [maximum effect (ME) = 104 ± 3 versus 103 ± 4%]. After exposure to KCl (60 mM), LA also induced concentration-dependent vasorelaxation (n = 7) compared to that under Phe-induced contraction (ME = 113.5 + 5.1 versus 104.5 + 4.0%). Furthermore, LA-induced vasorelaxation in vessels contracted with S(-)-BayK8644 (200 nM), a L-type Ca2+ channel agonist (ME = 91.4 + 4.3 versus 104.5 + 4.0%, n = 7). Lastly, LA (10-3 M) reduced NADPH-dependent superoxide accumulation in the heart (18 ± 1 versus 25 ± 1 MLU/min/μg protein, n = 4, p < 0.05) and kidney (82 ± 3 versus 99 ± 4 MLU/min/μg protein, n = 4, p < 0.05). Our data show that LA reduces blood pressure in normotensive and hypertensive rats. In SHR, this effect might involve Ca+2 channels in the resistance vessels and by its capability of reducing oxidative stress in heart and kidneys.

Autonomic Receptors and Nitric-Oxide Involvements in Mediating Vasorelaxation Effect Induced by Syzygium polyanthum Leaves Extract.

Context:Syzygium polyanthum (Wight) Walp leaves are traditionally used by Malays for treating hypertension. Our previous study showed that aqueous extract of S. polyanthum (AESP) and methanolic extract of S. polyanthum (MESP) extracts of S. polyanthum leaves significantly reduced blood pressure of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).Aims:This study aimed to investigate their vasorelaxation potential and the possible involvement of autonomic receptors and nitric oxide in mediating their effect.Settings and Design:Both extracts will be tested on isolated thoracic aorta rings of WKY and SHR. The involvement of autonomic receptors and nitric oxide will be elucidated using respective blockers.Materials and Methods:Isolated thoracic aorta rings from WKY and SHR were mounted onto myograph chambers to measure changes in the aorta tension. Increasing concentrations of AESP and MESP, from 1 μg/ml to 10 mg/ml were added onto the myograph chambers. Blockers such as atropine (1 μM), phentolamine (1 μM), propranolol (1 μM), and Nω-nitro-l-arginine methyl ester (100 μM) were preincubated before addition of extracts to check for involvement of muscarinic, α- and β-adrenergic receptors (AR) as well as nitric oxide, respectively.Statistical Analysis Used:Two-way ANOVA, followed by post hoc Bonferroni test was used, where P < 0.05 (two-tailed) was considered statistically significant.Results:AESP and MESP caused significant vasorelaxations through nitric oxide pathway. The former was mediated through α-AR while the latter was mediated by β-adrenergic and muscarinic receptors.Conclusion:Vasorelaxation effect by AESP and MESP involved nitric oxide pathway which is possibly mediated by the autonomic receptors.SUMMARY This is the first study that reveals significant vasorelaxation effect induced by Syzygium polyanthum leaves extract. Vasorelaxation maybe one of the possible mechanisms for its ability to reduce blood pressure. This study also suggested that the vasorelaxation effect by this plant extract may involve nitric oxide pathway mediated by the autonomic receptors. Abbreviations Used: AESP: Aqueous extract of Syzygium polyanthum leaves. MESP: Methanolic extract of Syzygium polyanthum leaves. SHR: spontaneously hypertensive rat, WKY: Wistar-Kyoto rat.

Vasorelaxation effect of Syzygium polyanthum (wight) walp. Leaves extract on isolated thoracic aorta rings of normal and hypertensive rats

Introduction: Syzygium polyanthum (Wight) Walp. leaves are traditionally consumed by the Malays as an alternative treatment for hypertension. Until now, effect of S. polyanthum leaves extract on blood vessel is not yet disclosed. Methods: This study investigated the effect of S. polyanthum leaves aqueous extract (AESP) (0.01, 0.1, 1 &amp; 10 mg/ml) on isolated thoracic aorta rings of normotensive Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR). Both rings were pre-contracted using phenylephrine (1 µM). Distilled water that dissolved AESP served as negative control. Results: As compared to negative control, AESP at 0.1, 1, and 10 mg/ml significantly relaxed aorta rings of WKY by 39.81 ± 2.99 % (P&lt;0.001), 59.55 ± 7.60 % (P&lt;0.001), and 72.58 ± 5.57 % (P&lt;0.001), respectively. In SHR, AESP at 1 and 10 mg/ml significantly relaxed the aorta rings of SHR by 40.53 ± 3.66 % (P&lt;0.001) and 65.73 ± 8.24 % (P&lt;0.001), respectively. There was a significant difference between AESP-induced vasorelaxation in WKY and in SHR at a concentration of 0.1 mg/ml (P&lt;0.001). The IC50 value for AESPinduced vasorelaxation on aorta rings of WKY (94.67 ± 1.41 µg/ml) was lower than of SHR (799.80 ± 1.69 µg/ml). Conclusions: The S. polyanthum leaves extract was able to cause significant relaxation on aorta rings of both normal and hypertensive rats. Thus, this finding is well-corroborated with the use of this plant as a traditional remedy for hypertension. It is suggested that an in-depth investigation of the mechanism of vasorelaxation of this plant is carried-out in the future.

0416 : Vascular smooth muscle cells are responsible for a prothrombotic phenotype of spontaneously hypertensive rat arteries

The hypothesis that hypertension confers a hypercoagulable state arises from the complications associated with hypertension, stroke and myocardial infarction. Our objective was to determine whether spontaneous hypertension causes changes in the thrombin generating capacity of the vascular wall. We used spontaneously hypertensive rats (SHR) compared with Wistar rats. The addition of thoracic aorta rings of SHR to a Wistar or SHR plasma pool resulted in a greater increase in thrombin generation compared to the addition of equivalent rings from Wistar. Comparison of 5 week-old and 12 week-old rats indicate that established hypertension is required to induce increased thrombin generation within the vessel wall. Whereas no difference was observed for endothelial cells, thrombin formation was higher at the surface of cultured aortic smooth muscle cells (SMCs) from SHR than from Wistar. Exposure of negatively-charged phospholipids was higher on SHR than on Wistar aortic rings as well as on SMCs. Tissue factor activity was higher in SHR SMCs. Twelve week-old SHR exhibited accelerated FeCl3-induced thrombus formation in carotid arteries and the resulting occlusive thrombi are disaggregated by blockade of glycoprotein Ibα-von Willebrand factor interactions. SHR SMCs were more sensitive to thrombininduced proliferation than Wistar SMCs. This cellular effect was totally abolished by a protease-activated receptor 1 inhibitor. The prothrombotic phenotype of the SHR vessel wall was due to the ability of SMCs to support greater thrombin generation and resulted in accelerated occlusive thrombus formation after arterial injury, which is sensitive to glycoprotein Ibα-von Willebrand factor inhibitors.

Vascular smooth muscle cells are responsible for a prothrombotic phenotype of spontaneously hypertensive rat arteries.

The hypothesis that hypertension induces a hypercoagulable state arises from the complications associated with hypertension: stroke and myocardial infarction. Here, we determine whether hypertension causes changes in the thrombin-generating capacity of the vascular wall. We used spontaneously hypertensive rats (SHR) compared with Wistar rats. The addition of thoracic aortic rings of SHR to a Wistar or SHR plasma pool resulted in a greater increase in thrombin generation compared with equivalent rings from Wistar. This increase occurred in 12- but not 5-week-old rats and was prevented by an angiotensin II-converting enzyme inhibitor, indicating that established hypertension is required to induce increased thrombin generation within the vessel wall. Whereas no difference was observed for endothelial cells, thrombin formation was higher on aortic smooth muscle cells (SMCs) from SHR than on those from Wistar. Exposure of negatively charged phospholipids was higher on SHR than on Wistar rings, as well as on cultured SMCs. Tissue factor activity was higher in SHR SMCs. Twelve-week-old SHR exhibited accelerated FeCl3-induced thrombus formation in carotid arteries, and the resulting occlusive thrombi were disaggregated by blockade of glycoprotein Ibα-von Willebrand factor interactions. SHR SMCs were more sensitive to thrombin-induced proliferation than Wistar SMCs. This effect was totally abolished by a protease-activated receptor 1 inhibitor. The prothrombotic phenotype of the SHR vessel wall was due to the ability of SMCs to support greater thrombin generation and resulted in accelerated occlusive thrombus formation after arterial injury, which was sensitive to glycoprotein Ibα-von Willebrand factor inhibitors.

Sesamin ameliorates arterial dysfunction in spontaneously hypertensive rats via downregulation of NADPH oxidase subunits and upregulation of eNOS expression

Sesamin is one of the major lignans in sesame seeds with antihyperlipidemic, antioxidative and antihypertensive activities. The aim of this study was to examine the effects of sesamin on arterial function in spontaneously hypertensive rats (SHRs). SHRs were orally administered sesamin (40, 80 and 160 mg·kg(-1)·d(-1)) for 16 weeks. After the rats were killed, thoracic aortas were dissected out. The vasorelaxation responses of aortic rings to ACh and nitroprusside were measured. The expression of eNOS and NADPH oxidase subunits p47(phox) and p22(phox) in aortas were detected using Western blotting and immunohistochemistry. Aortic nitrotyrosine was measured with ELISA. The total antioxidant capacity (T-AOC) and MDA levels in aortas were also determined. The aortic rings of SHRs showed significantly smaller ACh-induced and nitroprusside-induced relaxation than those of control rats. Treatment of SHRs with sesamin increased both the endothelium-dependent and endothelium-independent relaxation of aortic rings in a dose-dependent manner. In aortas of SHRs, the level of T-AOC and the expression of nitrotyrosine, p22(phox) and p47(phox) proteins were markedly increased, while the level of MDA and the expression of eNOS protein were significantly decreased. Treatment of SHRs with sesamin dose-dependently reversed these biochemical and molecular abnormalities in aortas. Long-term treatment with sesamin improves arterial function in SHR through the upregulation of eNOS expression and downregulation of p22(phox) and p47(phox) expression.

Endothelial Nitric Oxide Synthase-Independent Release of Nitric Oxide in the Aorta of the Spontaneously Hypertensive Rat

In the aorta of male spontaneously hypertensive rats (SHR), but not in that of normotensive Wistar-Kyoto rats (WKY), contractions to phenylephrine obtained in the presence of L-NAME [inhibitor of nitric oxide synthase (NOS)] and indomethacin (inhibitor of cyclooxygenase) are inhibited by an unknown endothelium-derived factor. The present study aimed to identify the mechanism underlying this endothelium-dependent inhibition in the SHR aorta. Aortic rings of male SHR and WKY, with and without endothelium, were suspended in organ chambers in the presence of indomethacin and L-NAME for the measurement of isometric tension. Contractions to phenylephrine were smaller in SHR aortae with endothelium than in those without, but were similar in the two types of preparations of WKY aortae. The endothelium-dependent, NOS-independent inhibition of phenylephrine-induced contraction was abolished by oxyhemoglobin [extracellular NO scavenger], carboxy-PTIO (NO scavenger) and ODQ (inhibitor of soluble guanylyl cyclase). It was unmasked not only by indomethacin but also by apocynin (antioxidant), but inhibited by diphenyleneiodonium (inhibitor of flavoproteins including cytochrome P450 reductase). The cytochrome P450 reductase protein expression was similar in SHR and WKY aortae. However, the level of nitrate and nitrite, substrates of cytochrome P450 reductase, were higher in SHR than WKY plasma and aortae. Therefore, in SHR but not WKY aortae, eNOS-independent NO is formed by cytochrome P450 reductase.

Abstract 390: Cardiovascular Effects of the Angiotensin-(1-7) Analogue A1317

Angiotensin-(1-7) (Ang-(1-7)) has been shown to have anti-hypertensive properties mediated through the receptor Mas . The beneficial effects of the Mas/Ang-(1-7) pathway prompted us to develop Ang-(1-7) analogues and peptides ligands for Mas. In the present study we evaluated the vasorelaxant and anti-hypertensive effect of the analogue A1317. To examine the NO production, chinese hamster ovary (CHO) cells transfected with Mas cDNA were incubated with A1317 at 10 -7 and 10 -6 mol/l for 60 minutes. Subsequently, the slices were mounted for evaluation by confocal microscopy. The vasorelaxant activity of the A1317 was measured in rings from Wistar rats and spontaneously hypertensive rats (SHR) descending thoracic aorta with functional endothelium pre-contracted with phenylephrine (0.1μM). To determine which receptor is involved in the vasorelaxant effect of the peptide experiment was performed in the presence of the Ang-(1-7) antagonist, A-779 (10 μm), in SHR rings. The acute effect of the peptide on arterial pressure was evaluated in male normotensive Wistar rats and SHR. Mean arterial pressure (MAP) and heart rate (HR) were monitored for 5 hours after in bolus administration of different doses of A1317 (0.1-12.5nmol/kg) in conscious animals. A1317 stimulated the NO production in CHO-Mas transfected cells and produced a concentration-dependent vasodilator effect in endothelium-containing aortic rings (Emax=18.97±1.33% in Wistar rings and 38.0±3.3% in SHR rings). The vasorelaxation produced by A1317 was attenuated by A779. Acute administration of A1317 reduced the MAP time and dose-dependently in conscious SHR. The reduction in MAP started to be significant after 130 minutes of the in bolus injection (Δ=-13±2.0mmHg; 0.5nmol/kg). The maximum change in MAP was observed after 4 hours of the in bolus injection (Δ=-21±4.0mmHg; 0.5nmol/kg). A1317 also produced a decrease in MAP of normotensive Wistar rats, however the magnitude of the MAP change was considerably smaller than that observed in SHR (Δ=-8.8±2.0mmHg). There was no significant effect of A1317 on HR of Wistar rats or SHR. These data suggest that the peptide A1317 presents antihypertensive and vasorelaxant properties and may have cardiovascular applications similar to those of Ang-(1-7).

Effect of ageing in the mechanisms of endothelium dependent and independent vasorelaxation of [Ru(terpy)(bdq)NO+]3+ (TERPY) in spontaneously hypertensive rats (SHR)

Isolated aortic rings with and without endothelium of young (4 months) and old (16 months) SHR were incubated for 30 minutes with different pharmacological tools and the potency of relaxation of the NO donor TERPY was evaluated in concentration response curves made in phenylephrine (0,1 μM) contracted rings. Endothelium removal decreased the potency of TERPY in both groups, but this reduction was lower in old SHR. L‐NAME (NOS inhibitor; 0,1mM) incubated rings showed reduced the potency of TERPY in both groups. TIRON (superoxide anions scavenger; 0,1mM) decreased the potency of TERPY in aortic rings with endothelium of both groups. In denuded aortic rings, incubation with ODQ (guanilate cyclase inhibitor; 1μM) reduced the potency in both groups. TEA (potassium channels blocker; 1mM) and TIRON (0,1mM) didn't alter the potency of TERPY in denuded aortic rings of both groups. This results show that endothelium potentiates the effect of TERPY in aortic rings of young and old SHR, but this effect is decreased in old SHR. eNOS and superoxide anions are involved in this process. In denuded aortic rings, guanilate cyclase is involved in the relaxing response of TERPY, but potassium channels and superoxide anions are not. We conclude that ageing decreases the endothelium dependent potentiation of TERPY in SHR aorta.

BPP-5a produces a potent and long-lasting NO-dependent antihypertensive effect

The bradykinin potentiating peptides (BPPs) are oligopeptides found in different animal venoms. BPPs isolated from Bothrops jararaca venom were the first natural inhibitors described for somatic angiotensin I-converting enzyme (ACE). They were used in the structural modeling for captopril development, a classical ACE inhibitor widely used to treat human hypertension. We evaluated the effect of BPP-5a on cardiovascular parameters of conscious Wistar (WTs) and spontaneously hypertensive rats (SHRs). In SHR, BPP-5a showed potent cardiovascular effects, at doses ranging from 0.47 to 710 nmol/kg. The maximal changes in mean arterial pressure (MAP) and heart rate (HR) were found at the dose of 2.37 nmol/kg (Δ MAP: -38 ± 4 mmHg, p < 0.01; Δ HR: -71 ± 17 bpm, p < 0.05). Reductions in MAP and HR occurred throughout 6 hours of post-injection period. In contrast to active site-directed ACE inhibitors, no ACE inhibition, evaluated by the Ang I pressor effect, or bradykinin potentiation was observed during the antihypertensive effect of the pentapeptide. In vitro assays showed no effects of BPP-5a upon argininosuccinate synthetase and B(1), B(2), AT(1), AT(2) or Mas receptors. Ex vivo assays showed that BPP-5a induced endothelium-dependent vasorelaxation in isolated aortic rings of SHRs and WTs. Although the BPP-5a is considered an ACE inhibitor, our results indicate that its antihypertensive effect is exerted via a unique target, a nitric-oxide-dependent mechanism.

Hypertension and the absence of EDHF‐mediated responses favour endothelium‐dependent contractions in renal arteries of the rat

Experiments were designed to determine the modulation by nitric oxide (NO) and endothelium-dependent hyperpolarizations (EDHF-mediated responses) of endothelium-dependent contractions in renal arteries of normotensive and hypertensive rats. Rings, with or without endothelium, of renal arteries of 8-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were suspended in myographs for isometric force recording. ACh evoked relaxations in preparations contracted with phenylephrine. L-NAME (inhibitor of NOS) attenuated (WKY) or abolished (SHR) these relaxations. TRAM-34 plus UCL 1684 (inhibitors of EDHF-mediated responses) did not decrease the relaxation, except in rings of WKY when L-NAME was also present. High concentrations of ACh caused a secondary increase in tension, augmented in rings of WKY by L-NAME or TRAM-34 plus UCL 1684. The increase in tension was prevented by indomethacin. Under baseline tension, ACh induced endothelium-dependent contractions, prevented by indomethacin (COX inhibitor) or terutroban (TP receptor antagonist). The calculated endothelium-dependent contractions were larger in rings of SHR compared with those of WKY. In preparations of SHR, the contractions were augmented by L-NAME in the presence of SC19220 (EP-1 receptor antagonist). In arteries of WKY, the endothelium-dependent contractions were augmented by TRAM-34 plus UCL 1684. The responses were reduced by SC19220. In the renal artery of the rat, EDCF-mediated contractions are augmented by hypertension. The endothelium-dependent contractions are facilitated by NOS inhibition (in the presence of an EP-1 receptor antagonist) and by the withdrawal of EDHF-mediated responses.

Raloxifene Modulates Pulmonary Vascular Reactivity in Spontaneously Hypertensive Rats

Selective estrogen receptor modulators (SERMs) reduce vascular tone in the systemic circulation. Their effects on the pulmonary circulation are unknown. The present study examined the effect of oral treatment with raloxifene (a second-generation SERM) on vasomotor reactivity in pulmonary arteries from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Pulmonary arterial rings were suspended in a multi-channel myograph, and changes in isometric tension were measured. WKY rings constricted less to U46619 than SHR rings, and the difference was eliminated after chronic treatment with raloxifene. More contraction to U46619 was obtained after inhibition of nitric oxide synthase (NOS) by L-NAME (as an index of basal NO release) in raloxifene-treated than in control SHR rings. Less U46619-induced contraction after raloxifene treatment occurred only in SHR rings with endothelium, and this effect was abolished upon removal of the endothelium. Raloxifene treatment did not enhance the contribution of basal NO to U46619-induced constriction in WKY rings. Raloxifene treatment did not modify endothelium-dependent relaxation to acetylcholine and endothelium-independent relaxation to nifedipine. The reduced relaxing sensitivity to sodium nitroprusside (SNP) in SHR rings was normalized by raloxifene treatment. Raloxifene treatment reduced CaCl2-induced tone in SHR but not in WKY rings. The results show that chronic treatment with raloxifene could improve pulmonary vascular function in hypertensive animals by (1) increasing basal NO release, (2) reducing vascular smooth muscle tone, and (3) improving the effect of NO on vascular smooth muscle in SHR. In contrast, raloxifene has little effect on vascular reactivity in pulmonary arteries from normotensive WKY rats.

Decreased Levels of Cytochrome P450 2E1–Derived Eicosanoids Sensitize Renal Arteries to Constrictor Agonists in Spontaneously Hypertensive Rats

We compared renal interlobar arteries of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in terms of cytochrome P450 (CYP) 4A and CYP2E1 protein expression; levels of 20-HETE, 19-HETE, and 18-HETE; and responsiveness to phenylephrine in the absence and presence of N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 mumol/L), a CYP4A inhibitor. Relative to data in WKY, arteries of SHR exhibited diminished (P<0.05) CYP2E1 and levels of 19-HETE (66.7+/-6.0 versus 44.9+/-2.8 pmol/mg) and 18-HETE (13.8+/-1.6 versus 7.9+/-0.5 pmol/mg), whereas CYP4A and 20-HETE levels (99.3+/-9.1 versus 98.9+/-12.8 pmol/mg) were unchanged. Phenylephrine contracted vascular rings of SHR and WKY; the R(max) was similar in both strains, but SHR vessels were more sensitive as denoted by the lower (P<0.05) EC50 (0.28+/-0.07 versus 0.71+/-0.12 mumol/L). DDMS decreased 20-HETE and, to a lesser extent, 19-HETE, while increasing (P<0.05) the EC50 for phenylephrine by 475% and 54% in vessels of SHR and WKY, respectively. The desensitizing effect of DDMS was reversed by 20-HETE. Notably, the minimal concentration of 20-HETE that decreased the EC50 for phenylephrine in DDMS-treated vessels was smaller in SHR (0.1 micromol/L) than WKY (10 micromol/L), and the sensitizing effect of 20-HETE was blunted (P<0.05) by the (R) stereoisomers of 19-HETE and 18-HETE. We conclude that the increased sensitivity to phenylephrine in arteries of SHR is attributable to a vasoregulatory imbalance produced by a deficit in vascular CYP2E1-derived products, most likely 19(R)-HETE and 18(R)-HETE, which condition amplification of the sensitizing action of 20-HETE.

Endothelium‐dependent contractions to acetylcholine, ATP and the calcium ionophore A 23187 in aortas from spontaneously hypertensive and normotensive rats

The present study was designed to determine whether or not an increase in endothelial intracellular concentration of calcium ([Ca2+]i) evokes endothelium-dependent contractions in the aorta from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Acetylcholine, adenosine triphosphate (ATP) and the calcium ionophore, A 23187, produced endothelium-dependent relaxations in isolated aortic rings of both WKY and SHR. These relaxations in response to the three agonists were significantly smaller in the SHR when compared with the WKY. Endothelium-dependent contractions to acetylcholine, ATP and A 23187 were observed only in the aorta isolated from the SHR. In the presence of NG-nitro-L-arginine, an NO synthase inhibitor, the endothelium-dependent contractions in response to acetylcholine, ATP and A 23187 were potentiated significantly in the aorta SHR and were unmasked in that of WKY. However, the contractions were still significantly greater in SHR than in WKY. These contractions were abolished by indomethacin and valeryl salicylate (two cyclo-oxygenase inhibitors) as well as by S 18886 (a TP-receptor antagonist), indicating that the endothelium-dependent contraction produced by the three agonists share the same characteristics. The results of the present study indicate that the release/generation of endothelium-derived contracting factor, requires an increase in endothelial [Ca2+]i.

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O(2)(-)) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O(2)(-)-generator, abolished relaxant responses to ACh. These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect.

Tempol selectively attenuates angiotensin II evoked vasoconstrictor responses in spontaneously hypertensive rats

To assess whether superoxide anions mediate vasoconstrictor responses to agonists in blood vessels of spontaneously hypertensive rats (SHRs). The effect of the superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol), on responses to angiotensin II (Ang II), endothelin-1, phenylephrine and potassium chloride was determined in aortic rings and perfused mesenteric vascular beds (MVB) of adult male rats of the Sprague-Dawley, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) strains. The effect of tempol on Ang II-evoked superoxide production was assessed in aortic rings. There were no differences in the maximum tension (Emax) attained in response to agonists, but the negative logarithm of the concentration required to produce 50% of the maximal response (EC50) for Ang II was lower (P < 0.05) in aortic rings of SHRs. In the MVBs of SHRs, the Emax but not the EC50 values attained in response to Ang II, endothelin-1 and phenylephrine were greater. Tempol significantly and selectively reduced the Emax of Ang II in both aorta and MVB preparations with intact endothelium. The reduction in Emax attained in response to Ang II was more pronounced in SHRs (P < 0.01) than in WKY rats (P < 0.05) or Sprague-Dawley rats (P < 0.05). The inhibitory effect of tempol was absent when a nitric oxide synthase inhibitor was included or endothelium was denuded. A significant increase in lucigenin chemiluminescence evoked by Ang II in both intact and endothelium-denuded aortic rings of SHRs was abolished when tempol was included in the buffer. These data suggest that increased superoxide anions mediate vasoconstrictor responses to Ang II, but not to other agonists, in an endothelium-dependent manner, by quenching vasodilatory mediator, nitric oxide. This may account for the exaggerated vasoconstrictor responses to Ang II in SHRs.

Losartan and enalapril therapies enhance vasodilatation in the mesenteric artery of spontaneously hypertensive rats

We studied the effects of 10-week long enalapril and losartan treatments (4 and 15 mg kg −1 day −1, respectively) on mesenteric arterial function in vitro in spontaneously hypertensive rats (SHR) and Wistar–Kyoto rats (WKY). The relaxations of noradrenaline-precontracted rings to acetylcholine, nitroprusside and cromakalim were similar in WKY and enalapril- and losartan-treated SHR, and more pronounced than in untreated SHR. The responses to acetylcholine were attenuated by N G-nitro- l-arginine methyl ester in WKY and drug-treated SHR, but were completely inhibited in untreated SHR. When hyperpolarization of smooth muscle was prevented by KCl-induced precontractions, no differences were found in the relaxations to acetylcholine and nitroprusside between the groups, and the dilatations to cromakalim were abolished. Moreover, in noradrenaline-precontracted rings of drug-treated SHR, the addition of tetraethylammonium attenuated the nitric oxide synthase and cyclooxygenase-resistant relaxations to acetylcholine and abolished the enhanced dilatations to nitroprusside. In conclusion, since the enhancement of vasorelaxation in enalapril- and losartan-treated SHR was abolished by conditions preventing hyperpolarization, the improved vasodilatation following these therapies could be attributed to enhanced vasodilatation via K + channels in this model of hypertension.

Losartan inhibits thromboxane A2-induced platelet aggregation and vascular constriction in spontaneously hypertensive rats.

Our recent studies have shown that the nonpeptide angiotensin II (Ang II) antagonist losartan interacts with thromboxane A2/prostaglandin H2 receptors and inhibits the thromboxane A2 (TxA2) analog U46619-induced vasoconstriction in canine coronary arteries. In this study, we further investigated whether losartan prevents TxA2-induced platelet aggregation and vasoconstriction in spontaneously hypertensive rats (SHRs). Pretreatment with losartan (10 microM) significantly reduced U46619-induced, concentration-dependent washed platelet aggregation. The inhibition is specific for losartan, because another Ang II AT1-receptor antagonist, CV11974 (10 microM), an active metabolite of TCV116, did not block the platelet aggregation caused by U46619. In addition, losartan (10 microM) augmented acetylcholine (ACH)-induced nitric oxide (NO)-dependent vasodilation and abolished the ACH-induced endothelium-derived contracting factor (EDCF)-mediated vasoconstriction in the aortic rings from adult SHRs. U46619 produced dose-dependent vasoconstriction in aortic vessels of SHRs, which was demonstrated to be blocked by the potent, selective TxA2/PGH2 receptor antagonist SQ29,548. Pretreatment with losartan (10(-6)-10(-5) M) inhibited the contractile response of U46619 and shifted the concentration-response curve to the right in a dose-dependent manner. The effective concentration at half maximal contraction (EC50) of U46619 was increased 2.5- and 7.6-fold in the presence of 1 and 10 microM losartan, respectively, without changes in maximal contraction. The active metabolite of losartan, EXP3174, at 1 microM also competitively inhibited U46619-induced contractions in aortic rings of SHRs. In contrast, neither the AT1-receptor antagonist CV11974, the AT2 antagonist PD123319, nor the angiotensin-converting enzyme inhibitor lisinopril, each at concentrations of 1 microM, had any effect on the U46619-induced constriction in aortic rings. In conclusion, losartan, acting as both AT1- and TxA2/PGH2-receptor antagonists, may enhance its therapeutic profile in the treatment of hypertension and cardiovascular disease.

Role of endothelium in the endothelin-1-mediated potentiation of the norepinephrine response in the aorta of hypertensive rats.

To investigate the role of the endothelium in the functional interaction between endothelin-1 and norepinephrine in the contractile response of aortas from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Thoracic aorta rings with and without endothelium from SHR and from WKY rats were suspended in an organ bath to record the isometric tension. After an equilibration period of 120 min, the preparations with and without endothelin-1 were subjected to single and cumulative additions of norepinephrine in different experiments. To characterize the mechanisms involved in the interaction between endothelin-1 and norepinephrine, the aortic rings were pretreated with a cyclooxygenase pathway inhibitor (piroxicam, SO29548), an inhibitor of NO synthase [NG-nitro-L-arginine (NLA)], or selective endothelin receptor blockers (BQ-123 or BQ-788). In some experiments we examined the contractile responses to norepinephrine in aortas pretreated either with angiotensin II (AII) or with U46619, an agonist of prostaglandin H2-thromboxane A2 receptors. Finally, we examined the effect of the combination of calcium-entry blockade by administration of nifedipine and treatment with either endothelin-1 or U46619 on the norepinephrine reactivity. Administration of 3 x 10(-10) mol/l endothelin-1 potentiated the contractile response to norepinephrine in SHR aortas with endothelium, irrespective of whether they had been treated with NLA. No endothelin-1-mediated enhancement of the response to norepinephrine was observed in SHR denuded rings and in untreated and NLA-treated WKY rat aortas. All did not affect the response to norepinephrine in SHR rings with endothelium. The amplification by endothelin-1 of the response to (1-100) x 10(-9) mol/l norepinephrine was abolished by blockade of the cyclooxygenase pathway with piroxicam or SO29548. In WKY rat and SHR denuded aortas, 10(-8) mol/l U46619 potentiated the contractile responses to norepinephrine. Administration of 3 x 10(-6) mol/l BQ-123 abolished the increase in reactivity to norepinephrine evoked by endothelin-1 in intact SHR aorta, whereas 3 x 10(-6) mol/l BQ-788 failed to modify this potentiating effect. Administration of 10(-8) mol/l nifedipine inhibited the potentiation of the norepinephrine-induced contractions evoked both by endothelin-1 in SHR aortic rings with endothelium and by U46619 in SHR denuded rings. Our results show that a low concentration of endothelin-1 induced potentiation of the contractile response to norepinephrine in SHR aortas but not in WKY rat aortas. This response was endothelium-dependent. Furthermore, our study affords functional arguments that both endothelial and smooth muscle pathways are involved in the potentiating interaction. We propose that endothelin-1 stimulates the production of endothelium- and cyclooxygenase-generated vasoconstrictor factors, which in turn may serve directly as priming stimuli at the vascular smooth muscle level, to activate the Ca(2+)-signal pathway and consequently to increase locally the vascular sensitivity to norepinephrine.