Ang II-induced Contraction Research Articles

Sevoflurane inhibits angiotensin II-induced Rho kinase-mediated contraction of vascular smooth muscle from spontaneously hypertensive rat

Angiotensin II (Ang II)-induced vasoconstriction is mediated by changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity. Protein kinase C- and Rho kinase-mediated signaling pathways are proposed for the regulation of the Ca(2+) sensitization mechanisms. We have demonstrated that sevoflurane inhibits Rho kinase-mediated contraction of isolated rat aortic smooth muscle. A recent study demonstrated that Rho-kinase mediated Ca(2+) sensitization was involved in the pathophysiology of hypertension. This study was designed to investigate the effects of sevoflurane on Ang II-induced Rho kinase-mediated vascular contraction in spontaneously hypertensive rats (SHR). The effects of sevoflurane on vasoconstriction, increase in [Ca(2+)](i), and membrane translocation of Rho kinase in response to Ang II were investigated in normotensive Wistar-Kyoto rats (WKY) and SHR, using an isometric force transducer, a fluorometer, and Western blotting, respectively. The inhibitory effects of sevoflurane on Ang II (10(-7)M)-induced contraction were greater (P < 0.05) in SHR than in WKY at the highest concentration of sevoflurane (5.1%). Y27632 (3 × 10(-7)M), a specific inhibitor of Rho kinase, inhibited the Ang II-induced contraction in SHR, but not in WKY. Sevoflurane did not affect the increases in [Ca(2+)](i) in response to Ang II in either strain. Ang II stimulated Rho kinase activity in SHR, which was almost abolished by sevoflurane at a concentration of 5.1% (P < 0.05). These findings suggest that the inhibition of the Ang II-induced contraction by sevoflurane in SHR may be, at least in part, due to the attenuation of the Rho kinase-mediated signaling pathway.

Vascular smooth muscle Jak2 mediates angiotensin II-induced hypertension via increased levels of reactive oxygen species

Angiotensin II (Ang II) type AT(1) receptors expressed on vascular smooth muscle cells (VSMCs) couple to the Jak2 signalling pathway. However, the importance of this tissue-specific coupling is poorly understood. The purpose of this investigation was to determine the importance of VSMC-derived Jak2 in angiotensin II-mediated hypertension. The Cre-loxP system was used to conditionally eliminate Jak2 tyrosine kinase expression within the smooth muscle cells of mice. Following chronic Ang II infusion, the resulting increase in mean arterial pressure (MAP) was significantly attenuated in the Jak2 null mice when compared with littermate controls. The VSMC Jak2 null mice were also protected from the Ang II-induced vascular remodelling. Aortic rings from the VSMC Jak2 null mice exhibited reduced Ang II-induced contraction and enhanced endothelial-dependent relaxation via increased nitric oxide (NO) bioavailability. When compared with controls, the VSMC Jak2 nulls also had lower levels of hydrogen peroxide, Rho kinase activity, and intracellular Ca(2+) in response to Ang II. The data indicate that VSMC Jak2 expression is involved in the pathogenesis of Ang II-dependent hypertension due to the increased presence of reactive oxygen species (ROS). As such, VSMC-derived Jak2 tyrosine kinase modulates overall vascular tone via multiple, non-redundant mechanisms.

Adenosine sensitization after angiotensin II stimulation in afferent arterioles from normal rats does not occur during two-kidney, one-clip hypertension

G protein-coupled receptors such as the AT(1a) R are frequently subject to desensitization, extensively studied in cell culture but to small extent in hypertensive models. Recently, angiotensin II (ANG II)-induced desensitization was shown to last 10 min in isolated afferent arterioles (AAs), suggesting impact on ANG II vasoactivity. In the present study, we explored ANG II desensitization and effects of adenosine (Ado) in AAs from two-kidney, one-clip (2K1C) hypertensive rats. Our main hypothesis was that Ado affects ANG II contractility differently in 2K1C, because of persistently elevated levels of ANG II. Afferent arterioles were isolated with the agarose-infusion/enzyme-treatment technique from normotensive and 2K1C hypertensive rats, and stimulated with ANG II (10(-7) M) at baseline and re-stimulated after 20 or 40 min, with or without Ado (2.5 × 10(-5) M) in the vessel bath. Afferent arterioles from normotensive rats re-stimulated with ANG II after 20 min displayed a blunted contraction (Δ12.8 ± 4.3%, P < 0.05), which disappeared when AAs were stimulated after 40 min (Δ2.7 ± 2.3%, NS), indicating that desensitization lasted for 30 ± 10 min. Ado augmented ANG II contractions after 20 min, but not after 40 min, suggesting that only de-sensitized vessels were affected. Similar experiments in AAs from the clipped and non-clipped kidneys revealed no desensitization when re-stimulated with ANG II after 20 and 40 min, and contractions were unaffected by Ado. Reduced duration of desensitization in AAs from 2K1C may cause vessels to be sensitized longer and increase vasoconstriction. The present study demonstrates that Ado does not augment ANG II-induced contractions in AAs from 2K1C as in normotensive rats, possibly because of a reduced period of desensitization.

Heat shock augments angiotensin II-induced vascular contraction through increased production of reactive oxygen species

A temporal increase in temperature triggers a series of stress responses and alters vascular smooth muscle (VSM) contraction induced by agonist stimulation. Here we examined the role of reactive oxygen species (ROS) in heat shock-dependent augmentation of angiotensin II (AngII)-induced VSM contraction. Endothelium-denuded rat aortic rings were treated with heat shock for 45 min at 42 °C and then subjected to assays for the production of force, ROS, and the expression of ROS-related enzymes. AngII-induced contraction was enhanced in heat shock-treated aorta. AngII-induced production of hydrogen peroxide and superoxide were elevated in response to the heat shock treatment. Pre-treatment with superoxide dismutases (SOD) mimetic and inhibitors for glutathione peroxidase and NADPH oxidase but not for xanthine oxidase eliminated an increase in the AngII-induced contraction in the heat shock-treated aorta. Heat shock increased the expression of p47phox, a cytosolic subunit of NADPH oxidase, but not Cu–Zn–SOD and Mn–SOD. In addition, heat shock increased contraction that was evoked by hydrogen peroxide and pyrogallol. These results suggest that heat shock causes an elevation of ROS as well as a sensitization of ROS signal resulting in an augmentation of VSM contraction in response to agonist.

Aging‐induced changes in angiotensin II‐induced contractions and tachyphylaxis of isolated carotid arteries

Angiotensin II (Ang II) is a potent vasoconstrictor, yet the potential aging‐ induced changes in the magnitude of responses have not yet been explored. We hypothesized that aging induces changes in the magnitude of vasomotor responses to Ang II. Thus carotid arteries from newborn (8 days: 8d), young (2 months: 2m) and old (24 months: 24m) rats were isolated and placed in a wire myograph to measure changes in their isometric tension. Two dose response curves to Ang II were obtained in a sequential manner (1‐, and 2‐administration (Adm). Contractions of vessels to 1‐Adm of Ang II in 8d vessels were significantly less compared to those of 2m and 24m, which were similar in magnitude (1.40±0.2 mN and 5.2±0.6 and 5.7±0.39 mN, respectively). Compared to the 1‐Adm, contractions of vessels to 2‐Adm of Ang II were significantly reduced in 2m and 24m vessels (4.4±0.54 and 0.5±0.19mN), whereas it did not change in vessels of 8d rats (1.28±0,2 mN).These findings suggest that in carotid arteries 1) Ang II‐induced contractions increase with age, 2) repeated administration of Ang II elicits tachyphylaxis and the magnitude of tachyphylaxis increases with aging.(Hungarian Nat. Sci. Res. Fund.‐OTKA K71591, K67984, AHA Founders Aff., 0555897T)

Short-term angiotensin-1 receptor antagonism in type 2 diabetic Goto–Kakizaki rats normalizes endothelin-1-induced mesenteric artery contraction

Endothelin (ET)-1 and angiotensin II (Ang II) are likely candidates for a key role in diabetic vascular complications. We demonstrated previously that an enhanced ET-1-induced contraction is present in mesenteric arteries from Goto–Kakizaki (GK) rats at the chronic stage of type 2 diabetes. Here, we investigated whether short-term treatment of such rats with losartan, an angiotensin type 1 receptor antagonist, might normalize the ET-1-induced contraction. In mesenteric arteries from GK rats at the chronic stage (34–38 weeks) (vs. those from age-matched control Wistar rats): (1) the ET-1-induced contraction was enhanced, (2) the levels of ET-1 and Ang II were increased, (3) ET-1-stimulated ERK2 phosphorylation was increased, and (4) the ACh-induced endothelium-dependent relaxation was reduced. Mesenteric arteries isolated from such GK rats following treatment with losartan (25 mg/kg/day for 2 weeks) exhibited reduced ET-1- and Ang II-induced contractions, suppressed ET-1-stimulated ERK phosphorylation, and increased ACh-induced relaxation, while the rats exhibited normalized plasma NO metabolism and their mesenteric arteries exhibited increased basal NO formation. However, such losartan treatment did not alter the increased levels of ET-1 and Ang II seen in GK mesenteric arteries. Our data suggest that within the timescale studied here, losartan normalizes ET-1-induced mesenteric artery contraction through a suppression of ERK activities and/or by normalizing endothelial function.

Abstract 5074: The Rho Exchange Factor Arhgef1 in Vascular Smooth Muscle Cells is Essential for Angiotensin II-induced Hypertension

Rho exchange factors (Rho-GEFs) are responsible for Rho protein activation by catalyzing the exchange of GDP for GTP. Although over-activation of RhoA and its effector Rho kinase is recognized as a critical component of the pathogenesis of hypertension in experimental models and in human, the molecular mechanisms and the RhoA-GEF(s) involved in this activation of RhoA signaling in vascular smooth muscle cells (VSMCs) are unknown. Among the Rho-GEFs family, Arhgef1 (p115) constitutes with Arhgef11 (PDZ-Rho GEF) and Arhgef12 (Larg) the subfamily of RGS-containing Rho-GEFs that have been suggested to couple G-protein associated receptors to RhoA activation. To establish the role of Arhgef1 in the regulation of vascular tone and hypertension in vivo , we generated mice lacking Arhgef1 specifically in SMCs (SM-Arhgef1-KO). We used Arhgef1 lox/lox mice, which were mated to SMMHC-CreER T2 . SM-Arhgef1-KO mice were then obtained by treatment with tamoxifen (1 mg/day, IP, 5 days). In VSMCs from SM-Arhgef1-KO mice, stimulation with angiotensin II (Ang II, 0.1 μ M) was unable to induce RhoA/Rho kinase activation whereas that produced by U46619, phenylephrine (PhE), and endothelin-1 (ET1) was similar to that recorded in control mice. Ex vivo analysis of the contractile properties showed that, in aortic segments from SM-Arhgef1-KO mice, KCl-, PhE-, U46619- and ET1-induced contractions were similar to those obtained in control mice. In contrast, Ang II-induced contractions were severely reduced. In vivo, acute pressor responses to U46619 (3–100 μ g/kg), ET1 (0.03–1 μ g/kg) and PhE (0.1–100 μ g/kg) were similar in control and SM-Arhgef1-KO mice, but responses to Ang II (0.1–30 μ g/kg) were abolished. Basal blood pressure levels were similar in SM-Arhgef1-KO and control mice. In control mice, chronic infusion of Ang II (1 mg/kg/day) increased blood pressure whereas SM-Arhgef1-KO mice were resistant to the development of Ang II-induced hypertension. We therefore conclude that Arhgef1, as the molecular link between Ang II, RhoA activation and vascular tone regulation, provides an alternative target to reducing elevated vascular tone under hypertensive conditions without affecting normal blood pressure regulation by other vasoactive factors.

Abstract 5278: Essential Role of the Rho Exchange Factor Arhgef1 for Angiotensin II-induced RhoA/Rho Kinase Activation and Arterial Tone Regulation

Rho exchange factors (Rho-GEFs) are signaling molecules responsible for Rho protein activation by catalyzing the exchange of GDP for GTP. Although over-activation of the small G protein RhoA and its effector Rho kinase is recognized as a critical component of the pathogenesis of hypertension in experimental models and in human, the molecular mechanisms and the RhoA-GEF(s) involved in this activation of RhoA signaling in vascular smooth muscle cells (VSMCs) are still unknown. Nevertheless, angiotensin II (Ang II) has been shown to play a key role in the over-activation of RhoA in hypertension. Here we identify the RhoA-GEF and the molecular mechanism responsible for the activation of RhoA and Rho kinase in response to Ang II type 1 receptor activation (AT1R) in rat aortic VSMCs. Systematic functional analysis by siRNA-mediated silencing of the 28 RhoA GEFs indicated that only Arhgef1 (p115, lsc) is involved in Ang II (0.1 μ M)-mediated RhoA/Rho kinase activation. Knock-down of Arhgef1 had no significant effect on the activation of RhoA/Rho kinase induced by the thromboxan analog U46619, endothelin-1 and noradrenaline. The potency and efficacy of noradrenaline, phenyl-ephrine, U46619 and endothelin-1 to elicit contraction of rat aortic rings were not modified by Arhgef1 silencing whereas the contractile effect of Ang II was abolished. Ang II-induced Arhgef1 activation was further confirmed by in vitro guanine nucleotide exchange assay on RhoA, and we demonstrated by the transfection of Arhgef1 mutants that Ang II-induced Arhgef1 activation was due to its phosphorylation on Tyr738 by the tyrosine kinase Jak2. To confirm the functional role of Arhgef1 in Ang II-mediated regulation of vascular tone and hypertension, we then determined the activity of Arhgef1 in arteries from Ang II treated rats by pull-down assay. Chronic infusion of Ang II (250 ng/kg/min, 14 days) through an osmotic minipump induced an increase in arterial pressure associated with enhanced RhoA/Rho kinase activity and activation of Arhgef1 in arteries. Our results thus show that Arhgef1 activation and its downstream RhoA signaling are essential for Ang II-induced contraction and identify Arhgef1 as a potential specific target for the treatment of Ang II-dependent hypertension.

Activation of TRPV1 inhibits angiotensin II induced vasoconstriction in mice

Objective We investigated the long-term effect of capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, on vascular reactivity in C57BL/6J mice. Design and methods Two-month-old male C57BL/6J mice were fed with regular diet (control group, n=12) and capsaicin-containing diet (capsaicin group, n=12). Tail-cuff systolic blood pressure (SBP) was examined at the baseline and at the end of the intervention. After 6-month treatment with capsaicin, invasive carotid artery blood pressure and heart rate were measured, and the aortic contraction was examined using isometric myograph (Danish Myotech Technology, Denmark). Plasma levels of renin, angiotensinII (AngII) and aldosterone were determined. Vascular smooth muscle cells (VSMCs) were obtained from thoracic aorta of mice and cultured by tissue explant method. AngiotensinII type 1 receptor (AT1R) protein expression was detected by western blotting. Cytosolic calcium level was detected in VSMCs using the fluorescent dye technique. Results SBP, invasive carotid artery blood pressure and heart rate had no difference between two groups. Norepinephrine-induced contraction response in thoracic aorta was not different; while AngII-induced contraction of aortic ring was lower in the capsaicin group than control group (P<0.05). Plasma levels of renin, AngII and aldosterone were not different between the two groups. Treatment with capsaicin significantly decreased AT1R protein expression and AngII-induced calcium entry in cultured VSMCs (P<0.05). Conclusion Long term activation of TRPV1 with dietary capsaicin significantly blunted AngII-induced contraction response in aortic segments, which was related to the down-regulation of AT1R expression and reduction of AngII-induced calcium entry in VSMCs. Objective We investigated the long-term effect of capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, on vascular reactivity in C57BL/6J mice. Design and methods Two-month-old male C57BL/6J mice were fed with regular diet (control group, n=12) and capsaicin-containing diet (capsaicin group, n=12). Tail-cuff systolic blood pressure (SBP) was examined at the baseline and at the end of the intervention. After 6-month treatment with capsaicin, invasive carotid artery blood pressure and heart rate were measured, and the aortic contraction was examined using isometric myograph (Danish Myotech Technology, Denmark). Plasma levels of renin, angiotensinII (AngII) and aldosterone were determined. Vascular smooth muscle cells (VSMCs) were obtained from thoracic aorta of mice and cultured by tissue explant method. AngiotensinII type 1 receptor (AT1R) protein expression was detected by western blotting. Cytosolic calcium level was detected in VSMCs using the fluorescent dye technique. Results SBP, invasive carotid artery blood pressure and heart rate had no difference between two groups. Norepinephrine-induced contraction response in thoracic aorta was not different; while AngII-induced contraction of aortic ring was lower in the capsaicin group than control group (P<0.05). Plasma levels of renin, AngII and aldosterone were not different between the two groups. Treatment with capsaicin significantly decreased AT1R protein expression and AngII-induced calcium entry in cultured VSMCs (P<0.05). Conclusion Long term activation of TRPV1 with dietary capsaicin significantly blunted AngII-induced contraction response in aortic segments, which was related to the down-regulation of AT1R expression and reduction of AngII-induced calcium entry in VSMCs.

TGF-β Inhibits Ang II-Induced MAPK p44/42 Signaling in Vascular Smooth Muscle Cells by Ang II Type 1 Receptor Downregulation

Vascular changes in diabetes are characterized by reduced vasoconstriction and vascular remodeling. Previously, we demonstrated that TGF-β1 impairs Ang II-induced contraction through reduced calcium mobilization. However, the effect of TGF-β1 on Ang II-induced vascular remodeling is unknown. Therefore, we investigated the effect of TGF-β1 on Ang II-induced activation of the MAPK p44/42 pathway in cultured rat aortic smooth muscle cells (RASMC). Activation of MAPK p44/42 was determined with a phospho-specific antibody. Angiotensin type 1 receptor (AT₁) and AT₁ mRNA levels were measured by [³H]candesartan-binding and real-time PCR, respectively. AT₁ gene transcription activity was assessed using AT₁ promoter-reporter constructs and by a nuclear runoff assay. In TGF-β1-pretreated cells, Ang II-induced phosphorylation of MAPK p44/42 was inhibited by 29 and 46% for p42 and p44, respectively, and AT₁ density was reduced by 31%. Furthermore, pretreatment with TGF-β1 resulted in a 64% reduction in AT₁ mRNA levels and decreased AT₁ mRNA transcription rate by 42%. Pretreatment with TGF-β1 blocked Ang II-induced proliferation of RASMC, while stimulating Ang II-induced upregulation of plasminogen activator inhibitor-1. In conclusion, TGF-β1 attenuates Ang II-mediated MAPK p44/42 kinase signaling in RASMC through downregulation of AT₁ levels, which is mainly caused by the inhibition of transcription of the AT₁ gene.

Upregulation of AT2 receptor and iNOS impairs angiotensin II-induced contraction without endothelium influence in young normotensive diabetic rats

Diabetes and insulin resistance are associated with an increased risk of hypertension and cardiovascular disease. Recent evidence demonstrates that AT2 receptors (AT2R) play an important role in the hemodynamic control of hypertension by vasodilation. The quantitative significance of AT2R in the establishment of diabetic vascular dysfunction, however, is not well defined and needs further investigation. Goto-Kakizaki (GK) rats, a polygenic model of spontaneous normotensive type 2 diabetes, were used to examine any abnormalities in cardiovascular function associated with AT2R at the early stage of the disease without endothelium influence. Using a myograph to measure the isometric force, we observed that ANG II-induced contraction was impaired in denuded GK aorta compared with control Wistar-Kyoto (WKY) aorta and exhibited a retarded AT1R antagonist response and enhanced Rho kinase signaling. When AT1R were blocked, ANG II induced a significant vasodilation of precontracted GK aorta via AT2R. The protein and mRNA of AT2R were increased in diabetic GK denuded aorta. Blocking AT2R restored the ANG II-induced contraction in the GK vasculature to control levels, demonstrating a counteractive role for AT2R in AT1R-induced contraction. Inhibition of inducible nitric oxide synthase (iNOS) by NG-monomethyl-L-arginine mimicked AT2R inhibition in denuded GK aorta, suggesting that AT2R-induced vasodilation was dependent on iNOS/NO generation. The protein and mRNA of iNOS were also increased in GK aorta. In conclusion, these results clearly demonstrate that enhanced AT2R and iNOS-induced, NO-mediated vasodilation impair ANG II-induced contraction in an endothelium-independent manner at the early stage of type 2 diabetes.

Regional Heterogeneity of Functional Changes in Conduit Arteries After High‐fat Diet

To determine effects of dietary fat content on vascular responses in different conduit arteries in mice. Vascular responses to reactive oxygen species (ROS)/hydroxyl radical (.OH), acetylcholine (ACh), endothelin-1 (ET-1), and angiotensin II (Ang II) were determined in carotid and femoral arteries of C57BL/6J mice fed with diets varying in fat content (low fat (LF), 12.3%; high fat (HF), 41%; and very high fat (VHF), 58% (kcal from fat)) for 15 weeks, beginning at 4 weeks of age. In precontracted rings of carotid and femoral artery, ROS/.OH-induced a rapid, transient vasodilation. In the carotid, but not in femoral artery, ROS/.OH-induced dilation increased with increasing dietary fat intake (P < 0.05 vs. LF diet), while contractile responses to ROS/.OH remained unaffected. In femoral arteries, ROS/.OH-induced contractions were reversed into relaxations after both HF and VHF diet (P < 0.05 vs. LF diet). Both ET-1 and Ang II induced strong contractions in the femoral artery that were unaffected by dietary fat intake. In contrast, in the carotid artery Ang II-induced contraction was attenuated after HF and VHF diets (P < 0.005 vs. LF diet), whereas ET-1-induced vasoconstriction was significantly increased (P < 0.05 VHF vs. LF and HF). Treatment with VHF diet enhanced ACh-mediated endothelium-dependent relaxation only in the femoral artery (P < 0.05 vs. HF). These findings demonstrate that dietary fat content has regional and distinct effects on vascular function in different vascular beds. The data also suggest the possibility that in selected conduit arteries ROS-dependent vasodilator mechanisms become activated in response to increased dietary fat intake.

Adenosine enhances long term the contractile response to angiotensin II in afferent arterioles

Adenosine (Ado) enhances ANG II-induced constrictions of afferent arterioles (Af) by receptor-dependent and -independent pathways. Here, we test the hypothesis that transient Ado treatment has a sustained effect on Af contractility, resulting in increased ANG II responses after longer absence of Ado. Treatment with Ado (cumulative from 10(-11) to 10(-4) mol/l) and consecutive washout for 10 or 30 min increased constrictions on ANG II in isolated, perfused Af. Cytosolic calcium transients on ANG II were not enhanced in Ado-treated vessels. Selective or global inhibition of A(1)- and A(2)-adenosine receptors did not inhibit the Ado effect. Nitrobenzylthioinosine (an Ado transport inhibitor) clearly reduced the Ado-mediated responses. Selective inhibition of p38 MAPK with SB-203580 also prevented the Ado effect. Inosine treatment did not influence arteriolar reactivity to ANG II. Contractile responses of Af on norepinephrine and endothelin-1 were not influenced by Ado. Phosphorylation of the p38 MAPK and of the regulatory unit of 20-kDa myosin light chain was enhanced after Ado treatment and ANG II in Af. However, phosphorylation of p38 MAPK induced by norepinephrine or endothelin-1 was reduced in vessels treated with Ado, whereas 20-kDa myosin light chain was unchanged. The results suggest an intracellular, long-lasting mechanism including p38 MAPK activation responsible for the increase of ANG II-induced contractions by Ado. The effect is not calcium dependent and specific for ANG II. The prolonged enhancement of the ANG II sensitivity of Af may be important for tubuloglomerular feedback.

Long-term effects of prenatal methylmercury exposure on associative learning in the rat

Methylmercury (MeHg) exposure results in increased risk of hypertension and cardiovascular diseases. In this study, we aimed to investigate whether the in vivo exposure of MeHg in mice affects blood pressure and basilar arterial responses to angiotensin II (Ang II) and acetylcholine (ACh), which are important modulators of cerebrovascular autoregulation.Mice were exposed to MeHg (40 ppm) in drinking water for 21 days. Blood pressure was measured in conscious mice by an indirect tail-cuff method. Functional studies of the isolated arteries' response to vasoactive substances were performed using a micro-organ-bath system.Systolic and mean blood pressures were significantly increased after 2 and 3 weeks of treatment with MeHg, respectively. Ang II-induced contraction in an isolated basilar artery, which is mediated via Rho-kinase activation, was increased in MeHg-treated mice. ACh-induced relaxation, which is dependent on NO production from the endothelial cells, was decreased in MeHg-treated mice. However, alterations of vascular responses to Ang II and ACh were not observed in the isolated thoracic aorta.This study demonstrated that the cerebral vasculature appears to be particularly sensitive to in vivo exposure of MeHg. Our results suggest that in vivo MeHg increases blood pressure and causes alterations in the cerebrovascular reactivity in response to Ang II and ACh through enhancement of Rho-kinase activity and inhibition of NO bioavailability, respectively.

The Mechanism Behind the Inhibitory Effect of Isoflurane on Angiotensin II-Induced Vascular Contraction Is Different from That of Sevoflurane

Angiotensin II (Ang II)-induced vascular contraction is mediated both by a Ca(2+)-mediated signaling pathway and a Ca(2+) sensitization mechanism. We recently demonstrated that sevoflurane inhibits the contractile response to Ang II, mainly by inhibiting protein kinase C (PKC) phosphorylation that regulates myofilament Ca(2+) sensitivity, without significant alteration of intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat aortic smooth muscle. The current study was designed to determine the mechanisms by which isoflurane inhibits Ang II-induced contraction of rat aortic smooth muscle. The effects of isoflurane on vasoconstriction, increase in [Ca(2+)](i), and phosphorylation of PKC in response to Ang II (10(-7) M) were investigated, using an isometric force transducer, a fluorometer, and Western blotting, respectively. Ang II elicited a transient contraction of rat aortic smooth muscle that was associated with an increase in [Ca(2+)](i) and PKC phosphorylation. Isoflurane (1.2%-3.5%) inhibited Ang II-induced contraction of rat aortic smooth muscle in a concentration-dependent manner (P < 0.05 at 1.2%, P < 0.01 at 2.3% and 3.5% isoflurane, n = 6). Isoflurane also inhibited elevation of [Ca(2+)](i) in response to Ang II (P < 0.01 at 2.3% and 3.5% isoflurane, n = 6), but failed to affect Ang II-induced phosphorylation of PKC at concentrations up to 3.5% (n = 7). These results suggest that, unlike sevoflurane, the inhibitory effect of isoflurane on Ang II-induced contraction is mainly mediated by attenuation of the Ca(2+)-mediated signaling pathway.

Different responses to angiotensin-(1-7) in young, aged and diabetic rabbit corpus cavernosum

We evaluated the ability of angiotensin-(1-7) [Ang-(1-7)] to produce relaxation of the corpus cavernosum of New Zealand White rabbits. The reactivity of corpus cavernosal strips isolated from young rabbits (8–10 months old) was assessed in organ-bath chambers. Cumulative concentration response curves for Ang-(1-7), angiotensin II (Ang II), carbachol and sodium nitroprusside (SNP) were established. Ang-(1-7) (10 −12 to 10 −5 M) produced a concentration-dependent relaxation of the corpus cavernosal strips with a pD 2 value of 9.8 ± 0.3. Ang-(1-7)-induced maximal relaxant response was reduced by 48 ± 2%, 57 ± 3% and 76 ± 2% in the presence of A-779 (10 −6 M), a selective Ang-(1-7) receptor (AT 1-7) antagonist, nitro- l-arginine methyl ester ( l-NAME) (10 −4M), an inhibitor of nitric oxide (NO) synthase, or iberiotoxin (5 × 10 −8 M), an inhibitor of calcium-activated potassium (BK) channels, respectively. In contrast, Ang II-induced contraction was increased in the presence of A-779. Carbachol-, SNP- and Ang-(1-7)-induced relaxations were significantly reduced whereas Ang-II induced contraction was significantly increased in the cavernosum strips from older (18–24 months old) and diabetic rabbits compared to the young. Pre-incubation of the cavernosum strips obtained from young, older or diabetic rabbits with Ang-(1-7) resulted in a significant attenuation of Ang II-induced contraction. In conclusion, these results demonstrate that Ang-(1-7) can produce nitric oxide-dependent relaxation of the corpus cavernosum through activation of AT 1-7 and BK channels. Older and diabetic animals showed impaired Ang-(1-7)-mediated relaxation suggesting that aging and diabetes related erectile dysfunction (ED) may be partly due to decreased Ang-(1-7)-mediated relaxation of the corpus cavernosum. Acute pre-incubation with Ang-(1-7) was effective in attenuating Ang II-induced contraction of rabbit corpus cavernosum suggesting that the possible role of Ang-(1-7) in treatment of ED should be investigated.

PPARγ agonists partially restores hyperglycemia induced aggravation of vascular dysfunction to angiotensin II in thoracic aorta isolated from rats with insulin resistance

Altered vascular responses to various vasopressors in animal models of insulin resistance (IR) and diabetes have been well documented. However, the precise mechanisms about vascular responses in IR with or without frank hyperglycemia (prediabetic state) are not available. Moreover, recently the role of peroxisome proliferators activated receptor-γ (PPARγ) has been linked to influence the vascular responses in hypertensive and diabetic state. Hence, the present study was conceived to determine the role of hyperglycemia on angiotensin II (Ang II) mediated vascular responses in the high fat diet (HFD) induced insulin resistance either with mild or frank hyperglycemia [induced by injection of low dose streptozotocin (STZ) to HFD fed rats (HFD + STZ)]. In addition, insulin-sensitizing agent such as rosiglitazone and pioglitazone were also studied on biochemical and vascular responses. Ang II-induced contractions were studied isometrically in thoracic aortic rings isolated from 4 weeks of normal pellet diet (NPD) fed control, HFD and HFD + STZ fed insulin resistant rats. Specific binding of Ang II receptors were carried out using radioligand ([ 3H]–Ang II) binding studies. After 4 weeks of HFD feeding, rats exhibited characteristics features of insulin resistance such as mild hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia and hypertension; whereas HFD + STZ treated rats showed all above parameters along with frank hyperglycemia. Maximal contractile response ( E max) to Ang II is increased in HFD fed rats as compared to control rats. Moreover, E max values are further elevated in HFD + STZ group where the frank hyperglycemia was induced by low dose of streptozotocin. Rosiglitazone (5 mg kg −1, p.o.) and pioglitazone (10 mg kg −1, p.o.) treatment significantly lowered the plasma glucose, triglycerides, insulin and cholesterol levels in insulin resistance rats. In addition, it also restored the elevated systolic, mean arterial, diastolic blood pressure and attenuated the enhanced contractile responses to Ang II in thoracic aortic rings obtained from both HFD and HFD + STZ treated rats. Specific binding of [ 3H]–Ang II is upregulated in HFD-fed and HFD + STZ treated rats. Treatment with pioglitazone and rosiglitazone significantly decreased the AT 1R specific binding in HFD fed rats. Our results indicate the role of hyperglycemia in the elevation of Ang II induced vascular responses in thoracic aorta isolated from insulin resistant rats and PPARγ agonists can attenuate these responses.

Enhanced AT1receptor-mediated vasocontractile response to ANG II in endothelium-denuded aorta of obese Zucker rats

In the present study, we tested the hypothesis that ANG II causes a greater vasoconstriction in obese Zucker rats, a model of type 2 diabetes, with mild hypertension. Measurement of isometric tension in isolated aortic rings with intact endothelium revealed a modest but not significantly greater ANG II-induced contraction in obese than lean rats. Removal of endothelium or inhibition of nitric oxide (NO) synthase by N(G)-nitro-L-arginine methyl ester (L-NAME) enhanced 1) ANG II-induced contraction in both lean and obese rats, being significantly greater in obese rats (E(max) g/g tissue, denuded: lean 572 +/- 40 vs. obese 664 +/- 16; L-NAME: lean 535 +/- 14 vs. obese 818 +/- 23) and 2) ANG II sensitivity in obese compared with lean rats, as revealed by the pD(2) values. Endothelin-1 and KCl elicited similar contractions in the aortic rings of lean and obese rats. ACh, a NO-dependent relaxing hormone, produced greater relaxation in the aortic rings of obese than lean rats, whereas sodium nitroprusside, an NO donor, elicited similar relaxations in both rat strains. The expression of the ANG type 1 (AT(1)) receptor protein and mRNA in the endothelium-intact aorta was significantly greater in obese than lean rats, whereas the endothelium-denuded rings expressed modest but not significantly greater levels of AT(1) receptors in obese than lean rats. The endothelial NO synthase protein and mRNA expression levels were higher in the aorta of obese than lean animals. We conclude that, although ANG II produces greater vasoconstriction in obese rat aortic rings, enhanced endothelial AT(1) receptor-mediated NO production appears to counteract the increased ANG II-induced vasoconstriction, suggesting that arterial AT(1) receptor may not be a contributing factor to hypertension in this model of obesity.

Interactions between apelin and angiotensin II on rat portal vein

Apelin (AP), an endogenous ligand of the APJ receptor, is involved in the regulation of cardiovascular homeostasis. Regardless the multiple similarities between AP and angiotensin II (Ang II), their roles seem to be different. We studied both the interactions between Apelin 13 (AP13) and Ang II and to what extent, if any, nitric oxide (NO) is involved. The experiments were performed in endothelium-denuded or endothelium-intact rat portal vein in the presence of 10 microM N(G)-nitro L-arginine methyl ester or 10 microM aminoguanidine. AP13 did not modify the isolated rat portal vein tone by itself, but inhibited the Ang II-induced contractions acting mainly by a NO-dependent mechanism. Our results sustain the hypothesis that AP13 could increase the activity of both constitutive and inducible NO synthase on either endothelium intact or endothelium denuded rat portal vein rings.

Hydrogen peroxide modulates angiotensin II-induced contraction of mesenteric arteries from streptozotocin-induced diabetic rats

Hydrogen peroxide (H 2O 2) contributes in the regulation of vascular tone, especially in pathological states. The role of H 2O 2 and superoxide anion free radicals in angiotensin II (Ang II)-induced contraction of diabetic tissues was examined with the aim of elucidating the underlying mechanisms. Isometric tension in response to various drug treatments was measured in isolated superior mesenteric arteries of streptozotocin (STZ)-induced diabetic WKY rats using the Mulvany wire myograph. Compared to the normal (euglycaemic) arteries, the Ang II-induced contraction was significantly reduced in diabetic arteries. Superoxide dismutase (SOD; converts superoxide to H 2O 2) significantly reduced the contraction in both types of arteries — an effect abolished by catalase (H 2O 2 scavenger), suggesting that the SOD effect was mediated by H 2O 2. Treatment with catalase had no effect on the Ang II contraction in euglycaemic arteries, but it raised the contraction in diabetic arteries to euglycaemic levels. This increase was similar to that observed with diabetic arteries incubated with l-NAME. Combined catalase and l-NAME treatment further enhanced the contraction in diabetic arteries, suggesting that the catalase effect was not mediated by nitric oxide (NO). The catalase effect was abolished by indomethacin treatment. These results suggest that attenuation of Ang II-induced contraction in diabetic tissues is modulated by endogenous H 2O 2, the scavenging of which unmasks an indomethacin-sensitive (and therefore cyclooxygenase product-mediated) Ang II-induced contraction.