Ang II-induced Contraction Research Articles

Angiotensin II induces vascular dysfunction without exacerbating blood pressure elevation in a mouse model of menopause-associated hypertension

Follitropin-receptor knockout (FORKO) mice are estrogen-deficient, hyperandrogenic and exhibit features of menopause and elevated blood pressure (BP). Because the renin-angiotensin system has been implicated in menopause-associated hypertension, we questioned whether angiotensin II (Ang II) challenge would further increase BP in FORKO mice and whether this is associated with cardiovascular remodeling and inflammation. Ang II (400 ng/kg per min) increased BP, assessed by radiotelemetry, similarly in female FORKO and wild-type (WT) mice. Acetylcholine-induced vasodilation was attenuated and Ang II-induced contraction was enhanced in FORKO mice (P < 0.05). This was associated with increased expression of vascular Ang type 1 receptors (AT1R) and estrogen receptor alpha (ERalpha). Vascular structure (media/lumen ratio) was similar in both groups. Abundance of gp91, nitrotyrosine formation and superoxide production, indices of inflammation and cardiac collagen content were increased in Ang II-treated FORKO compared to Ang II-treated WT mice (P < 0.05). Thus, in FORKO mice Ang II exacerbates endothelial dysfunction, augments contractility, increases oxidative stress, and promotes cardiac fibrosis without worsening vascular remodeling or BP elevation compared to Ang II-treated WT controls. Our findings suggest that in FORKO mice Ang II may be more important in influencing vascular tone and endothelial function, possibly through oxidative stress and altered ERalpha signaling, than in arterial remodeling and BP elevation.

Exaggerated vasomotor response to ANG II in rats with fetal programming of hypertension associated with exposure to a low-protein diet during gestation

The renin-angiotensin system plays a key role in the initiation and maintenance of elevated blood pressure associated with altered intrauterine milieu. The current studies were undertaken to verify whether vascular response to ANG II is increased in adult offspring of low-protein fed dams (LP) compared with control (CTRL) and if so, to examine underlying mechanism(s). ANG II-induced contraction of carotid rings was increased in LP (E(max), the maximum asymptote of the curve, relative to maximal response to KCl 80 mM: 230 +/- 3% LP vs. 201 +/- 2% CTRL, P < 0.05). In both groups, contraction to ANG II was mediated solely by AT1R. Responses to thromboxane A2 analog U-46619 and to KCl 80 mM under step increases in tension were similar between groups. Endothelium depletion enhanced contraction to ANG II in both groups, more so in LP. Blockade of endothelin formation had no effect on response to ANG II, and ANG-(1-7) did not elicit vasomotor response in either group. Superoxide dismutase (SOD) analog Tempol normalized LP without modifying CTRL response to ANG II. Basal levels of superoxide (aortic segments, lucigenin-enhanced chemiluminescence and fluorescent dye hydroethidine) were higher in LP. ANG II further increased superoxide production in LP only, and this was inhibited by coincubation with diphenylene iodonium or apocynin (inhibitor of NADPH oxidase complex). AT1R expression in carotid arteries was increased in LP, whereas SOD expression was unchanged. In conclusion, vasoconstriction to ANG II is exaggerated in this model of developmental programming of hypertension, secondary to enhanced vascular production of superoxide anion by NADPH oxidase with concomitant increase of AT1R expression.

Modulation of cerebral vascular tone by NADPH oxidase

The aims of this study were to compare the activity and function of NADPH oxidase in rat intracranial cerebral (including basilar, BA) and systemic (including carotid artery, CA; mesenteric artery, MA; and aorta, AO) arteries, and to investigate potential physiological roles for NADPH oxidase in cerebral arteries. NADPH oxidase-derived superoxide was measured using lucigenin-enhanced chemiluminescence and dihydroethidium. The effect of NADPH oxidase activation on vascular tone was investigated in vitro and in vivo. NADPH- and angiotensin II-stimulated superoxide production was up to 100-fold greater in cerebral versus systemic arteries. The NADPH oxidase inhibitors, diphenyleneiodonium (DPI), gp91ds-tat, and apocynin attenuated superoxide production. This was associated with 10-fold greater expression of Nox4 protein in the BA versus AO, CA and MA. In vitro, NADPH relaxed arteries with the efficacy: BA>MA>AO=CA. Catalase or apocynin inhibited NADPH-induced relaxation. Contraction to angiotensin II was weaker in BA versus AO and CA, but was selectively potentiated by catalase and apocynin. In vivo, flow-dependent dilatation of the BA was associated with an increase in superoxide production in BA endothelial cells. Catalase and DPI inhibited flow-dependent dilatation. Thus, NADPH oxidase activity and function is profoundly higher in cerebral versus systemic arteries. In the BA, H2O2 generated from NADPH oxidase–derived superoxide offsets Ang II-induced contraction and partially mediates flow-dependent dilatation. These findings provide evidence supporting a physiological role for NADPH oxidase in modulating cerebral vascular tone.

Effect of tempol on altered angiotensin II and acetylcholine-mediated vascular responses in thoracic aorta isolated from rats with insulin resistance

The altered vascular responses to various vasopressors and relaxants have been well reported in various animal models of hypertension, insulin resistance and diabetes. Though the role of oxidative stress (increased superoxide levels) associated with these altered vascular responses in hyperglycemic/diabetic state is well documented, the role of the same remains to be largely unknown in vascular dysfunction coupled with prediabetic insulin resistant state. The objective of the present study was therefore to elucidate the role of free radicals particularly superoxides if any associated with vascular dysfunction in diet-induced insulin resistance of rats. In this regard, the effect of tempol (a membrane permeable superoxide dismutase mimetic/free radical scavenger) on the enhanced Ang II-induced contraction and impaired-ACh mediated relaxation in thoracic aorta of rats with insulin resistance was studied. Ang II-induced contraction and ACh-mediated relaxation responses were recorded isometrically in endothelium intact and denuded thoracic aortic ring preparations isolated from male Sprague–Dawley rats which were fed with either normal pellet diet (NPD) (control group) or high fat diet (HFD) (insulin resistant group) for 4 weeks. The HFD-fed rats exhibited characteristic features of insulin resistance syndrome viz., obesity, hyperinsulinaemia, mild hyperglycemia, hypertriglyceridemia, hypercholesterolemia, glucose intolerance and hypertension. Maximal contractile response ( E max) to Ang II was increased in endothelium intact aortic ring preparations obtained from HFD-fed rats as compared to NPD-fed control rats. Denudation of endothelium significantly increased Ang II-mediated E max responses in thoracic aortic rings of NPD-fed rats, whereas it produced only minimal alteration to the E max in the HFD-fed rats. In addition, ACh-mediated relaxation response was impaired in endothelium intact aortic rings isolated from HFD-fed rats. Tempol (30–300 μM) significantly and dose dependently inhibited enhanced vascular responses ( E max) of Ang II in endothelium intact, but not in endothelium denuded aortic ring preparations. Tempol (30 μM) reversed the impaired acetylcholine (ACh)-mediated relaxations in endothelium intact aortic ring preparations of HFD-fed rats. Endothelium independent vasorelaxations (EIV) to sodium nitroprusside (SNP) were similar for both NPD and HFD. In conclusion, our results indicate that superoxide radicals play crucial role in enhanced contractile and impaired vasodilatory responses to Ang II and ACh, respectively, in thoracic aortic rings isolated from diet-induced insulin resistant rats.

Adrenomedullin inhibits angiotensin II-induced contraction in human aortic smooth muscle cells

The vasodilating peptide adrenomedullin (AM) has been reported to regulate vascular tone as well as proliferation and differentiation of various cell types in an autocrine/paracrine manner. Our study was designed to investigate the effect of AM on Ang II-induced contraction on human aortic smooth muscle cells (HASMC) in vitro, evaluating the signal pathways involved. Our findings indicate that AM was able to inhibit HASMC Ang II-induced contraction (IC 50 19 nM). AM stimulated cAMP production in a dose-dependent fashion as well. SQ 22.536, an adenylate cyclase inhibitor, and KT5720, a PKA inhibitor, blunted the AM effect, suggesting that it was mediated by the activation of the cAMP transduction pathway. Our results suggest that AM plays a role in the regulation of HASMC contraction by antagonizing the Ang II effects and may be involved in conditions of altered regulation of the blood vessels.

The Inhibitory Effects of Sevoflurane on Angiotensin II- Induced, p44/42 Mitogen-Activated Protein Kinase-Mediated Contraction of Rat Aortic Smooth Muscle

Sevoflurane dilates blood vessels and reduces arterial blood pressure in a dose-dependent manner. Angiotensin II (Ang II) is one of the primary regulators of vascular tension and arterial blood pressure, and the p44/42 mitogen-activated protein kinases (p44/42 MAPK) are involved in Ang II-mediated vascular smooth muscle contraction. We designed this study to examine the effects of sevoflurane on Ang II-induced, p44/42 MAPK-mediated contraction of rat aortic smooth muscle. The effects of the p44/42 MAPK kinase (MEK1/2) inhibitor, PD 098059 (10(-5) molar [M], 5 x 10(-5) M and 10(-4) M), and sevoflurane (1.7%, 3.4%, and 5.1%) on Ang II-induced contraction and p44/42 MAPK phosphorylation were tested in rat aortic smooth muscle, using isometric force measurement and Western blot analysis, respectively. Ang II induced both a transient contractile response and phosphorylation of p44/42 MAPK, which were significantly attenuated by PD 098059 (P < 0.05-0.01). Sevoflurane inhibited Ang II-induced contractile response in a dose-dependent manner (P < 0.05 and 0.01 in response to 3.4% and 5.1% sevoflurane, respectively). Sevoflurane also dose-dependently depressed Ang II-elicited p44/42 MAPK phosphorylation (P < 0.01 in response to 3.4% and 5.1% sevoflurane). These results suggest that the inhibitory effect of sevoflurane on Ang II-induced vasoconstriction is, at least in part, caused by the inhibition of the p44/42 MAPK-mediated signaling pathway. The present study demonstrates that sevoflurane can dose-dependently inhibit both angiotensin II (Ang II)-induced contraction and p44/42 MAPK phosphorylation of rat aortic smooth muscle. These data suggest that sevoflurane-produced inhibition of Ang II-induced vasoconstriction is, at least in part, caused by depression of the p44/42 MAPK-mediated signaling pathway.

Cardiovascular and Renal Regulation by the Angiotensin Type 2 Receptor

The renin-angiotensin system is a coordinated hormonal cascade of major importance in cardiovascular and renal regulation. The principle effector of this system is the octapeptide angiotensin II (Ang II), which acts at 2 cell membrane receptors, AT1 and AT2. The majority of the actions of Ang II have been demonstrated to be mediated by the AT1 receptor, including growth promotion, vasoconstriction, antinatriuresis, aldosterone secretion, salt appetite, thirst, sympathetic outflow and inhibition of renin biosynthesis, and secretion.1 The AT2 receptor has been less well understood. Past studies, however, clearly demonstrated that the AT2 receptor mediates cellular differentiation and growth, opposing the actions of Ang II through the AT1 receptor.2 Studies in the mid to late 1990s demonstrated that AT2 receptor stimulation engenders an autacoid vasodilator cascade composed of bradykinin (BK), nitric oxide (NO), and guanosine cyclic 3′, 5′-monophosphate (cGMP).3–5 This discovery turned attention to the possibility that the AT2 receptor may mediate vasodilation, opposing the vasoconstrictor actions of Ang II at the AT1 receptor.6 Parallel cell signaling studies indicated that the AT2 receptor is G-protein–coupled (through Giα) and that receptor stimulation is accompanied by an increase in phosphotyrosine phosphatase activity and an inhibition of MAP kinase enzymes (p42 and p44) composing the extracellular signal-related kinase (ERK1/2). AT2 receptor-mediated inhibition of the extracellular signal-regulated kinase pathway opposed the actions of Ang II, resulting in extracellular signal-regulated kinase phosphorylation via the AT1 receptor.1,2,6,7 Work during the late 1990s through 2002 suggested that the AT2 receptor might serve as a vasodilator counterforce to the AT1 receptor.2,6,7 However, vasorelaxation was difficult to elicit in some experimental models, attributed to the relatively low level of AT2 receptor vascular expression compared with that of the …

AT receptors and L-type calcium channels: functional coupling in supersensitivity to angiotensin II in diabetic rats

The study was designed to investigate the role of calcium channels in enhanced angiotensin II (Ang II)-induced contraction in thoracic aortic rings from diabetic rats. Ang II-induced isometric tension was studied in thoracic aortic rings isolated from control or streptozotocin-induced (8 weeks) diabetic rats. Saturation binding studies at AT1 receptors and L-type calcium channels were performed using [3H] Ang II and [3H] PN200110, respectively. Ang II-induced calcium influx was studied in fura-2-loaded single vascular smooth muscle cells isolated from thoracic aorta of control and diabetic rats. Ang II did not induce contraction in calcium-free Krebs. In the presence of extracellular calcium, increased Emax (mg/mm2) and pD2 to Ang II was observed in aortic rings from diabetic (795.54+/-38.19; 8.27+/-0.12) compared to control (230.09+/-25.45; 7.68+/-0.22) rats, respectively. Nimodipine but not verapamil or diltiazem dose-dependently blocked the Ang II-induced contractions in a noncompetitive manner and its -log IC50 was significantly lower in aortic rings from diabetic (8.81+/-0.10) compared to control (9.34+/-0.11) rats. The Ang II-induced increase in intracellular calcium levels was significantly enhanced (2.5-fold) in vascular smooth muscle cells from diabetic rats. AT1 receptor saturation binding with [3H] Ang II revealed a significantly higher affinity (nM) and Bmax (pmol/mg protein) in aortic vascular membrane preparation from diabetic (0.31+/-0.04; 64.18+/-2.4) compared to control (0.52+/-0.02; 47.81+/-2.8) rats, respectively, while L-type calcium channel saturation binding with [3H] PN200110 showed a higher affinity (nM) with no change in the Bmax (fmol/mg protein) in diabetic (0.74+/-0.08; 4.52+/-0.40) compared to control (1.49+/-0.32; 5.43+/-0.60) aortic membranes, respectively. Our results suggest that Ang II-induced contraction is dependent on extracellular calcium, and enhanced functional coupling of AT1 receptors and DHP-sensitive L-type calcium channels results in supersensitivity to Ang II in thoracic aorta isolated from diabetic rats.

Modulatory role of the adventitia on noradrenaline and angiotensin II responsesRole of endothelium and AT2 receptors

We have studied the modulatory role of the adventitia on vascular tone and nitric oxide (NO) availability in response to noradrenaline (NA) and angiotensin II (Ang II). Changes in isometric tension were determined in carotid arteries from 3-month-old Sprague-Dawley rats denuded from adventitia (-A) and compared to intact rings (+A). NO availability was assessed by the fluorescent NO indicator, 4,5-diaminofluorescein diacetate (DAF-2). Responses to NA (10(-10) to 10(-6) M) were: (i) significantly lower in -A compared to +A rings; (ii) equally enhanced in +A and -A rings without endothelium; and (iii) reduced in +A and -A rings incubated with superoxide dismutase (SOD; 15 U/ml). Responses to Ang II (10(-10) to 10(-7) M) were: (i) similar between +A and -A segments; (ii) equally reduced in both groups by SOD; and (iii) increased by endothelial denudation in both +A and -A arteries. Blockade of AT2 receptors with PD 123,319 (10(-7) M) significantly increased Ang II-induced contractions in +A rings. In segments preincubated with losartan (10(-5) M) and precontracted with NA (10(-7) M), Ang II elicited a relaxation that was abolished by l-NAME (10(-4) M), PD 123,319 (10(-7) M), and endothelium or adventitial removal. NO availability was increased in carotid rings stimulated with Ang II, but not with NA. This NO release was blocked by PD 123,319 (10(-7) M) and endothelium denudation. These results suggest that the adventitia differently modulates responses to vasoconstrictors and that it is a key layer in Ang II-induced contractions, mediating NO release from the endothelium via AT2 receptors. This increase in NO counterbalances basal superoxide release.

Ca2+/Calmodulin-Dependent and -Independent Mechanisms are Involved in Angiotension II-induced Contraction of Rat Aortic Smooth Muscle

Background: Angiotensin II (Ang II) appears to play important roles in the pathogenesis of hypertension. However, the mechanism by which Ang II induces vascular smooth muscle contraction is not fully understood. The phosphorylation of myosin light chain (MLC) is an essential trigger of the cascade that initiates of smooth muscle contraction. In this study, we investigated the role of MLC phosphorylation on Ang II-induced vascular smooth muscle contraction. Methods: Rat thoracic aortas were used as an experimental substrates. We measured isometric tension, myosin light chain phosphorylation, intracellular concentration, mitogen-activated protein kinase phosphorylation, and tyrosine phosphorylation. Results: 100 nM Ang II increased smooth muscle contraction transiently in rat thoracic aorta. Ang II also increased intracellular and 20 kDa MLC phosphorylation. Pretreatment with 10M verapamil and 30M abolished the contraction developed at 30 seconds by Ang II, whereas pretreatment with 10M verapamil and 30M abolished the contraction and the intracellular increase induced at 2 minutes by Ang II. Moreover, pretreatment of 10M verapamil, 30M and 1M thapsigargin abolished the contraction as well as intracellular M increase developed at 30 seconds and 2 minutes by Ang II. However, MLC phosphorylation was not affected. GF109203X attenuated Ang II-induced contraction more so than ML-7. 100 nM Ang II increased tyrosine phosphorylation of mitogen-activated protein kinase, 68 and 125 kDa proteins. The 125 kDa protein was confirmed as paxillin in primary vascular smooth muscle cell culture. Conclusions: Ang II-induced contraction involves -dependent and independent components, and -dependent contraction by Ang II is mediated by voltage-dependent channel. Moreover, protein kinase C and the mitogen-activated protein kinase activation pathway are involved in Ang II-induced contraction.

The vascular relaxing effects of sevoflurane and isoflurane are more important in hypertensive than in normotensive rats

The vascular response to anesthetics is altered in hypertensive patients since the functional and structural integrities of vascular smooth muscle and endothelium are deranged. The effects of anesthetics on angiotensin II (Ang II)-induced changes in vascular tone are not well understood. We investigated the effects of sevoflurane and isoflurane on Ang II-induced vasoconstriction in spontaneously hypertensive rats (SHR). The dose-dependent effects of sevoflurane and isoflurane on the Ang II-induced contraction of aortic rings, in the presence and absence of an intact endothelium, were investigated in normotensive Wistar-Kyoto rats (WKY) and SHR and compared using isometric force transducers. Ang II (10(-9)-10(-6) M) induced a similar transient phasic contraction of endothelium-intact rings from the two rat strains in a dose-dependent manner. Removal of the endothelium augmented the Ang II-elicited phasic contraction, to a greater extent in the SHR group than in the WKY group. Sevoflurane and isoflurane (1-3 minimum alveolar concentration) concentration-dependently inhibited the Ang II-induced contraction of endothelium-intact rings from WKY; an effect that was greatly enhanced following removal of the endothelium. A greater degree of attenuation of the Ang II-induced contraction of both endothelium-intact and -denuded rings by the two anesthetics was observed in the SHR group. The inhibitory effects of isoflurane on the Ang II-induced contraction of aortic rings from both strains appeared to be stronger than that of sevoflurane at equipotent concentrations. Our finding that the inhibitory effects of isoflurane and sevoflurane on Ang II-induced vasoconstriction are enhanced in SHR may, at least in part, account for the anesthesia-induced systemic hypotension frequently seen in hypertensive patients.

The suitability of iodinated Angiotensin-(1–7) peptides as pharmacological tools

Introduction: The heptapeptide Angiotensin-(1–7) [(Ang-(1–7)] is a biologically active component of the Renin–Angiotensin System. Pharmacological studies often involve Ang-(1–7) radioactively labelled with 125I. Given the small size of the original peptide, we investigated whether introduction of a rather bulky iodine label interferes with the biological activity of Ang-(1–7). Methods: Ang-(1–7) was labelled with nonradioactive iodine with the chloramine-T method. The reaction products were separated on HPLC and analysed with mass spectrometry. The products were tested for biological activity in two ways: The ability of labelled Ang-(1–7) to block Ang II-induced contraction in rat aortic rings was tested in an organ bath setup. The affinity of labelled angiotensin for ACE in rat plasma was examined in vitro. Results: Iodination of Angiotensin-(1–7) resulted in two main products: monoiodinated and diiodinated Ang-(1–7) that could be easily separated on HPLC. In an organ bath experiment, monoiodinated Ang-(1–7) blocked Ang II responses identical to the native compound, whereas diiodinated Ang-(1–7) had lost its ability to block Ang II responses. Likewise, monoiodinated Ang-(1–7) had retained its affinity for ACE, while the affinity of diiodinated Ang-(1–7) was greatly reduced. Discussion: Monoiodinated Ang-(1–7) has a biological activity identical to the native compound, whereas this is lost in diiodinated Ang-(1–7). Therefore, only the monoiodinated radioactive form seems suited for pharmacological studies.

ANG II type 2 receptor regulates smooth muscle growth and force generation in late fetal mouse development.

Although evidence from culture studies implicates the angiotensin II (ANG II) type 2 receptor (AT(2)R) in the regulation of growth and differentiation of arterial smooth muscle (SM) cells (SMC), the lack of its expression in adult arteries has precluded direct investigation of its role in vivo. The goal of the present study was to determine the role of AT(2)R in the control of fetal SMC growth, contractility, and differentiation during vascular development. Determination of isometric tension in fetal aortas showed potentiated ANG II-induced contraction by treatment with the selective AT(2)R antagonist PD-123319, demonstrating the presence of functional AT(2)Rs that mediate reduced force development in vascular SMC. In direct contrast to numerous cell culture studies, proliferation indexes were decreased rather than increased in aortic SMC of fetal homozygous AT(2)R knockout compared with wild-type or heterozygous knockout mice. Experiments using SMC tissues from heterozygous female AT(2)R knockout mice, which are naturally occurring chimeras for AT(2)R expression, showed that AT(2)R mRNA expression was exactly 50% of that of wild type. This indicated that loss of AT(2)R expression did not confer a selective advantage or disadvantage for SMC lineage determination and expansion. Real time RT-PCR analyses showed no significant difference in expression of SM-alpha-actin, SM myosin heavy chain, and myocardin in various SM tissues from all three genotypes, suggesting that knockout of AT(2)R had no effect on subsequent SMC differentiation. Taken together, results indicate that functional AT(2)R are expressed in fetal aorta and mediate reduced force development but do not significantly contribute to regulation of SMC differentiation.

Design, synthesis, structural studies, biological evaluation, and computational simulations of novel potent AT(1) angiotensin II receptor antagonists based on the 4-phenylquinoline structure.

Novel AT(1) receptor antagonists bearing substituted 4-phenylquinoline moieties instead of the classical biphenyl fragment were designed and synthesized as the first step of an investigation devoted to the development of new antihypertensive agents and to the understanding of the molecular basis of their pharmacodynamic and pharmacokinetic properties. The newly synthesized compounds were tested for their potential ability to displace [(125)I]Sar(1),Ile(8)-Ang II specifically bound to AT(1) receptor in rat hepatic membranes. These AT(1) receptor binding studies revealed nanomolar affinity in several of the compounds under study. The most potent ligands 4b,t were found to be equipotent with losartan and possessed either a 3-tetrazolylquinoline or a 2-amino-3-quinolinecarboxylic moiety, respectively. Moreover, some selected compounds were evaluated for antagonism of Ang II-induced contraction in rabbit aortic strips, and the most potent compounds in the binding test 4b,t were slightly more potent than losartan in inhibiting Ang II-induced contraction. Finally, the most relevant structure-affinity relationship data were rationalized by means of computational studies performed on the isolated ligands as well as by computational simulations on the ligands complexed with a theoretical AT(1) receptor model.

Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II.

The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents. We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK). Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody. Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein. Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein. The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels.

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.

Role of rho-kinase activity in angiotensin II-induced contraction of rabbit clitoral cavernosum smooth muscle.

Isometric tension measurement using a selective Rho-kinase inhibitor (+)- (R)-trans4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632) and a selective myosin light chain kinase (MLCK) inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7) were used in rabbit clitoral cavernosum smooth muscle (CSM). N(G)-nitro-L-arginine methyl ester (L-NAME) was used to evaluate the relationship between NO release and Rho-kinase. Y-27632 significantly attenuated contractions induced by ANG II, dose-dependently. However, ML7 did not affect the contractile response to ANG II except in the high concentrations of ML7. Y-27632 inhibited contraction with phenylephrine (PhE), but ML7 did not inhibit contraction with PhE. Nitric oxide synthase inhibitor (NAME) did not affect the Y-27632-induced relaxation in the pre-contracted strip with PhE. The present study demonstrates that G-protein-coupled increase in myofilament Ca(2+) sensitivity mediated through the RhoA/Rho-kinase signal pathway is involved in the control by ANG II of the clitoral CSM tone. RhoA/Rho-kinase pathway acts in the ANG II-induced contraction independently of the NO pathway.

Angiotensin II stimulates contraction and growth of testicular peritubular myoid cells in vitro.

Seminiferous tubule contraction, an important step in the regulation of spermatogenesis and testicular sperm output, is regulated by several agonists. In the present paper, we investigated whether angiotensin II (Ang II) may have a place among them. In binding experiments performed to assess the presence of specific receptors in rat peritubular myoid cells (TPMC), binding of (125)I-Ang II to TPMC was saturable in a time-dependent manner. Competition binding experiments performed with Losartan and PD 123319 showed that Losartan was able to inhibit the binding of (125)I-Ang II, whereas PD 123319 was ineffective. Ang II induced a dose-dependent rise in intracellular Ca(2+). Depletion of intracellular calcium stores by thapsygargin resulted in a lower rise of intracellular calcium, and the L-type voltage-operated calcium channel (VOCC-L) blocker verapamil abolished the Ca(2+) influx in rat TPMC. Altogether, these findings indicate that the Ang II-induced increase in [Ca(2+)](i) involves both extracellular influx and Ca(2+) release from intracellular stores. Ang II induced a dose-dependent TPMC contraction, and Losartan and not PD 123319 inhibited the response. Ang II-induced contraction was inhibited by adrenomedullin, previously shown to antagonize endothelin 1-provoked contraction in those cells. Ang II elicited (3)H-thymidine DNA incorporation and proliferation in a dose-dependent manner in TPMC. Losartan and both MAPK inhibitor PD 98059 and tyrosine kinase inhibitor AG18 were able to inhibit Ang II-induced (3)H-thymidine uptake and cell proliferation. In conclusion, the present study documents that angiotensin II, the active mediator of the tissue and circulating renin-angiotensin system present in the mammalian testis, induces contraction, growth and rise in intracellular calcium in rat peritubular myoid cells via angiotensin II type 1 receptors, and suggests that Ang II is involved in the paracrine regulation of the seminiferous tubule function.

Role of pp60(c-src) and p(44/42) MAPK in ANG II-induced contraction of rat tonic gastrointestinal smooth muscles.

We examined the role of mitogen-activated protein kinase (p(44/42) MAPK) in ANG II-induced contraction of lower esophageal sphincter (LES) and internal anal sphincter (IAS) smooth muscles. Studies were performed in the isolated smooth muscles and cells (SMC). ANG II-induced changes in the levels of phosphorylation of different signal transduction and effector proteins were determined before and after selective inhibitors. ANG II-induced contraction of the rat LES and IAS SMC was inhibited by genistein, PD-98059 [a specific inhibitor of MAPK kinases (MEK 1/2)], herbimycin A (a pp60(c-src) inhibitor), and antibodies to pp60(c-src) and p(120) ras GTPase-activating protein (p(120) rasGAP). ANG II-induced contraction of the tonic smooth muscles was accompanied by an increase in tyrosine phosphorylation of p(120) rasGAP. These were attenuated by genistein but not by PD-98059. ANG II-induced increase in phosphorylations of p(44/42) MAPKs and caldesmon was attenuated by both genistein and PD-98059. We conclude that pp60(c-src) and p(44/42) MAPKs play an important role in ANG II-induced contraction of LES and IAS smooth muscles.

Inhibition of mitogen-activated protein/extracellular signal-regulated kinase improves endothelial function and attenuates Ang II-induced contractility of mesenteric resistance arteries from spontaneously hypertensive rats.

Extracellular signal-regulated kinases (ERK1/2) modulate vascular smooth muscle cell (VSMC) growth and contractility, important factors in blood pressure regulation. In the present in vivo study, we investigated whether short-term inhibition of ERK1/2-dependent signaling pathways influences vascular function and blood pressure (BP) in spontaneously hypertensive rats (SHR). SHR and Wistar-Kyoto (WKY) rats were injected subcutaneously with either PD98059, selective MEK1/2 inhibitor (20 mg/kg), or vehicle. BP was measured by telemetry. Rats were killed 24 h after injection and small mesenteric arteries mounted as pressurized systems for morphometric analysis and assessment of endothelial function and angiotensin II (Ang II)-induced contractility. ERK1/2 phosphorylation was measured by Western blots, using protein extracts from mesenteric arteries, aorta, heart and kidneys. BP was higher (P < 0.01) in SHR than in WKY rats. PD98059 did not influence BP in either group. Endothelial-dependent relaxation (acetylcholine-induced), which was impaired in SHR, was improved by PD98059 (P < 0.05). Ang II increased contraction, with greater responses in SHR (Emax = 25 +/- 4%) than WKY (Emax = 9 +/- 3%) (P < 0.01). PD98059 reduced Ang II-induced contraction in SHR (Emax = 5.8 +/- 0.4%) and WKY (Emax = 4 +/- 0.4%). Vascular structure was unaltered by PD98059. Vascular and renal ERK1/2 phosphorylation, which was higher in SHR than WKY, was decreased by PD98059 in SHR. Short-term treatment with PD98059 improves endothelial function and vascular contractility without influencing BP in SHR. These findings provide evidence that vascular ERK1/2 activity is upregulated and that MEK1/2-sensitive signaling pathways play an important role in the regulation of vascular function in SHR. Acute inhibition of MEK1/2 does not alter blood pressure despite improved endothelial function and reduced arterial reactivity to Ang II.