AT2 Receptor Antagonist PD123319 Research Articles

Cerebral ischemia enhances vascular angiotensin AT1 receptor-mediated contraction in rats.

The aim of the study was to examine how focal cerebral ischemia affects the expression and function of vascular angiotensin II receptors. We used an intraluminal filament occlusion technique to occlude the right middle cerebral artery (MCA) of the rat. Myographs were used for functional studies of the MCA and real-time polymerase chain reaction, for determination of relative mRNA levels. The contractile responses to angiotensin II were stronger in the right occluded MCA compared with the left MCA and the MCA from sham-operated rats 48 hours after MCA occlusion (P<0.05). The angiotensin II type 1 (AT1) receptor antagonists candesartan and losartan abolished the enhanced responses to angiotensin II (P<0.05), whereas the AT2 receptor antagonist PD123319 had no effect. The amount of AT1 receptor mRNA was lower in the occluded MCAs compared with nonoccluded MCAs 48 hours after occlusion (P<0.05), whereas the mRNA levels of angiotensin converting enzyme (ACE) were higher in the occluded arteries. The mRNA levels of the AT2 receptor and nuclear factor-kappaB were unchanged. Focal cerebral ischemia in the rat upregulated the contractile responses to angiotensin II in the ipsilateral MCA, and this contraction was mediated by AT1 receptors. Real-time polymerase chain reaction revealed decreased AT1 receptor mRNA levels in the occluded MCA, whereas the amount of ACE mRNA was increased, suggesting locally enhanced angiotensin II production. These results support a role for AT1 receptors in cerebral ischemia, and we think that AT1 receptors might be a future therapeutic target in ischemic stroke.

Increased renal vascular endothelial growth factor and angiopoietins by angiotensin II infusion is mediated by both AT1 and AT2 receptors.

A link between angiotensin II and cell proliferation has previously been reported. However, there remains controversy as to the role of the individual angiotensin II receptor subtypes in mediating these effects and their link to angiogenic cytokines and their receptors. Male Sprague-Dawley rats were infused with either angiotensin II or vehicle for 14 d at a dose of 58.3 ng/min. Angiotensin II-infused rats received no treatment, an AT(1) receptor antagonist valsartan (30 mg/kg per d), or an AT(2) receptor antagonist PD123319 (830 ng/min). Gene expression of vascular endothelial growth factor (VEGF) and receptor VEGF-R2, as well as Tie-2 and its ligands angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) were assessed by reverse transcription-PCR. Protein expression was assessed by Western blotting and immunohistochemistry. Gene and protein expression of VEGF, Ang-1, and Ang-2 were increased by angiotensin II infusion. Valsartan and PD123319 attenuated angiotensin II-associated increases in VEGF gene and protein expression. Ang-1 and Ang-2 gene but not protein expression were reduced by both treatments. These changes occurred in the context of attenuation of angiotensin II-induced glomerular cell proliferation by both valsartan and PD123319. In situ hybridization and immunohistochemical studies localized VEGF, Ang-1, and Ang-2 expression to the epithelial cells of the glomerulus, and VEGF-R2 and Tie-2 receptors to the endothelial cells of the kidney. These findings extend the increasing evidence that the AT(2) receptor, in addition to the AT(1) receptor subtype, plays an important role in mediating the proliferative actions of angiotensin II in the kidney.

Angiotensin II accelerates functional recovery in the rat sciatic nerve in vivo: role of the AT2 receptor and the transcription factor NF-kappaB.

The AT2 receptor regulates several functions of nerve cells, e.g., ionic fluxes, cell differentiation, and axonal regeneration, but also modulates programmed cell death. We tested the hypothesis that angiotensin II (ANG II) via its AT2 receptor not only promotes regeneration but also functional recovery after sciatic nerve crush in adult rats. ANG II (10(-7), 10(-9), 10(-11) M) applied locally via osmotic minipumps promoted functional recovery with maximal effects after the lowest concentration. The toe spread distance as a parameter for re-innervation after 20 days was significantly (P<0.01) greater (10.2+/-10.27 mm) compared with the control group (8.73+/-0.16 mm). The response to local electrical stimulation (return of sensorimotor function) was reduced to 14.6 days vs. 17.9 days in the control group (P<0.01). The AT2 receptor antagonist PD 123319 administered alone or in combination with ANG II completely prevented the ANG II-induced recovery, whereas the AT1 receptor antagonist losartan had no effect. Furthermore, ANG II induces, via the AT2 receptor, activation of the transcription factor NF-kappaB in Schwann cells. Histological criteria, morphometric analyses, and electron microscopy confirmed the functional data. These results are the first to present direct evidence for an involvement of the AT2 receptor and NF-kappaB in peripheral nerve regeneration.

Effects of angiotensin-(1-7) and other bioactive components of the renin-angiotensin system on vascular resistance and noradrenaline release in rat kidney.

Angiotensin (Ang) is broken down enzymatically to several different metabolites which, in addition to Ang II, may have important biological effects in the kidney. This study investigates the role of Ang metabolites on vascular resistance and noradrenaline release in the rat kidney. In rat isolated kidney Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I induced pressor responses and enhanced noradrenaline release to renal nerve stimulation (RNS) in an concentration-dependent manner, with the following rank order of potency (EC(50)): Ang II >or= Ang III > Ang I = des-Asp-Ang I > Ang IV. All effects were blocked by the AT(1)-receptor antagonist EXP 3174 (0.1 micromol/l) but not by the AT(2)-receptor antagonist PD 123319 (1 micromol/l). Angiotensin-converting enzyme (ACE) inhibition by captopril (10 micromol/l) abolished the effect of Ang I and des-Asp-Ang I but had no influence on the effect of the other metabolites. Ang-(1-7) blocked the effects of Ang I and Ang II, being 10 times more potent against Ang I than Ang II. The selective Ang-(1-7) receptor blocker d-Ala7-Ang-(1-7) (10 micromol/l) did not influence the inhibitory effects of Ang-(1-7). Ang-(1-7) (10 micromol/l) by itself had no influence on vascular resistance and RNS-induced noradrenaline release. Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I regulate renal vascular resistance and noradrenaline release by activation of AT(1) receptors. In the case of Ang I and des-Asp-Ang I this depends on conversion by ACE. Ang-(1-7) may act as a potent endogenous inhibitor/antagonist of ACE and the AT(1)-receptors, respectively.

No involvement of the AT2-receptor in angiotensin II-enhanced sympathetic transmission in vitro

Angiotensin II (Ang II) enhances sympathetic neurotransmission via AT(1)-receptors located on sympathetic nerve terminals. We recently demonstrated that inhibition of Ang II-mediated facilitation in the pithed rat by irbesartan resulted in a U-shaped dose response curve, which was not observed when PD 123319, at a concentration that selectively blocks the AT(2)-receptor, was co-administered. Hence, the irbesartan-mediated upstroke might be explained by the involvement of the AT(2)-receptor after AT(1) blockade with high-dose irbesartan. In the present study, we further investigated the possible role of the AT(2)-receptor in Ang II-mediated facilitation in vitro. We studied the effect of the AT(2)-receptor antagonist PD 123319 (10 nM) on Ang II-enhanced sympathetic outflow evoked by electrical field stimulation (EFS) in the rat isolated inferior vena cava. Additionally, we investigated the effect of the AT(1)-receptor blocker irbesartan (0.1 nM 1 M) on the sequelae of Ang II-enhanced, EFS-evoked sympathetic nerve traffic in the presence or absence of PD 123319 (10 nM). PD 123319 did not influence Ang II-enhanced sympathetic outflow. Irbesartan dose-dependently attenuate Ang II-augmented transmitter release (pIC50 7.99+0.03), whereas no U-shaped concentration-response relationship for irbesartan was observed. Co-administration of PD 123319 with irbesartan proved unable to influence Ang II-mediated facilitation differently compared with irbesartan alone. The experimental observations indicate that the AT(2)-receptor is not involved in Ang II-mediated enhancement of sympathetic nerve traffic in the present in vitro study.

Control of dendritic cell differentiation by angiotensin II.

Here we analyze the role of the angiotensinergic system in the differentiation of dendritic cells (DC). We found that human monocytes produce angiotensin II (AII) and express AT1 and AT2 receptors for AII. DC differentiated from human monocytes in the presence of AT1 receptor antagonists losartan or candesartan show very low levels of CD1a expression and poor endocytic and allostimulatory activities. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in the development of nonadherent cells with CD1a expression and endocytic and allostimulatory activities higher than control DC. Similar contrasting effects were observed in mouse DC obtained from bone marrow cultures supplemented with granulocyte-monocyte colony-stimulating factor. DC differentiated in the presence of the AT1 receptor antagonist losartan express lower levels of CD11c, CD40, and Ia and display a lower ability to endocyte horseradish peroxidase (HRP) and to induce antibody responses in vivo, compared with controls. By contrast, DC differentiation in the presence of either the AT2 receptor antagonist PD 123319 or exogenous AII results in cells with high levels of CD11c, CD40, and Ia, as well as high ability to endocyte HRP and to induce antibody responses in vivo. Our results support the notion that the differentiation of DC is regulated by AII.

Angiotensin II type 2 receptor blockade partially negates antihypertrophic effects of type 1 receptor blockade on pressure-overload rat cardiac hypertrophy.

We investigated the effects of angiotensin II type 2 (AT2) receptor blockade on the antihypertrophic effects of type 1 receptor (AT1) blockade in pressure-overload cardiac hypertrophy in adult rats. Cardiac hypertrophy was induced by banding the abdominal aorta above the renal arteries. The rats were treated with either an AT1 receptor antagonist TCV-116 (TCV, 10 mg/kg/day), an AT2 receptor antagonist PD123319 (PD, 20 mg/kg/day), or both for 4 weeks after the aortic banding. We measured systolic and diastolic blood pressure (BP), body weight (BW), left ventricular weight (LVW), and serum and cardiac angiotensin converting enzyme (ACE) activities. Aortic banding increased BP and LVW/BW, and TCV reversed both these increases. PD affected neither BP nor LVW/BW. TCV+PD reversed the increase in BP but not LVW/BW. Thus, PD was considered to counteract the antihypertrophic effect of TCV without affecting BP. All three treatments reduced cardiac ACE activity without affecting serum ACE activity. Our data demonstrated that AT2 receptor blockade negates the antihypertrophic effects of AT1 receptor blockade in an adult rat model of pressure-overload cardiac hypertrophy. AT2 receptors may mediate the signaling pathways involved in growth inhibition, which could counteract mediation of the cellular growth signaling pathways by AT1 receptors.

The angiotensin II receptor type 2 agonist CGP 42112A stimulates NO production in the porcine jejunal mucosa

BackgroundThis study was conducted to elucidate if nitric oxide is released by the porcine jejunal mucosa upon selective stimulation of AT2 receptors and the possible involvement of iNOS, and to investigate the presence of jejunal AT1 and AT2 receptors. Young landrace pigs were anaesthetized with ketamine and α-chloralose. Jejunal luminal NO output was assessed by intraluminal tonometry and analysed by chemiluminescense. Western blot analysis quantified mucosal iNOS and detected AT1 and AT2 receptor protein expression. AT1 and AT2 receptor RNA expression was detected by rtPCR.ResultsBaseline luminal NO output correlated significantly to baseline mucosal iNOS-protein content. In animals treated with the AT2-receptor agonist CGP42112A (n = 11) luminal NO output increased significantly (at 0.1 micrograms kg-1 min-1 and 1.0 micrograms kg-1 min-1), but not in animals simultaneously treated with the AT2-receptor antagonist PD123319 (bolus 0.3 mgkg-1, infusion 0.03 mg kg-1 h-1) (n = 7). No differences in iNOS protein expression were found between groups or before/after the administration of drugs. Western blot and rtPCR recognised expression of the AT1 and AT2 receptors in jejunal tissue.ConclusionThe results suggest that activation of AT2 receptors increases jejunal luminal NO output. This response was not due to an increase in the expression of the iNOS protein in the mucosa.

AVE 0991, a nonpeptide mimic of the effects of angiotensin-(1-7) on the endothelium.

Recently, we demonstrated that the heptapeptide angiotensin-(1-7) (Ang-[1-7]) exhibits a favorable kinetic of nitric oxide (NO) release accompanied by extremely low superoxide (O2-) production. In this report we describe AVE 0991, a novel nonpeptide compound that evoked effects similar to Ang-(1-7) on the endothelium. AVE 0991 and unlabeled Ang-(1-7) competed for high-affinity binding of [125I]-Ang-(1-7) to bovine aortic endothelial cell membranes with IC50 values of 21+/-35 and 220+/-280 nmol/L, respectively. Stimulated NO and O2- release from bovine aortic endothelial cells was directly and simultaneously measured on the cell surface by selective electrochemical nanosensors. Peak concentrations of NO and O2- release by AVE 0991 and Ang-(1-7) (both 10 micromol/L) were not significantly different (NO: 295+/-20 and 270+/-25 nmol/L; O2-: 18+/-2 and 20+/-4 nmol/L). However, the released amount of bioactive NO was approximately 5 times higher for AVE 0991 in comparison to Ang-(1-7). The selective Ang-(1-7) antagonist [D-Ala(7)]-Ang-(1-7) inhibited the AVE 0991-induced NO and O2- production by approximately 50%. A similar inhibition level was observed for the Ang II AT1 receptor antagonist EXP 3174. In contrast, the Ang II AT2 receptor antagonist PD 123,177 inhibited the AVE 0991-stimulated NO production by approximately 90% but without any inhibitory effect on O2- production. Both NO and O2- production were inhibited by NO synthase inhibition ( approximately 70%) and by bradykinin B2 receptor blockade (approximately 80%). AVE 0991 efficiently mimics the effects of Ang-(1-7) on the endothelium, most probably through stimulation of a specific, endothelial Ang-(1-7)-sensitive binding site causing kinin-mediated activation of endothelial NO synthase.

Stimulation of collagen gel contraction by angiotensin II and III in cardiac fibroblasts.

The aim of the present study was to investigate whether angiotensin II (Ang II), angiotensin III (Ang III) or Ang II (2-8), angiotensin IV (Ang IV) or Ang II (3-8) and Ang II (1-7), Ang II (4-8), Ang II (5-8) and Ang II (1-4) can stimulate collagen gel contraction in cardiac fibroblasts in serum-free conditions. Cardiac fibroblasts (from male adult Wistar rats) from passage 2 were cultured to confluency and added to a hydrated collagen gel in a Dulbecco's Modified Eagle's Medium, with or without foetal bovine serum, for one, two or three days. The area of the collagen gels embedded with cardiac fibroblasts was determined by a densitometric analysis. Collagen gel contraction was characterised by a decrease in the gel area. Ang II dose-dependently stimulated the contraction of collagen mediated by cardiac fibroblasts after one, two or three days of incubation in a serum-free medium. Telmisartan completely blocked the Ang II-induced collagen contraction by cardiac fibroblasts. PD 123319 and des-Asp(1)-Ile(8)-Ang II had no effect on the Ang II-induced collagen contraction by cardiac fibroblasts. Ang III also stimulated the contraction of collagen mediated by cardiac fibroblasts after one, two or three days of incubation in a serum-free medium. des-Asp(1)-Ile(8)-Ang II and telmisartan completely blocked the Ang III-induced collagen gel contraction by cardiac fibroblasts. des-Asp(1)-Ile(8)-Ang II, however, had no effect on the Ang II-induced collagen gel contraction by cardiac fibroblasts. Ang IV and Ang II (4-8), (5-8), (1-7) and (1-4), however, had no effect on collagen gel contraction by cardiac fibroblasts. Addition of telmisartan, PD 123319 or des-Asp(1)-Ile(8)-Ang II alone did not affect collagen gel contraction by cardiac fibroblasts. Our data demonstrate that the effects of Ang II on the collagen gel contraction by adult rat cardiac fibroblasts in serum-free conditions are Ang II type 1(AT(1))-receptor- mediated, because they are abolished by the specific AT(1)-receptor antagonist, telmisartan, and not by the AT(2)-receptor antagonist PD 123319 or by the Ang III antagonist des-Asp(1)-Ile(8)-angiotensin. The Ang III- stimulated contraction of collagen by cardiac fibroblasts is completely blocked by the Ang III receptor antagonist, des-Asp(1)-Ile(8)-angiotensin II, and by telmisartan.

AT2 receptor-mediated relaxation is preserved after long-term AT1 receptor blockade.

Angiotensin II type 2 receptor (AT2R) stimulation may cause vasodilation per se and may contribute to the antihypertensive effect produced by Angiotensin II type 1 receptor (AT1R) antagonists, given that AT1R blockade increases endogenous levels of Ang II, suggesting a physiological role for the unblocked AT2R. Thus, we first directly assessed whether or not there is desensitization to AT2R-mediated vasorelaxation because this is an important consideration, given the raised Ang II levels and the marked desensitization that is known to occur after AT1R stimulation. Second, we examined if AT2R-mediated vasorelaxation is preserved after long-term treatment with the AT1R antagonist candesartan cilexetil. Consecutive concentration-response curves to AT2R stimulation, with either Ang II (with AT1R blockade) or the selective agonist CGP42112, were studied in rat isolated mesenteric resistance arteries mounted in an arteriograph. AT2R stimulation with Ang II induced a concentration-dependent relaxation without desensitization. Similarly, CGP42112 evoked highly reproducible relaxation, which, like Ang II, was abolished by the AT2R antagonist PD123319. By contrast, AT1R-mediated contraction exhibited marked desensitization. In rats treated with candesartan cilexetil (2 mg/kg per day for 2 weeks), AT1R-mediated contraction was abolished, whereas AT2R-mediated relaxation evoked by either Ang II or CGP42112 was highly reproducible, PD123319-sensitive, and of a magnitude similar to that observed in naïve animals. Therefore, this study has provided unequivocal evidence for the reproducible nature of AT2R-mediated vasorelaxation during short-term and long-term AT1R blockade. Such preservation of AT2R function is a prerequisite for the consideration of physiological role(s) of AT2R during AT1R blockade.

Angiotensin type 2 receptor antagonism confers renal protection in a rat model of progressive renal injury.

The role of the angiotensin type 2 (AT(2)) receptor in the pathogenesis of progressive renal injury has not been previously elucidated. The renal expression of the AT(1) and AT(2) receptors in subtotally nephrectomized rats (STNx) and the effects of AT(2) receptor blockade on renal injury were explored. Reduced renal expression of the AT(1) but not the AT(2) receptor was observed in STNx by reverse transcription-PCR, by in vitro autoradiography, and by immunohistochemical staining. The STNx rats were randomly assigned to AT(1) receptor antagonist valsartan, AT(2) receptor antagonist PD123319, or the combination of both for 4 wk. Increased proteinuria in STNx rats was reduced by PD123319 but to a lesser degree when compared with valsartan. Reduced gene and protein expression of the slit diaphragm protein nephrin was prevented by either valsartan or PD123319. Expression of osteopontin, proliferating cell nuclear antigen, and monocyte/macrophage infiltration was increased in STNx rats and was reduced by both AT(1) and AT(2) receptor antagonists. These effects of AT(2) receptor antagonism were observed in the presence of increased BP in STNx rats. These findings suggest that blockade of the AT(2) receptor alone confers a degree of renal protection; in particular, it seems that the combination of the AT(1) and AT(2) receptor antagonists may confer additive renal effects than either receptor antagonist as monotherapy.

Angiotensin II activates nuclear transcription factor-κB through AT1 and AT2 receptors

Recent evidence suggests that angiotensin II (Ang II) induces a variety of proinflammatory mediators including chemokines. Nuclear factor-kappaB (NF-kappaB) activation plays an important role in Ang II-mediated inflammation. The present study investigated which Ang II receptor subtype is involved in NF-kappaB activation. We focused particularly on the Ang II subtype 2 (AT2) receptor because we previously observed that Ang II-induction of the chemokine RANTES in vitro and in vivo is mediated through AT2 receptors.AT1 or AT2 receptors were selectively overexpressed in COS7 cells that normally do not express Ang II receptors. In addition, rat glomerular endothelial cells (GER) that express AT1 and AT2 receptors and PC12 cells that exclusively exhibit AT2 receptors were studied also. Ang II-receptor expression was confirmed by Western blots of membrane lysates. NF-kappaB DNA binding in vitro was detected by electrophoretic shift assays. In addition, in vivo transactivation of a reporter gene construct with kappa enhancer coupled to luciferase also was investigated. Expression of the inhibitor of kappaB alpha (IkappaB-alpha) was detected by Western blots.In AT1 or AT2 receptor transfected cells, but not untransfected COS7 cells, 10-7 mol/L Ang II induced NF-kappaB DNA binding in vitro, as detected by electrophoretic shift assays and in vivo transactivation of a reporter gene construct. The AT2 receptor antagonist PD 123319 but not losartan attenuated Ang II-mediated NF-kappaB activation in COS7 cells transfected with AT2 receptors. While Ang II also induced NF-kappaB activation in PC12 cells, this activation was blocked by PD 123319. Finally, stimulation of GERs with Ang II led to the activation of NF-kappaB through both subtypes of Ang II receptors. Nuclear extracts from COS7 cells transfected with AT2 receptors and PC12 cells with NF-kappaB DNA-binding activity consisted of p50/p65 complexes. There was no difference in subunit composition of nuclear proteins from Ang II-stimulated AT1 receptor transfected COS7 cells. An artificial peptide (p-Amino-Phe6-Ang II) with a high affinity for the AT2 receptor also activated NF-kappaB. Ang II-induced activation of NF-kappaB was associated with degradation of IkappaB-alpha in all studied cell lines.Our results clearly demonstrate in various cell lines that Ang II induces NF-kappaB activation through AT2 receptors. These data may have important therapeutic consequences, because potential Ang II-mediated proinflammatory renal and cardiovascular effects may not be totally antagonized by the currently increased clinical use of AT1 receptor antagonists.

Effects of Angiotensin II on the Renal Interstitial Concentrations of NO2 /NO3 and Cyclic GMP in Anesthetized Rats

The present study was conducted to determine whether exogenous angiotensin II (Ang II) may increase the renal interstitial fluid concentrations of NO2/NO3 (NOx) and cyclic guanosine monophosphate (cGMP) concomitantly and which Ang II receptor subtypes may induce these changes in anesthetized rats, using a microdialysis method. Ang II (50 ng/kg per min, i.v.) significantly increased mean blood pressure (MBP), extraction rates of renal interstitial NOx from 23.9+/-1.0 to 31.2+/-1.9 pmol/min, and cGMP from 4.1+/-0.3 to 6.4+/-0.5 fmol/min, and decreased renal blood flow (RBF). The AT1-receptor antagonist CV11974 alone significantly increased RBF, but did not alter MBP, renal interstitial concentrations of NOx and cGMP. A superimposition of Ang II on CV11974 did not affect MBP and RBF, but significantly increased renal interstitial concentrations of NOx and cGMP. The AT2-receptor antagonist PD123319 alone did not change any of the parameters. However, superimposition of Ang II on PD123319 increased MBP and decreased RBF without any effects on renal interstitial concentrations of NOx and cGMP. These results suggest that Ang II stimulates NO production via the AT2-receptor in the kidney.

Angiotensin type 2 receptor-mediated hypotension in angiotensin type-1 receptor-blocked rats.

The type-2 (AT(2)) angiotensin (Ang) II receptor has been characterized as potentially counterregulatory to the actions of Ang II at its type-1 (AT(1)) receptor. We investigated the effects of Ang II and CGP-42112A (CGP), a selective peptide AT(2) receptor agonist, on blood pressure (BP) in rats with or without pharmacological blockade of the AT(1) receptor with losartan (LOS) or valsartan (VAL). In anesthetized rats (n=5 per group) receiving normal sodium intake, Ang II (200 pmol/kg per minute IV) alone increased BP from a control of 112+/-3 to 168+/-7 mm Hg (P<0.001) and LOS (30 mg/kg) alone decreased BP to 89+/-7 mm Hg (P<0.0001 from control). Ang II administered together with LOS decreased BP further to 71+/-4 mm Hg (P<0.00001 from control and LOS alone). AT(2) receptor antagonist PD 123,319 (PD) completely blocked the hypotensive response to LOS combined with Ang II (P=NS from control). In conscious rats (n=5 per group) receiving normal sodium intake, VAL (10 mg/kg) alone decreased BP from a control of 98+/-5 to 86+/-3 mm Hg (P<0.00001). Ang II combined with VAL induced a consistent, highly significant decline in BP for 6 days to a nadir of 69+/-3 mm Hg (P<0.01 versus daily VAL alone). PD completely blocked the chronic hypotensive response to the combination of Ang II and VAL to control levels before VAL administration. In another study in conscious rats (n=5 per group), CGP (70 microg/kg per minute) also decreased BP in VAL-treated conscious rats. BP was 119+/-3 mm Hg during the control period, decreased to 86+/-6 mm Hg during 3 days of VAL alone, (P<0.00001) and decreased further to 65+/-7 mm Hg (P<0.001 from daily VAL alone) with 7 days of CGP in the presence of VAL. In the absence of VAL, CGP decreased BP for 4 consecutive days, and this response was blocked by PD. Also, the CGP-induced decrease in BP over a 7-day period was blocked by N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. The results strongly suggest that the AT(2) receptor induces a systemic vasodilator response mediated by NO that counterbalances the vasoconstrictor action of Ang II at the AT(1) receptor.

Angiotensin-Induced Enhancement of Excitatory Junction Potentials Evoked by Periarteriolar Nerve Stimulation and Vasoconstriction in Rat Mesenteric Arteries Are Both Mediated by the Angiotensin AT1 Receptor

The aims of the present study were to determine the angiotensin II (AngII) receptor subtype(s) involved in vasoconstriction and enhancement of sympathetic neurotransmission in rat isolated mesenteric arteries. Vasoconstriction was assessed in mesenteric artery ring preparations suspended under 0.5 g of tension in a myograph. In control arteries, with an intact endothelium, AngII (1 nmol/l–3 µmol/l) caused a concentration-dependent contraction. The pEC₅₀ for AngII was 7.6 ± 0.2 and the maximum response of 0.24 ± 0.07 g was reached with 100 nmol/l. In the presence of indomethacin (3.0 µmol/l) and N^ω-nitro-L-arginine (NOLA) (100 µmol/l) to remove the influence of endothelium-derived prostaglandins and nitric oxide, the maximum response evoked by AngII was increased to 0.48 ± 0.1 g and the pEC₅₀ was 7.6 ± 0.3. The AT1 receptor antagonist losartan (30 nmol/l) competitively blocked the AngII-induced contractions with an estimated pA₂ of 8.2 in both the control arteries and in arteries treated with indomethacin and NOLA. The AT2 receptor antagonist PD 123319 (1 µmol/l) did not affect AngII-induced contractions under either condition. Conventional intracellular microelectrode recording techniques were used to investigate the effects of AngII on excitatory junction potentials (EJP) evoked by stimulation of periarteriolar sympathetic nerves. Stimulation with trains of 10 pulses delivered at 0.9 Hz evoked EJP which were blocked by tetrodotoxin (0.1 µmol/l), guanethidine (30 µmol/l) and the P_2X receptor desensitizing agent α,β-methylene ATP (30 µmol/l) suggesting the EJP were mediated by ATP, or a related purine, released from sympathetic nerves. AngII (0.3– 100 nmol/l) did not affect the resting membrane potential or the amplitude of the first EJP, but did enhance the amplitude of the plateau EJP later in the train. A maximum 49.2 ± 3.9% enhancement of the plateau EJP amplitude was elicited by 10 nmol/l AngII and the pEC₅₀ was 9.1 ± 0.1. The facilitatory effect of AngII on EJP amplitude was not altered in the presence of indomethacin and NOLA. Losartan (30 nmol/l) competitively blocked the AngII-induced enhancement of plateau EJP amplitude, with an estimated pA₂ of 8.6. PD 123319 did not alter the enhancement of plateau EJP amplitude by AngII. The results from the present study show that both the vasoconstriction and enhancement of plateau EJP amplitude by AngII in rat mesenteric arteries are blocked by the AT1 receptor antagonist losartan and are unaffected by the AT2 receptor antagonist PD 123319.

Inhibition of baroreflex by angiotensin II via Fos expression in nucleus tractus solitarii of the rat.

We evaluated the modulatory action of angiotensin II at the nucleus tractus solitarii on spontaneous baroreceptor reflex response, the angiotensin subtype receptors involved, and the role of Fos protein in this process, using Sprague-Dawley rats anesthetized with pentobarbital sodium. Microinjection bilaterally of angiotensin (Ang ) II (5, 10, 20, or 40 pmol) into the nucleus tractus solitarii significantly suppressed the spontaneous baroreceptor reflex, as represented by the magnitude of transfer function between systemic arterial pressure and heart rate signals. There also was a concomitant increase in Fos-like immunoreactivity in the nucleus tractus solitarii. Both the suppression of spontaneous baroreceptor reflex and Fos expression in nucleus tractus solitarii neurons elicited by Ang II were discernibly attenuated by pretreatment with or comicroinjection into the bilateral nucleus tractus solitarii of a 15-mer antisense c-fos oligonucleotide that targets against the initiation codon of c-fos mRNA. In addition, those 2 actions of Ang II were reversed by the coadministration of the nonpeptide Ang II type 1 (AT(1)) receptor antagonist losartan (1.6 nmol) but not by the nonpeptide AT(2) receptor antagonist PD 123,319 (1.6 nmol). Control treatments with artificial cerebrospinal fluid, sense cDNA, or antisense oligonucleotide with a scrambled sequence were ineffective. We conclude that under minimal cardiovascular perturbation, Fos expression mediated via activation of AT(1) subtype receptors may underlie the inhibitory modulation of beat-to-beat baroreflex control of blood pressure by Ang II at the nucleus tractus solitarii.

Coronary hemodynamic and ventricular responses to angiotensin type 1 receptor inhibition in SHR: interaction with angiotensin type 2 receptors.

This study was designed to determine the effects of angiotensin II type 1 (AT(1)) receptor inhibition on coronary hemodynamics and ventricular mass and hydroxyproline content and the additive effects of angiotensin II type 2 (AT(2)) receptor inhibition in spontaneously hypertensive rats (SHR). The selective AT(1) receptor antagonist candesartan (10 mg/kg per day) was administered alone or in combination with the AT(2) receptor antagonist PD 123319 (50 mg/kg per day) for 12 weeks. Control SHR received placebo for the same period. Left and right ventricular coronary blood flow, blood flow reserve, and minimal coronary vascular resistance were determined by using radiomicrospheres in male 35-week-old rats. Mean arterial pressure; total peripheral resistance; left and right ventricular, renal, and aortic weights; and hydroxyproline concentration were also determined. Candesartan reduced mean arterial pressure and left ventricular, renal, and aortic masses, as well as hydroxyproline concentration and minimal coronary vascular resistance of both ventricles. PD 123319 partially prevented the hypotensive effect of AT(1) receptor inhibition and reversed the effect on myocardial hydroxyproline concentration. These data suggest that AT(2) receptors contribute to the hypotensive and antifibrotic effects but not the coronary hemodynamic improvement or reduced left ventricular mass of AT(1) receptor inhibition in these adult SHR.

Differential effects of angiotensin AT1and AT2receptors on the expression, translation and function of the Na+-H+exchanger and Na+-HCO3−symporter in the rat heart after myocardial infarction

OBJECTIVESThis study investigated the role of angiotensin receptor subtype 1 (AT1) and angiotensin receptor subtype 2 (AT2) in the regulation of Na+-H+exchanger (NHE) and Na+-HCO3−symporter (NBC) in the infarcted myocardium.BACKGROUNDThe cardiac renin-angiotensin system is activated after myocardial infarction (MI), and both angiotensin AT1and AT2receptors are upregulated in the myocardium.METHODSNa+-H+exchanger isoform-1 and NBC-1 gene expression were determined by reverse transcription polymerase chain reaction and Northern blot analysis; protein levels by Western blot analysis; and activity by measurement of H+transport in left ventricular (LV) free wall, interventricular septum (IS) and right ventricle (RV) after induction of MI. Rats were treated with placebo, the angiotensin-converting enzyme inhibitor ramipril (1 mg/kg/day), the AT1receptor antagonist valsartan (10 mg/kg/day) or the AT2receptor antagonist PD 123319 (30 mg/kg/day). Treatment was started seven days before surgery.RESULTSNa+-H+exchanger isoform-1 and NBC-1 messenger RNA (mRNA) expression and protein levels were increased twofold in the LV free wall after MI, whereas no changes were observed in the IS and RV. Na+-dependent H+flux was increased in the LV free wall. Ramipril inhibited mRNA and protein upregulation of both transporters. Valsartan inhibited the upregulation of NHE-1 mRNA and protein but had no effect on NBC-1 mRNA expression and translation. In contrast, PD 123319 abolished the upregulation of NBC-1 mRNA and protein but had no effect on NHE-1 upregulation. Ramipril and valsartan prevented post-MI increase in NHE-1 activity, whereas ramipril and PD 123319 decreased NBC-1 activity.CONCLUSIONSAngiotensin II via its AT1and AT2receptors differentially controls transcriptional and translational regulation as well as the activity of NHE-1 and NBC-1 in the ischemic myocardium and contributes to the control of pH regulation in cardiac tissue.

Identification, Distribution, and Expression of Angiotensin II Receptors in the Normal Human Prostate and Benign Prostatic Hyperplasia

The tissue distribution, cellular localization, and level of expression of angiotensin II (Ang II) receptors were examined in the normal human prostate and benign prostatic hyperplasia (BPH) by in vitro autoradiography, immunohistochemistry, and radioligand binding studies. In the normal human prostate, Ang II receptors were of the AT1 subtype and localized predominantly to periurethral stromal smooth muscle. The AT1 receptor antagonist losartan totally displaced specific[ 125I]-[Sar1,Ile8]Ang II binding, in a concentration-dependent manner, whereas the AT2 receptor antagonist PD123319 was without effect. There was no significant difference in receptor affinity, but AT1 receptor density was markedly reduced in BPH compared with that in normal prostate. In rat prostate, Ang II (0.01–1 μm) produced a concentration-dependent increase in[ 3H]-noradrenaline release from sympathetic nerves. The findings of the present study suggest that angiotensin AT1 receptors predominate in the human prostate. The high concentration of AT1 receptors in the periurethral region suggests a role for Ang II in modulating cell growth, smooth muscle tone, and possibly micturition. Furthermore, down-regulation of AT1 receptors in BPH may be due to receptor hyperstimulation by increased local levels of Ang II in BPH. Finally, Ang II may play a functional role in modulating sympathetic transmission in the prostate. These data support the novel concept that activation of the renin-angiotensin system may be involved in the pathophysiology of BPH.