ANG II AT2 receptor modulates AT1 receptor-mediated descending vasa recta endothelial Ca2+ signaling

We tested whether the respective angiotensin type 1 (AT(1)) and 2 (AT(2)) receptor subtype antagonists losartan and PD-123319 could block the descending vasa recta (DVR) endothelial intracellular calcium concentration ([Ca(2+)](i)) suppression induced by ANG II. ANG II partially reversed the increase in [Ca(2+)](i) generated by cyclopiazonic acid (CPA; 10(-5) M), acetylcholine (ACh; 10(-5) M), or bradykinin (BK; 10(-7) M). Losartan (10(-5) M) blocked that effect. When vessels were treated with ANG II before stimulation with BK and ACh, concomitant AT(2) receptor blockade with PD-123319 (10(-8) M) augmented the suppression of endothelial [Ca(2+)](i) responses. Similarly, preactivation with the AT(2) receptor agonist CGP-42112A (10(-8) M) prevented AT(1) receptor stimulation with ANG II + PD-123319 from suppressing endothelial [Ca(2+)](i). In contrast to endothelial [Ca(2+)](i) suppression by ANG II, pericyte [Ca(2+)](i) exhibited typical peak and plateau [Ca(2+)](i) responses that were blocked by losartan but not PD-123319. DVR vasoconstriction by ANG II was augmented when AT(2) receptors were blocked with PD-123319. Similarly, AT(2) receptor stimulation with CGP-42112A delayed the onset of ANG II-induced constriction. PD-123319 alone (10(-5) M) showed no AT(1)-like action to constrict microperfused DVR or increase pericyte [Ca(2+)](i). We conclude that ANG II suppression of endothelial [Ca(2+)](i) and stimulation of pericyte [Ca(2+)](i) is mediated by AT(1) or AT(1)-like receptors. Furthermore, AT(2) receptor activation opposes ANG II-induced endothelial [Ca(2+)](i) suppression and abrogates ANG II-induced DVR vasoconstriction.     

Angiotensin II type 2 receptor-mediated duodenal mucosal alkaline secretion in the rat

The aims of this study were to elucidate the distribution of angiotensin receptors (AT(1) and AT(2)) in the duodenal wall and to investigate whether AT(2) receptors are involved in the regulation of duodenal mucosal alkaline secretion, which is of importance for the mucosal defense against gastric acid. Immunohistochemistry was used to locate AT(1) and AT(2) receptors in chloralose-anesthetized rats. Duodenal mucosal alkaline output was measured by use of in situ pH-stat titration. Immunohistochemistry demonstrated a distinct staining for both AT(1) and AT(2) receptors in the lamina propria of the villi and also for AT(1) receptors in the muscularis interna. When angiotensin II was infused in the presence of the AT(1) receptor antagonist losartan, mucosal alkaline secretion increased by ~50%. This response was inhibited by the AT(2) receptor antagonist PD-123319. The AT(2) receptor agonist CGP-42112A increased mucosal alkaline secretion by ~50%. This increase was absent in the presence of PD-123319 but not in the presence of losartan or the local anesthetic lidocaine. We conclude that angiotensin II stimulates duodenal mucosal alkaline secretion by activation of AT(2) receptors located in the duodenal mucosa/submucosa.     

Central angiotensin II AT1 receptors mediate fetal swallowing and pressor responses in the near-term ovine fetus

Swallowed volumes in the fetus are greater than adult values (per body weight) and serve to regulate amniotic fluid volume. Central ANG II stimulates swallowing, and nonspecific ANG II receptor antagonists inhibit both spontaneous and ANG II-stimulated swallowing. In the adult rat, AT1 receptors mediate both stimulated drinking and pressor activities, while the role of AT2 receptors is controversial. As fetal brain contains increased ANG II receptors compared with the adult brain, we sought to investigate the role of both AT1 and AT2 receptors in mediating fetal swallowing and pressor activities. Five pregnant ewes with singleton fetuses (130 +/- 1 days) were prepared with fetal vascular and lateral ventricle (LV) catheters and electrocorticogram and esophageal electromyogram electrodes and received three studies over 5 days. On day 1 (ANG II), following a 2-h basal period, 1 ml artificial cerebrospinal fluid (aCSF) was injected in the LV. At time 4 h, ANG II (6.4 microg) was injected in the LV, and the fetus was monitored for a final 2 h. On day 3, AT1 receptor blocker (losartan 0.5 mg) was administered at 2 h, and ANG II plus losartan was administered at 4 h. On day 5, AT2 receptor blocker (PD-123319; 0.8 mg was administered at 2 h and ANG II plus PD-123319 at 4 h. In the ANG II study, LV injection of ANG II significantly increased fetal swallowing (0.9 +/- 0.1 to 1.4 +/- 0.1 swallows/min; P < 0.05). In the losartan study, basal fetal swallowing significantly decreased in response to blockade of AT1 receptors (0.9 +/- 0.1 to 0.4 +/- 0.1 swallows/min; P < 0.05), while central injection of ANG II in the presence of AT1 receptor antagonism did not increase fetal swallowing (0.6 +/- 0.1 swallows/min). In the PD-123319 study, basal fetal swallowing did not change in response to blockade of AT2 receptor (0.9 +/- 0.1 swallows/min), while central injection of ANG II in the presence of AT2 blockade significantly increased fetal swallowing (1.5 +/- 0.1 swallows/min; P < 0.05). ANG II caused significant pressor responses in the control and PD-123319 studies but no pressor response in the presence of AT1 blockade. These data demonstrate that in the near-term ovine fetus, AT1 receptor but not AT2 receptors accessible via CSF contribute to dipsogenic and pressor responses.     

Role of AT2 receptor in the brain in regulation of blood pressure and water intake

The effects of intracerebroventricular (ICV) injection of angiotensin II (ANG II) on blood pressure and water intake were examined with the use of ANG II receptor-deficient mice. ICV injection of ANG II increased systolic blood pressure in a dose-dependent manner in wild-type (WT) mice and ANG type 2 AT(2) receptor null (knockout) (AT(2)KO) mice; however, this increase was significantly greater in AT(2)KO mice than in WT mice. The pressor response to a central injection of ANG II in WT mice was inhibited by ICV preinjection of the selective AT(1) receptor blocker valsartan but exaggerated by the AT(2) receptor blocker PD-123319. ICV injection of ANG II also increased water intake. It was partly but significantly suppressed both in AT(2)KO and AT(1)aKO mice. Water intake in AT(2)/AT(1)aKO mice did not respond to ICV injection of ANG II. Both valsartan and PD-123319 partly inhibited water intake in WT mice. These results indicate an antagonistic action between central AT(1)a and AT(2) receptors in the regulation of blood pressure, but they act synergistically in the regulation of water intake induced by ANG II.     

ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells

Angiotensin II(ANG II) has long been known for its pressor and growth-promotingeffects, which are both mediated by theAT₁ receptor. By contrast, theAT₂ receptor has recently beenreported to mediate inhibition of proliferation through as yetundefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks thecells in G₁ phase. Consistent withthis, ANG II inhibits cyclin D₁ expression and cyclinD₁-associated kinase activity. Theantimitogenic effect of ANG II is partly mimicked by theAT₂-selective agonist CGP-42112.It is also blocked partly and in an additive fashion by theAT₁- andAT₂-selective antagonists losartanand PD-123319, indicating the contribution of both receptor subtypes tothis response. AT₁-dependentantiproliferation is selectively blocked by the cyclooxygenaseinhibitor indomethacin and restored by prostaglandin E₂, whereasAT₂-receptor-mediated inhibitionof growth is suppressed by the tyrosine phosphatase inhibitorsorthovanadate and bpV(pic). Both pathways are, however,pertussis toxin sensitive. We hypothesize that, in fasciculatacells, the AT₁ receptor inhibitsbFGF-induced proliferation by stimulating prostaglandin synthesis,whereas the AT₂ receptor mediatesits effect through a pathway that requires protein tyrosine phosphataseactivation.

     

Angiotensin II potentiates adrenergic and muscarinic modulation of guinea pig intracardiac neurons

The intrinsic cardiac plexus represents a major peripheral integration site for neuronal, hormonal, and locally produced neuromodulators controlling efferent neuronal output to the heart. This study examined the interdependence of norepinephrine, muscarinic agonists, and ANG II, to modulate intrinsic cardiac neuronal activity. Intracellular voltage recordings from whole-mount preparations of the guinea pig cardiac plexus were used to determine changes in active and passive electrical properties of individual intrinsic cardiac neurons. Application of either adrenergic or muscarinic agonists induced changes in neuronal resting membrane potentials, decreased afterhyperpolarization duration of single action potentials, and increased neuronal excitability. Adrenergic responses were inhibited by removal of extracellular calcium ions, while muscarinic responses were inhibited by application of TEA. The adrenergic responses were heterogeneous, responding to a variety of receptor-specific agonists (phenylephrine, clonidine, dobutamine, and terbutaline), although α-receptor agonists produced the most frequent responses. Application of ANG II alone produced a significant increase in excitability, while application of ANG II in combination with either adrenergic or muscarinic agonists produced a much larger potentiation of excitability. The ANG II-induced modulation of firing was blocked by the angiotensin type 2 (AT(2)) receptor inhibitor PD 123319 and was mimicked by the AT(2) receptor agonist CGP-42112A. AT(1) receptor blockade with telmasartin did not alter neuronal responses to ANG II. These data demonstrate that ANG II potentiates both muscarinically and adrenergically mediated activation of intrinsic cardiac neurons, doing so primarily via AT(2) receptor-dependent mechanisms. These neurohumoral interactions may be fundamental to regulation of neuronal excitability within the intrinsic cardiac nervous system.     

Acute and chronic angiotensin-(1-7) restores vasodilation and reduces oxidative stress in mesenteric arteries of salt-fed rats

This study determined the effect of ANG-(1-7) on salt-induced suppression of endothelium-dependent vasodilatation in the mesenteric arteries of male Sprague-Dawley rats. Chronic intravenous infusion of ANG-(1-7), oral administration of the nonpeptide mas receptor agonist AVE-0991, and acute preincubation of the arteries with ANG-(1-7) and AVE-0991 all restored vasodilator responses to both ACh and histamine that were absent in the arteries of rats fed a high-salt (4% NaCl) diet. The protective effects of ANG-(1-7) and AVE-0991 were inhibited by acute or chronic administration of the mas receptor antagonist A-779, the ANG II type 2 (AT(2)) receptor blocker PD-123319, or N-nitro-l-arginine methyl ester, but not the ANG II type 1 receptor antagonist losartan. Preincubation with the antioxidant tempol or the nitric oxide (NO) donor diethylenetriamine NONOate and acute and chronic administration of the AT(2) receptor agonist CGP-42112 mimicked the protective effect of ANG-(1-7) to restore vascular relaxation. Acute preincubation with ANG-(1-7) and chronic infusion of ANG-(1-7) ameliorated the elevated superoxide levels in rats fed a high-salt diet, but the expression of Cu/Zn SOD and Mn SOD enzyme proteins in the vessel wall was unaffected by ANG-(1-7) infusion. These results indicate that both acute and chronic systemic administration of ANG-(1-7) or AVE-0991 restore endothelium-dependent vascular relaxation in salt-fed Sprague-Dawley rats by reducing vascular oxidant stress and enhancing NO availability via mas and AT(2) receptors. These findings suggest a therapeutic potential for mas/AT(2) receptor activation in preventing the vascular oxidant stress and endothelial dysfunction associated with elevated dietary salt intake.     

Both AT₁ and AT₂ receptors mediate proliferation and migration of porcine vascular smooth muscle cells

Angiotensin receptor antagonists have shown clinical promise in modulating vascular disease, in part by limiting smooth muscle cell proliferation and migration. The majority of studies examining the contribution of these receptors have been undertaken in cells derived from rat aorta, which primarily express the ANG II type 1 (AT(1)) receptor. This investigation studied the relative contribution of AT(1) and ANG II type 2 (AT(2)) receptors to the mitogenic program of porcine smooth muscle cells. Smooth muscle cells were derived from porcine coronary artery explants. The presence of both AT(1) and AT(2) receptors was demonstrated through ligand binding and RT-PCR analysis. Biochemical and cellular markers of proliferation were monitored in the presence of selective receptor antagonists. Smooth muscle cell migration was measured using both wound healing and Boyden chamber migration assays. Visualization of the AT(1) and AT(2) receptors in growing and quiescent porcine smooth muscle cells with epifluorescence microscopy demonstrated that their subcellular distribution varied with growth state. An examination with several growth assays revealed that both AT(1)-specific losartan and AT(2)-specific PD-123319 receptor antagonists inhibited ANG II-stimulated RNA and DNA synthesis, PCNA expression, and hyperplasia. ANG II induced both directional and nondirectional cell migration. AT(1) receptor antagonist treatment significantly decreased ANG II-induced directional migration only, whereas AT(2) receptor antagonist treatment significantly reduced both modes of migration. Interestingly, the focal adhesion kinase inhibitor PF-573228 also blocked migration but not proliferation. Furthermore, focal adhesion kinase activation in response to ANG II was prevented only by PD-123319, indicating that this activation is downstream of the AT(2) receptor. The observed role of the AT(2) receptor in ANG II-induced migration was confirmed with smooth muscle cells depleted of the AT(2) receptor with short hairpin RNA treatment.     

Angiotensin-II subtype 2 receptor agonist (CGP-42112) inhibits catecholamine biosynthesis in cultured porcine adrenal medullary chromaffin cells

Angiotensin II subtype 2 receptor (AT(2)-R) is abundantly expressed in adrenal medullary chromaffin cells. However, the physiological roles of AT(2)-R in chromaffin cells remain to be clarified. Therefore, we investigated the effects of CGP42112 (AT(2)-R agonist) on catecholamine biosynthesis in cultured porcine adrenal medullary cells. We initially confirmed AT(2)-R was predominantly expressed in porcine adrenal medullary cells by [(125)I]-Ang II binding studies. CGP42112 (>==1 nM) significantly inhibited cGMP production from the basal value. Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the biosynthesis of catecholamine, and its activity is regulated by both TH-enzyme activity and TH-synthesis. CGP42112 (>==1 nM) significantly inhibited TH-enzyme activity from the basal value. These inhibitory effects of CGP42112 on TH-enzyme activity and-cGMP production were abolished by PD123319 (AT(2)-R antagonist) while CV-11974 (AT(1)-R antagonist) was ineffective. We also tested whether decrease of cGMP is involved in the inhibitory effect of CGP42112 on TH-enzyme activity. Pretreatment of 8-Br-cGMP (membrane-permeable cGMP analogue) prevented the inhibitory effect of CGP 42112 on TH-enzyme activity. Similar to that of TH-enzyme activity, CGP42112 (>==1 nM) significantly reduced TH-mRNA and TH-protein level from the basal value, and these inhibitory effects were abolished by PD123319 but not CV-11974. These findings demonstrate that CGP 42112 reduces both TH-enzyme activity and TH-synthesis and that these inhibitory effects could be mediated by decrease of cGMP production.     

Biochemical Characterization of Two Distinct Angiotensin AT2 Receptor Populations in Murine Neuroblastoma N1E-115 Cells

Abstract: The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT₂ subtype. However, displacement of ¹²⁵I-Angll with the AT₂ nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT₂ receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound ¹²⁵I-Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ～80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT₂ receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT₁-selective antagonist Losartan. However, whereas the nonpeptidic AT₂-selective antagonist PD-123319 completely displaced the binding of ¹²⁶I-Angll from peak I in a monophasic fashion (IC₅₀= 9.1 ± 4.1 nM; mean ± SEM; n = 3), PD-123319 was much less effective in displacing ¹²⁵I-Angll from peak III (IC₅₀= 196 β 27 nM; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in ¹²⁵I-Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT₂ receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated ¹²⁵I-Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT₂ receptors.     

Functional involvement of angiotensin AT2 receptor in adrenal catecholamine secretion in vivo.

The aim of the present study was to analyse modulations of adrenal catecholamine secretion from the adrenal gland of anesthetized dogs in response to locally administered angiotensin II (AngII) in the presence of either PD 123319 or CGP 42112, both of which are highly specific and selective ligands to angiotensin AT2 receptor. Plasma concentrations of epinephrine and norepinephrine in adrenal venous and aortic blood were quantified by a high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) method. Adrenal venous blood flow was measured by gravimetry. Local administration of AngII (0.05 microg, 0.1 microM) to the left adrenal gland increased adrenal gland catecholamine output more than 30 times that found in nonstimulated states. Administration of either PD 123319 (0.085 microg (0.23 microM) to 8.5 microg (23 microM)) or CGP 42112 (0.005 microg (0.01 microM) to 5 microg (10 microM)) did not affect the basal catecholamine output significantly. The increase in adrenal catecholamine output in response to AngII was inhibited by approximately 80% following the largest dose of PD 123319. CGP 42112 significantly attenuated the catecholamine response to AngII by approximately 70%. PD 123319 and CGP 42112 were devoid of any agonist actions with respect to catecholamine output by the adrenal gland in vivo. Furthermore, both PD 123319 and CGP 42112 inhibited the increase in adrenal catecholamine secretion induced by local administration of AngII. The present study suggests that AT2 receptors play a role in mediating catecholamine secretion by the adrenal medulla in response to AngII receptor agonist administration in vivo.     

AT(1) and AT(2) receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts

We assessed ANG II type 1 (AT(1)) and type 2 (AT(2)) receptor (R) expression and functional recovery after ischemia-reperfusion with or without AT(1)R/AT(2)R blockade in isolated working rat hearts. Groups of six hearts were subjected to global ischemia (30 min) followed by reperfusion (30 min) and exposed to no drug and no ischemia-reperfusion (control), ischemia-reperfusion and no drug, and ischemia-reperfusion with losartan (an AT(1)R antagonist; 1 micromol/l), PD-123319 (an AT(2)R antagonist; 0.3 micromol/l), N(6)-cyclohexyladenosine (CHA, a cardioprotective adenosine A(1) receptor agonist; 0.5 micromol/l as positive control), enalaprilat (an ANG-converting enzyme inhibitor; 1 micromol/l), PD-123319 + losartan, ANG II (1 nmol/l), or ANG II + losartan. Compared with controls, ischemia-reperfusion decreased AT(2)R protein (Western immunoblots) and mRNA (Northern immunoblots, RT-PCR) and impaired functional recovery. PD-123319 increased AT(2)R protein and mRNA and improved functional recovery. Losartan increased AT(1)R mRNA (but not AT(1)R/AT(2)R protein) and impaired recovery. Other groups (except CHA) did not improve recovery. The results suggest that, in isolated working hearts, AT(2)R plays a significant role in ischemia-reperfusion and AT(2)R blockade induces increased AT(2)R protein and cardioprotection.     

Angiotensin II type 1 receptor blockade attenuates renal fibrogenesis in an immune-mediated nephritic kidney through counter-activation of angiotensin II type 2 receptor

The relative roles of angiotensin II (Ang II) type 1 receptor (AT(1)R) and Ang II type 2 receptor (AT(2)R) in immune-mediated nephritis are unknown, and the effect of the blockade of AT(1)R and its indirect counter-activation of AT(2)R relative to the anti-fibrotic action in this disease is unclear. To address this question, we studied the role of AT(1)R and AT(2)R in anti-glomerular basement membrane nephritis in SJL mice. Groups of mice were treated with either an AT(1)R antagonist (CGP-48933; CGP group), an AT(2)R antagonist (PD-123319; PD group), both (CGP/PD group), or a vehicle (PCt group) from Day 29 to 56. At Day 56 post-treatment, fibrosis-related parameters such as interstitial matrix deposition, and the expression of genes of TGF-beta1, plasminogen activator inhibitor-1, and type I collagen were significantly reduced in the kidney in the CGP group. There were no significant effects on these parameters in the PD group. However, this anti-fibrotic action by CGP-48933 was totally abolished by co-treatment with PD-123319 in the CGP/PD group. The gene expression of renin was significantly increased in the kidneys in the CGP and CGP/PD groups, suggesting that CGP-48933 had increased Ang II generation in those groups. In conclusion, counter-activation of AT(2)R by increased Ang II under AT(1)R blockade likely conferred an anti-fibrotic protection in this model.     

Mechanisms of angiotensin II-induced expression of B2 kinin receptors

Although the primary roles of the kallikreinkinin system and the renin-angiotensin system are quite divergent, they are often intertwined under pathophysiological conditions. We examined the effect of ANG II on regulation of B(2) kinin receptors (B2KR) in vascular cells. Vascular smooth muscle cells (VSMC) were treated with ANG II in a concentration (10(-9)-10(-6) M)- and time (0-24 h)-dependent manner, and B2KR protein and mRNA levels were measured by Western blots and PCR, respectively. A threefold increase in B2KR protein levels was observed as early as 6 h, with a peak response at 10(-7) M. ANG II (10(-7) M) also increased B2KR mRNA levels twofold 4 h after stimulation. Actinomycin D suppressed the increase in B2KR mRNA and protein levels induced by ANG II. To elucidate the receptor subtype involved in mediating this regulation, VSMC were pretreated with losartan (AT(1) receptor antagonist) and/or PD-123319 (AT(2) receptor antagonist) at 10 microM for 30 min, followed by ANG II (10(-7) M) stimulation. Losartan completely blocked the ANG II-induced B2KR increase, whereas PD-123319 had no effect. In addition, expression of B2KR mRNA levels was decreased in AT(1A) receptor knockout mice. Finally, to determine whether ANG II stimulates B2KR expression via activation of the MAPK pathway, VSMC were pretreated with an inhibitor of p42/p44(mapk) (PD-98059) and/or an inhibitor of p38(mapk) (SB-202190), followed by ANG II (10(-7) M) for 24 h. Selective inhibition of the p42/p44(mapk) pathway significantly blocked the ANG II-induced increase in B2KR expression. These findings demonstrate that ANG II regulates expression of B2KR in VSMC and provide a rationale for studying the interaction between ANG II and bradykinin in the pathogenesis of vascular dysfunction.     

Angiotensin II AT(2) receptor inhibits smooth muscle cell migration via fibronectin cell production and binding

To explore the vascular function of the angiotensin II (ANG II) AT(2) receptor subtype (AT(2)R), we generated a vascular smooth muscle cell (SMC) line expressing the AT(2)R (SMC-vAT(2)). The involvement of AT(2)R in the motility response of SMCs was examined in SMC-vAT(2) cells and their controls (SMC-v) cultured on either laminin or fibronectin matrix proteins with the agarose drop technique. All experiments were conducted in the presence of a saturating concentration of losartan to inactivate the AT(1)R subtype. Under basal conditions, both cell lines migrated outside drops, but on laminin only. Treatment with ANG II significantly inhibited the migration of SMC-vAT(2) but not SMC-v cells, and this effect was prevented by the AT(2)R antagonist CGP-42112A. The decreased migration of SMC-vAT(2) was not associated with changes in cell growth, cytoskeleton stiffness, or smooth muscle actin, desmin, and tenascin expression. However, it was correlated with increased synthesis and binding of fibronectin. Both responses were prevented by incubation with selective AT(2)R antagonists. Addition of GRGDTP peptide, which prevents cell attachment of fibronectin, reversed the AT(2)R inhibitory effect on SMC-vAT(2) migration. These results suggest that activated ANG II AT(2)R inhibits SMC migration via cellular fibronectin synthesis and associated cell binding.     

Angiotensin receptor subtype AT(1) mediates alveolar epithelial cell apoptosis in response to ANG II

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT(1) and AT(2) (Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 276: L885-L889, 1999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT(1)-selective receptor antagonists L-158809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of 10(-8) M and 10(-7) M, respectively. The inhibition was concentration dependent with IC(50) of 10(-12) M and 10(-11) M on the primary rat AEC. In contrast, the AT(2)-selective antagonists PD-123319 or PD-126055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT(1), despite the expression of mRNAs for both AT(1) and AT(2).     

Localized accumulation of angiotensin II and production of angiotensin-(1-7) in rat luteal cells and effects on steroidogenesis

These studies aim to investigate subcellular distribution of angiotensin II (ANG II) in rat luteal cells, identify other bioactive angiotensin peptides, and investigate a role for angiotensin peptides in luteal steroidogenesis. Confocal microscopy showed ANG II distributed within the cytoplasm and nuclei of luteal cells. HPLC analysis showed peaks that eluted with the same retention times as ANG-(1-7), ANG II, and ANG III. Their relative concentrations were ANG II >or= ANG-(1-7) > ANG III, and accumulation was modulated by quinapril, an inhibitor of angiotensin-converting enzyme (ACE), Z-proprolinal (ZPP), an inhibitor of prolyl endopeptidase (PEP), and parachloromercurylsulfonic acid (PCMS), an inhibitor of sulfhydryl protease. Phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, did not affect peptide accumulation. Quinapril, ZPP, PCMS, and PMSF, as well as losartan and PD-123319, the angiotensin receptor type 1 (AT1) and type 2 (AT2) receptor antagonists, were used in progesterone production studies. ZPP significantly reduced luteinizing hormone (LH)-dependent progesterone production (P < 0.05). Quinapril plus ZPP had a greater inhibitory effect on LH-stimulated progesterone than either inhibitor alone, but this was not reversed by exogenous ANG II or ANG-(1-7). Both PCMS and PMSF acutely blocked LH-stimulated progesterone, and PCMS blocked LH-sensitive cAMP accumulation. Losartan inhibited progesterone production in permeabilized but not intact luteal cells and was reversed by ANG II. PD-123319 had no significant effect on luteal progesterone production in either intact or permeabilized cells. These data suggest that steroidogenesis may be modulated by angiotensin peptides that act in part through intracellular AT1 receptors.     

Renal effects of angiotensin II receptor subtype 1 and 2-selective ligands injected into the paraventricular nucleus of conscious rats.

We determined the effects of losartan and CGP42112A (selective ligands of the AT1 and AT2 angiotensin receptors, respectively) and salarasin (a relatively nonselective angiotensin receptor antagonist) on urinary volume and urinary sodium and potassium excretion induced by administration of angiotensin II (ANG II) into the paraventricular nucleus (PVN) of conscious rats. Both the AT1 and AT2 ligands and salarasin administered in the presence of ANG II elicited a concentration-dependent inhibition of urine excretion, but losartan inhibited only 75% of this response. The IC50 for salarasin, CGP42112A, and losartan was 0.01, 0.05, and 6 nM, respectively. Previous treatment with saralasin, CGP42112A and losartan competitively antagonized the natriuretic responses to PVN administration of ANG II, and the IC50 values were 0.09, 0.48, and 10 nM, respectively. The maximum response to losartan was 65% of that obtained with saralasin. Pretreatment with saralasin, losartan, and CGP42112A injected into the PVN caused shifts to the right of the concentration-response curves, but the losartan concentrations were disproportionately greater compared with salarasin or CGP42112A. The IC50 values were 0.06, 0.5, and 7.0 for salarasin, CGP42112A, and losartan, respectively. These results suggest that both AT1 and AT2 receptor subtypes in the PVN are involved in ANG II-related urine, sodium, and potassium excretion, and that the inhibitory responses to AT2 blockade are predominant.     

Glial angiotensinogen regulates brain angiotensin II receptors in transgenic rats TGR(ASrAOGEN)

TGR(ASrAOGEN)680, a newly developed transgenic rat line with specific downregulation of astroglial synthesis of angiotensinogen, exhibits decreased brain angiotensinogen content associated with a mild diabetes insipidus and lower blood pressure. Autoradiographic experiments were performed on TGR(ASrAOGEN) (TG) and Sprague-Dawley (SD) control rats to quantify AT(1) and AT(2) receptor-binding sites in different brain nuclei and circumventricular organs. Dose-response curves for drinking response to intracerebroventricular injections of ANG II were compared between SD and TG rats. In most of the regions inside the blood-brain barrier [paraventricular nucleus (PVN), piriform cortex, lateral olfactory tract (LOT), and lateral preoptic area (LPO)], AT(1) receptor binding (sensitive to CV-11974) was significantly higher in TG compared with SD. In contrast, in the circumventricular organs investigated [subfornical organ (SFO) and area postrema], AT(1) receptor binding was significantly lower in TG. AT(2) receptors (binding sensitive to PD-123319) were detected at similar levels in the inferior olive (IO) of both strains. Angiotensin-binding sites sensitive to both CV-11974 and PD-123319 were detected in the LPO of SD rats and specifically upregulated in LOT, IO, and most notably PVN and SFO of TG. The dose-response curve for water intake after intracerebroventricular injections showed a higher sensitivity to ANG II of TG (EC(50) = 3.1 ng) compared with SD (EC(50) = 11.2 ng), strongly suggesting that the upregulation of AT(1) receptors inside the blood-brain barrier of TG rats is functional. Finally, we showed that downregulation of angiotensinogen synthesized by astroglial cells differentially regulates angiotensin receptor subtypes inside the brain and in circumventricular organs.