The subtype 2 (AT2) angiotensin receptor mediates renal production of nitric oxide in conscious rats.

The angiotensin AT2 receptor modulates renal production of cyclic guanosine 3',5'-monophosphate (cGMP; J. Clin. Invest. 1996. 97:1978-1982). In the present study, we hypothesized that angiotensin II (Ang II) acts at the AT2 receptor to stimulate renal production of nitric oxide leading to the previously observed increase in cGMP. Using a microdialysis technique, we monitored changes in renal interstitial fluid (RIF) cGMP in response to intravenous infusion of the AT2 receptor antagonist PD 123319 (PD), the AT1 receptor antagonist Losartan, the nitric oxide synthase (NOS) inhibitor nitro--arginine-methyl-ester (-NAME), the specific neural NOS inhibitor 7-nitroindazole (7-NI), or Ang II individually or combined in conscious rats during low or normal sodium balance. Sodium depletion significantly increased RIF cGMP. During sodium depletion, both PD and -NAME caused a similar decrease in RIF cGMP. Combined administration of PD and -NAME decreased RIF cGMP to levels observed with PD or -NAME alone or during normal sodium intake. During normal sodium intake, Ang II caused a twofold increase in RIF cGMP. Neither PD nor -NAME, individually or combined, changed RIF cGMP. Combined administration of Ang II and either PD or -NAME produced a significant decrease in RIF cGMP compared with that induced by Ang II alone. Combined administration of Ang II, PD, and -NAME blocked the increase in RIF cGMP produced by Ang II alone. During sodium depletion, 7-NI decreased RIF cGMP, but the reduction of cGMP in response to PD alone or PD combined with 7-NI was greater than with 7-NI alone. During normal sodium intake, 7-NI blocked the Ang II-induced increase in RIF cGMP. PD alone or combined with 7-NI produced a greater inhibition of cGMP than did 7-NI alone. During sodium depletion, 7-NI (partially) and -NAME (completely) inhibited RIF cGMP responses to -arginine. These data demonstrate that activation of the renin- angiotensin system during sodium depletion increases renal nitric oxide production through stimulation by Ang II at the angiotensin AT2 receptor. This response is partially mediated by neural NOS, but other NOS isoforms also contribute to nitric oxide production by this pathway.     

The subtype-2 (AT2) angiotensin receptor regulates renal cyclic guanosine 3', 5'-monophosphate and AT1 receptor-mediated prostaglandin E2 production in conscious rats.

The renal effects of angiotensin II(AII) are attributed to AT1 receptors. In contrast, the function of renal AT2 receptors in unknown. Using a microdialysis technique, we monitored changes in renal interstitial fluid (RIF) prostaglandin E2 (PGE2) and cyclic guanosine 3', 5'-monophosphate (cGMP) in response to dietary sodium (Na) depletion alone, or Na depletion or normal Na diet combined with the AT1 receptor blocker, Losartan, the AT2 receptor blocker, PD 123319 (PD), or angiotensin II, individually or combined in conscious rats. Na depletion significantly increased PGE2 and cGMP. During Na depletion, Losartan decreased PGE2 and did not change cGMP. In contrast, PD significantly increased PGE2 and decreased cGMP. Combined administration of Losartan and PD decreased PGE2 and cGMP. During normal Na diet, RIF PGE2 and cGMP increased in response to angiotensin II. Neither Losartan nor PD, individually or combined, changed RIF PGE2 or cGMP. Combined administration of angiotensin II and Losartan or PD produced a significant decrease in response of PGE2 and cGMP to angiotensin II, respectively. These data demonstrate that activation of the reninangiotensin system during Na depletion increases renal interstitial PGE2 and cGMP. The AT1 receptor mediates renal production of PGE2. The AT2 receptor mediates cGMP. AT2 blockade potentiates angiotensin-induced PGE2 production at the AT1 receptor.     

Angiotensin receptor subtype 1 mediates angiotensin II enhancement of isoproterenol-induced cyclic AMP production in preglomerular microvascular smooth muscle cells

In a previous study, we found that angiotensin (Ang) II enhances beta-adrenoceptor-induced cAMP production in cultured preglomerular microvascular smooth muscle cells (PMVSMCs) obtained from spontaneously hypertensive rats. The purpose of the present investigation was to identify the Ang receptor subtypes that mediate this effect. In our first study, we compared the ability of Ang II, Ang III, Ang (3-8), and Ang (1-7) to increase cAMP production in isoproterenol (1 microM)-treated PMVSMCs. Each peptide was tested at 0.1, 1, 10, 100, and 1000 nM. Both Ang II and Ang III increased intracellular (EC50s, 1 and 11 nM, respectively) and extracellular (EC50s, 2 and 14 nM, respectively) cAMP levels in a concentration-dependent fashion. In contrast, Ang (3-8) and Ang (1-7) did not enhance either intracellular or extracellular cAMP levels at any concentration tested. In our second study, we examined the ability of L 158809 [a selective Ang receptor subtype 1 (AT1) receptor antagonist] to inhibit Ang II (100 nM) and Ang III (100 nM) enhancement of isoproterenol (1 microM)-induced cAMP production in PMVSMCs. L 158809 (10 nM) abolished or nearly abolished (p <.001) Ang II and Ang III enhancement of isoproterenol-induced intracellular and extracellular cAMP levels. In contrast, PD 123319 (300 nM; a selective AT2 receptor antagonist) did not significantly alter Ang II enhancement of isoproterenol-induced intracellular or extracellular cAMP levels. We conclude that AT1 receptors, but not AT2, Ang (3-8), nor Ang (1-7) receptors mediate Ang II and Ang III enhancement of beta-adrenoceptor-induced cAMP production in cultured PMVSMCs.     

Modification of the pulmonary renin-angiotensin system and lung structural remodelling in congestive heart failure

Lung structural remodelling, characterized by myofibroblast proliferation and collagen deposition, contributes to impaired functional capacity in CHF (congestive heart failure). As the lung is the primary site for the formation of Ang II (angiotensin II), local modifications of this system could contribute to lung remodelling. Rats with CHF, induced following myocardial infarction (MI) via coronary artery ligation, were compared with sham-operated controls. The MI group developed lung remodelling as confirmed by morphometric measurements and immunohistochemistry. Pulmonary Ang II concentrations increased more than 6-fold (P<0.01), and AT1 (Ang II type 1) receptor expression was elevated by 3-fold (P<0.01) with evidence of distribution in myofibroblasts. AT2 (Ang II type 2) receptor expression was unchanged. In isolated lung myofibroblasts, AT1 and AT2 receptors were expressed, and Ang II stimulated proliferation as measured by [3H]thymidine incorporation. In normal rats, chronic intravenous infusion of Ang II (0.5 mg.kg(-1) of body weight.day(-1)) for 28 days significantly increased mean arterial pressure (P<0.05), without pulmonary hypertension, lung remodelling or a change in AT1 receptor expression. We conclude that there is a modification of the pulmonary renin-angiotensin system in CHF, with increased Ang II levels and AT1 receptor expression on myofibroblasts. Although this may contribute to lung remodelling, the lack of effect of increased plasma Ang II levels alone suggests the importance of local pulmonary Ang II levels combined with the effect of other factors activated in CHF.     

Angiogenesis and angiotensin II: differential postnatal angiogenic activities unmasked by gene-engineered mouse models of angiotensin AT1 and AT2 receptor subtypes

We tested angiogenic activities of angiotensin II(Ang II) in ischemic hindlimbs using AT1 receptor(AT1R)-knock out(KO), AT2R-KO, wild-type(WT) mice. METHODS AND RESULTS: Angiogenesis was evaluated three weeks after unilateral hindlimb ischemia by laser Doppler perfusion(LDP) and capillary density. The ischemia/normal LDP ratio was markedly(p < 0.001) decreased in AT1R-KO(54 +/- 5% recovery) and AngII infusion-AT1R-KO(43 +/- 3%) than in WT(71 +/- 6%). In contrast, ischemia/normal LDP ratio was significantly(p < 0.01) increased in AT2R-KO(82 +/- 5%) and AngII infusion-AT2R-KO(96 +/- 6%) than in WT(71 +/- 6%). AT1R-KO and AngII infusion -AT1R-KO mice displayed lower capillary densities than WT(15 +/- 3, 11 +/- 3 vs 24 +/- 3 per field; p < 0.001). CONCLUSION: Ischemia in skeletal muscle causes upregulation of AT1R and AT2R expression, which positively and negatively modulates VEGF expression. This VEGF regulation via AngII receptor subtypes is closely involved in postnatal angiogenesis in ischemic limbs.     

Cardiac-specific overexpression of AT1 receptor mutant lacking G alpha q/G alpha i coupling causes hypertrophy and bradycardia in transgenic mice

Ang II type 1 (AT1) receptors activate both conventional heterotrimeric G protein-dependent and unconventional G protein-independent mechanisms. We investigated how these different mechanisms activated by AT1 receptors affect growth and death of cardiac myocytes in vivo. Transgenic mice with cardiac-specific overexpression of WT AT1 receptor (AT1-WT; Tg-WT mice) or an AT1 receptor second intracellular loop mutant (AT1-i2m; Tg-i2m mice) selectively activating G(alpha)q/G(alpha)i-independent mechanisms were studied. Tg-i2m mice developed more severe cardiac hypertrophy and bradycardia coupled with lower cardiac function than Tg-WT mice. In contrast, Tg-WT mice exhibited more severe fibrosis and apoptosis than Tg-i2m mice. Chronic Ang II infusion induced greater cardiac hypertrophy in Tg-i2m compared with Tg-WT mice whereas acute Ang II administration caused an increase in heart rate in Tg-WT but not in Tg-i2m mice. Membrane translocation of PKCepsilon, cytoplasmic translocation of G(alpha)q, and nuclear localization of phospho-ERKs were observed only in Tg-WT mice while activation of Src and cytoplasmic accumulation of phospho-ERKs were greater in Tg-i2m mice, consistent with the notion that G(alpha)q/G(alpha)i-independent mechanisms are activated in Tg-i2m mice. Cultured myocytes expressing AT1-i2m exhibited a left and upward shift of the Ang II dose-response curve of hypertrophy compared with those expressing AT1-WT. Thus, the AT1 receptor mediates downstream signaling mechanisms through G(alpha)q/G(alpha)i-dependent and -independent mechanisms, which induce hypertrophy with a distinct phenotype.     

Angiotensin II-induced hypertension in the rat. Effects on the plasma concentration, renal excretion, and tissue release of prostaglandins.

We examined in rats the effects of intraperitoneal angiotensin II (AII) infusion for 12 d on urinary excretion, plasma concentration, and in vitro release of prostaglandin (PG) E2 and 6-keto-PGF1 alpha, a PGI2 metabolite. AII at 200 ng/min increased systolic blood pressure (SBP) progressively from 125 +/- 3 to 170 +/- 9 mmHg (P less than 0.01) and elevated fluid intake and urine volume. Urinary 6-keto-PGF1 alpha excretion increased from 38 +/- 6 to 55 +/- 5 and 51 +/- 7 ng/d (P less than 0.05) on days 8 and 11, respectively, of AII infusion, but urinary PGE2 excretion did not change. Relative to a control value of 129 +/- 12 pg/ml in vehicle-infused (V) rats, arterial plasma 6-keto-PGF1 alpha concentration increased by 133% (P less than 0.01) with AII infusion. Aortic rings from AII-infused rats released more 6-keto-PGF1 alpha (68 +/- 7 ng/mg) during 15-min incubation in Krebs solution than did rings from V rats (40 +/- 3 ng/mg); release of PGE2, which was less than 1% of that of 6-keto-PGF1 alpha, was also increased. Slices of inner renal medulla from AII-infused rats released more 6-keto-PGF1 alpha (14 +/- 1 ng/mg) during incubation than did slices from V rats (8 +/- 1 ng/mg, P less than 0.05), but PGE2 release was not altered. In contrast, AII infusion did not alter release of 6-keto-PGF1 alpha or PGE2 from inferior vena cava segments or from renal cortex slices. Infusion of AII at 125 ng/min also increased SBP, plasma 6-keto-PGF1 alpha concentration, and in vitro release of 6-keto-PGF1 alpha from rings of aorta and renal inner medulla slices; at 75 ng/min AII had no effect. SBP on AII infusion day 11 correlated positively with both 6-keto-PGF1 alpha plasma concentration (r = 0.54) and net aortic ring release (r = 0.70) when data from all rats were combined. We conclude that augmentation of PGI2 production is a feature of AII-induced hypertension. The enhancement of PGI2 production may be an expression of nonspecific alteration in vascular structure and metabolic functions during AII-induced hypertension, as well as the result of a specific effect of the peptide on the arachidonate-prostaglandin system.     

Interleukin‐10 negatively modulates extracellular signal‐regulated kinases 1 and 2 in aorta from hypertensive mouse induced by angiotensin II infusion

The activation of extracellular signal‐regulated kinase 1 and 2 (ERK 1/2) pathway promotes increased vascular contractility in angiotensin II (Ang II)‐induced hypertensive mice. Interleukin‐10 (IL‐10) is an immune‐regulatory cytokine with the ability to prevent vascular hypercontractility during hypertension. We hypothesized that IL‐10 would downregulate vascular ERK 1/2 activation during Ang II‐induced hypertension. Wild‐type (WT) or IL‐10 knockout (IL‐10^?/?) mice received Ang II infusion (90 ηg.min) or vehicle (saline), via osmotic mini‐pumps (0.25 μL/h for 14 days), whereas another WT group were infused with exogenous IL‐10 (0.5 ηg/min, 14 days) simultaneously, or not, with Ang II. Aortic rings were mounted in a myograph, and concentration‐response curves to phenylephrine were evaluated, in the presence or absence of ERK 1/2 inhibitor (PD98059, 10 μm , 40 min). Protein expression of vascular ERK 1/2 was determined by Western blot. Ang II infusion increased the maximal contractile response in both WT and IL‐10^?/? mice. Concomitant infusion of IL‐10 and Ang II prevented hypercontractility in the vasculature. Exogenous IL‐10 infusion prevented ERK 1/2 activation and hypercontractility, induced by Ang II. These findings suggest that IL‐10 negatively modulates ERK 1/2 activation and prevents hypercontractility during Ang II‐induced hypertension.     

Angiotensin II upregulates type-1 angiotensin II receptors in renal proximal tubule.

Angiotensin II (Ang II) is an important regulator of proximal tubule salt and water reabsorption. Recent studies indicate that rabbit proximal tubule angiotensin II receptors are the type-1 (AT1R) subtype. We studied the effect of Ang II on proximal tubule receptor expression. Rabbits were treated with either angiotensin converting enzyme inhibitors or a low salt diet to modulate endogenous Ang II levels. In captopril-treated rabbits, liver and glomerular AT1R mRNA levels increased 242 +/- 125 and 141 +/- 60%, respectively (n = 6-7; P < 0.05), as determined by quantitative PCR. In contrast, proximal tubule AT1R mRNA levels decreased 40 +/- 11% (n = 6; P < 0.05). Binding of 125I Ang II to renal cortical basolateral membranes of captopril-treated rabbits decreased from 2.9 +/- 0.55 to 1.4 +/- 0.17 fmol/mg protein (n = 6; P < 0.025). In rabbits fed a sodium chloride-deficient diet for 4 wk, AT1R mRNA levels decreased 52 +/- 11% in liver and 43 +/- 7% in glomeruli (n = 4-5; P < 0.05), whereas they increased 141 +/- 85% (n = 5; P < 0.05) in proximal tubule. In basolateral membranes from rabbits on the sodium chloride-deficient diet, specific binding of 125I Ang II increased from 2.1 +/- 0.2 to 4.3 +/- 1.1 fmol/mg protein (n = 7; P < 0.05). To determine whether Ang II directly regulates expression of proximal tubule AT1 receptors, further studies were performed in cultured proximal tubule cells grown from microdissected S1 segments of rabbit proximal tubules and immortalized by transfection with a replication-defective SV40 vector. Incubation of these cells with Ang II (10(-11) to 10(-7) M) led to concentration-dependent increases in both AT1R mRNA levels and specific 125I Ang II binding. Pretreatment with pertussis toxin inhibited Ang II stimulation of AT1R mRNA. AT1R mRNA expression was decreased by either forskolin or a nonhydrolyzable cAMP analogue (dibutryl cAMP). Simultaneous Ang II administration overcame the inhibitory effect of forskolin but not dibutryl cAMP. These results indicate that proximal tubule AT1R expression is regulated by ambient Ang II levels, and Ang II increases AT1R mRNA at least in part by decreasing proximal tubule cAMP generation through a pertussis toxin-sensitive mechanism. Upregulation of proximal tubule AT1R by Ang II may be important in mediating enhanced proximal tubule sodium reabsorption in states of elevated systemic or intrarenal Ang II.     

Neuropeptide Y stimulates renal prostaglandin synthesis in the isolated rat kidney: contribution of Ca++ and calmodulin

We have investigated the effects of neuropeptide Y (NPY) on vascular tone and renal output of prostaglandins (PGs) and the mechanism underlying these actions by examining the effects of Ca++ depletion, Ca++ channel blockers and calmodulin inhibitors in the isolated Tyrode's perfused rat kidney. Administration of NPY (0.23-2.3 nmol) into the kidney produced a dose-related renal vasoconstriction and an increase in the output of PGE2 and 6-keto-PGF1 alpha, the stable hydrolysis product of PGI2. Omission of Ca++ (1.8 mM) or addition of Ca++ channel blockers, diltiazem (60 microM) or nifedipine (1.4 microM), to the perfusion fluid abolished the effects of NPY to promote renal vasoconstriction and PG synthesis. Infusion of calmodulin inhibitors, trifluoperazine (2 microM), W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide] (20 microM) or calmidazolium (0.2 microM), attenuated the renal vasoconstriction and the increase in PG output produced by NPY (0.7 nmol). In kidneys perfused with normal Tyrode's solution, infusion of NPY, in a concentration (1.7 X 10(-8) M) that produced only a small transient increase in renal vascular tone and failed to alter the renal output of PGs, enhanced the rise in PGE2 and 6-keto-PGF1 alpha elicited by norepinephrine (0.25 nmol) but not by arginine vasopressin (0.004 nmol) or angiotensin II (0.09 nmol). The renal vasoconstriction elicited by norepinephrine and arginine vasopressin as well as by angiotensin II was enhanced by NPY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for distinct prostaglandin I2 and D2 receptors in human platelets.

Incubation of human platelet-rich plasma with prostaglandin I2 (PGI2), results in a marked increase in adenosine 3':5'-monophosphate (cAMP) that persists for at least 60 min. The persistent stimulation of cAMP levels by PGI2 can be rapidly reversed by the addition of either prostaglandin E1 or E2 (PGE1, PGE2), but not by prostaglandin D2 (PGD2). Studies of agonist-specific desensitization of cAMP accumulation show that PGE1 or PGE2 can desensitize for subsequent PGE or PGI2 activation, and that subthreshold levels of PGI2 desensitize for subsequent PGE1 stimulation. PGD2 desensitizes for consequent PGD2 activation, but not for PGE1, PGE2 or PGI2, and PGE compounds and PGI2 do not desensitize for subsequent PGD2 activation. Agonist-specific desensitization for PGI2 is not dependent on cAMP accumulation, but appears to be a consequence of receptor occupation. Support of the desensitization experiments was obtained through the use of the prostaglandin antagonist N-0164 [sodium-p-benzyl-4-[-oxo-2-(4-chlorobenzyl)-3-phenyl-propyl]phenyl phosphonate). This compound proved to be a potent antagonist of PGD2 and a weak antagonist of PGI2-stimulated cAMP accumulation. These data indicate that human platelets have distinct pharmacological receptors for both PGI2 and PGD2, and that PGE compounds may actually interact with a PGI2 receptor.     

Antihypertensive effects of selective prostaglandin E2 receptor subtype 1 targeting

Clinical use of prostaglandin synthase-inhibiting NSAIDs is associated with the development of hypertension; however, the cardiovascular effects of antagonists for individual prostaglandin receptors remain uncharacterized. The present studies were aimed at elucidating the role of prostaglandin E2 (PGE2) E-prostanoid receptor subtype 1 (EP1) in regulating blood pressure. Oral administration of the EP1 receptor antagonist SC51322 reduced blood pressure in spontaneously hypertensive rats. To define whether this antihypertensive effect was caused by EP1 receptor inhibition, an EP1-null mouse was generated using a "hit-and-run" strategy that disrupted the gene encoding EP1 but spared expression of protein kinase N (PKN) encoded at the EP1 locus on the antiparallel DNA strand. Selective genetic disruption of the EP1 receptor blunted the acute pressor response to Ang II and reduced chronic Ang II-driven hypertension. SC51322 blunted the constricting effect of Ang II on in vitro-perfused preglomerular renal arterioles and mesenteric arteriolar rings. Similarly, the pressor response to EP1-selective agonists sulprostone and 17-phenyltrinor PGE2 were blunted by SC51322 and in EP1-null mice. These data support the possibility of targeting the EP1 receptor for antihypertensive therapy.     

Angiotensin II type 2 receptor overexpression activates the vascular kinin system and causes vasodilation

Angiotensin II (Ang II) is a potent vasopressor peptide that interacts with 2 major receptor isoforms - AT1 and AT2. Although blood pressure is increased in AT2 knockout mice, the underlying mechanisms remain undefined because of the low levels of expression of AT2 in the vasculature. Here we overexpressed AT2 in vascular smooth muscle (VSM) cells in transgenic (TG) mice. Aortic AT1 was not affected by overexpression of AT2. Chronic infusion of Ang II into AT2-TG mice completely abolished the AT1-mediated pressor effect, which was blocked by inhibitors of bradykinin type 2 receptor (icatibant) and nitric oxide (NO) synthase (L-NAME). Aortic explants from TG mice showed greatly increased cGMP production and diminished Ang II-induced vascular constriction. Removal of endothelium or treatment with icatibant and L-NAME abolished these AT2-mediated effects. AT2 blocked the amiloride-sensitive Na(+)/H(+) exchanger, promoting intracellular acidosis in VSM cells and activating kininogenases. The resulting enhancement of aortic kinin formation in TG mice was not affected by removal of endothelium. Our results suggest that AT2 in aortic VSM cells stimulates the production of bradykinin, which stimulates the NO/cGMP system in a paracrine manner to promote vasodilation. Selective stimulation of AT2 in the presence of AT1 antagonists is predicted to have a beneficial clinical effect in controlling blood pressure.     

Comparison of equimolar concentrations of iloprost, prostacyclin, and prostaglandin E1 on human platelet function

Prostaglandins E1 and I2 (PGE1 and PGI2) have been shown to be potent inhibitors of platelet aggregation. We compared the antiaggregatory effect of equimolar concentrations of these two agents with that of a newly synthesized prostacyclin analogue, iloprost, and measured the effects of these agents on intracellular levels of cyclic adenosine monophosphate (cAMP) in human platelets. In addition, because the platelet inhibitory properties of prostanoids are associated with increased vasoactivity, we assessed the effects of each prostanoid on coronary flow in isolated perfused rat hearts. Concentrations ranging from 0.0001 mumol/L to 1 mumol/L of iloprost, PGI2, and PGE1 were incubated with either platelet-rich plasma or gel-filtered platelets. Greater than 90% inhibition of platelet aggregation in response to threshold concentrations of adenosine diphosphate (n = 6) and epinephrine (n = 6) was observed in all donors when 0.01 mumol/L iloprost, 0.1 mumol/L PGI2, and 1 mumol/L PGE1 were added to platelet-rich plasma. In gel-filtered platelets, at threshold concentrations of thrombin (n = 6), 90% inhibition was observed with 0.01 mumol/L iloprost. In contrast, similar inhibition to thrombin required 0.1 mumol/L PGI2, and with PGE1 it was never achieved in two donors. At 0.01 mumol/L of prostanoid, cAMP levels (n = 6) rose from a baseline value of 439 +/- 99 pmol/10(9) platelets to 1857 +/- 454 pmol/10(9) platelets for iloprost, 758 +/- 99 pmol/10(9) platelets for PGI2, and 692 +/- 199 pmol/10(9) platelets for PGE1. In addition, at 6 mumol/L, alterations in coronary flow (P greater than 0.05) were noted to be 127% of baseline values for iloprost (n = 5) and 153% for PGI2 (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV.

Recent studies from this laboratory have demonstrated that angiotensin II (Ang II) stimulates the expression of plasminogen activator inhibitor 1 (PAI-1) in cultured endothelial cells. This response does not appear to be mediated via an interaction with either the AT1 or the AT2 receptor subtype. Since a novel angiotensin receptor has been identified in a variety of tissues that specifically binds the hexapeptide Ang IV (Ang II, [3-8]), we therefore examined the effects of Ang IV on the expression of PAI-1 mRNA in bovine aortic endothelial cells. Ang IV stimulated dose- and time-dependent increases in the expression of PAI-1 mRNA. The effect of Ang IV (10 nM) was not inhibited by Dup 753 (1.0 microM), a highly specific antagonist of the AT1 receptor, or by PD123177 (1.0 microM), a highly specific antagonist of the AT2 receptor. In contrast, the AT4 receptor antagonist, WSU1291 (1.0 microM), effectively prevented PAI-1 expression. Although larger forms of angiotensin (i.e., Ang I, Ang II, and Ang III) are capable of inducing PAI-1 expression, this property is lost in the presence of converting enzyme or aminopeptidase inhibitors. These results indicate that the hexapeptide Ang IV is the form of angiotensin that stimulates endothelial expression of PAI-1. This effect appears to be mediated via the stimulation of an endothelial receptor that is specific for Ang IV.     

Reduced angiogenesis and delay in wound healing in angiotensin II type 1a receptor-deficient mice

Angiotensin II (Ang II) is a bioactive peptide that plays important roles in blood pressure regulation and salt–water homeostasis. Recently, Ang II was reported to function in the promotion of angiogenesis. Since the wound healing process is highly dependent upon angiogenesis, we employed Ang II receptor knockout mice (AT1a^−/−) to investigate whether or not Ang II facilitates angiogenesis and wound healing via AT1a receptor signaling. In comparison to wild-type (WT) mice, wound healing and wound-induced angiogenesis were significantly suppressed in AT1a^−/− mice, and these mice exhibited reduced expression of CD31 in wound granulation tissues. In comparison to vehicle-treated mice, wound healing was delayed significantly in mice treated with an AT1-R antagonist and this delay was accompanied by the reduced expression of vascular endothelial growth factor in wound granulation tissues. These findings suggest that Ang II–AT1a signaling plays a crucial role in wound healing and wound-induced angiogenesis.     

Chronic blockade of AT2-subtype receptors prevents the effect of angiotensin II on the rat vascular structure.

Angiotensin II (Ang II) is both a vasoactive and a potent growth-promoting factor for vascular smooth muscle cells. Little is known about the in vivo contribution of AT1 and AT2 receptor activation to the biological action of Ang II. Therefore, we investigated the effect of AT1 or AT2 subtype receptor chronic blockade by losartan or PD123319 on the vascular hypertrophy in rats with Ang II-induced hypertension. Normotensive rats received for 3 wk subcutaneous infusions of Ang II (120 ng/kg per min), or Ang II + PD 123319 (30 mg/kg per d), or Ang II + losartan (10 mg/kg per d) or PD 123319 alone, and were compared with control animals. In normotensive animals, chronic blockade of AT2 receptors did not affect the plasma level of angiotensin II and the vascular reactivity to angiotensin II mediated by the AT1 receptor. Chronic blockade of AT1I in rats receiving Ang II resulted in normal arterial pressure, but it induced significant aortic hypertrophy and fibrosis. Chronic blockade of AT2 receptors in Ang II-induced hypertensive rats had no effect on arterial pressure, but antagonized the effect of Ang II on arterial hypertrophy and fibrosis, suggesting that in vivo vasotrophic effects of Ang II are at least partially mediated via AT2 subtype receptors.