Roles of angiotensin type 1 and 2 receptors in pregnancy-associated blood pressure change.

BACKGROUND: Activation of the renin-angiotensin system with increased levels of renin and angiotensin (Ang) II in pregnancy has been reported, but the vascular responsiveness to Ang II seems to be decreased, thereby keeping maternal blood pressure (BP) constant. We postulated that the balance of angiotensin type 1 (AT1) and angiotensin type 2 (AT2) receptor expression, which would exert antagonistic actions on vasoconstriction and cell growth, might control BP in pregnancy. METHODS: Using wild type (C57BL/6J), AT1a receptor null and AT2 receptor null mice, we examined the changes in BP, expression and localization of AT1 and AT2 receptors in placenta, umbilical cord, and uterus by immunohistochemical staining and urinary albumin measurement during pregnancy. RESULTS: Wild type mice did not show any significant change in BP throughout pregnancy. The BP in AT1a receptor null mice declined significantly in the second trimester of pregnancy, whereas BP in AT2 receptor null mice increased significantly in the third trimester. We did not observe any significant differences in albuminuria, litter size, or body weight of neonates among the three groups. Vascular smooth muscle cells in blood vessels of the umbilical cord and placenta specifically expressed AT2 receptors, which are minimally expressed in adult vessels. In contrast, AT1 receptors were dominantly expressed in the cytotrophoblast and chorionic plate as well as blood vessels in placenta and umbilical cord. CONCLUSIONS: Our results suggested that disturbance of the balance of the AT1 and AT2 receptors could trigger pregnancy induced hypertension.

Expression of endothelial angiotensin II receptor mRNA in pregnancy-induced hypertension.

OBJECTIVE: The normal suppression of vascular sensitivity to angiotensin II (Ang II) in pregnancy is lost in pregnancy-induced hypertension (PIH). To examine the mechanism, we investigated Ang II receptor subtype 1 (AT1R) and 2 (AT2R) expression in human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC). METHODS: The HUVEC and VSMC were incubated with serum from normal pregnant women and PIH patients for 0 to 12 h. The AT1R and AT2R mRNA were semiquantified as the ratio to glyceraldehyde-3-phosphate dehydrogenase mRNA, using multiplex reverse transcription-polymerase chain reaction. The AT1R expression was also evaluated by immunocytochemistry. RESULTS: Serum from PIH patients significantly increased AT1R mRNA of HUVEC (1.48 +/- 0.44) after a 12-h incubation compared with that from normal pregnant women (0.25 +/- 0.14). On the other hand, AT2R mRNA of HUVEC incubated with serum from PIH patients (0.14 +/- 0.02) was significantly decreased compared with HUVEC incubated with serum from normal pregnant women (0.31 +/- 0.08). The AT1R mRNA of VSMC was significantly increased by serum from both PIH patients and normal pregnant women. The AT1R-to-AT2R mRNA ratio increased by serum from PIH patients was significantly reduced by anti-tumor necrosis factor-alpha (TNF-alpha) antibody (20 microg/mL). Valsartan (an AT1R antagonist, at 1 to 10 nmol/L) significantly increased AT2R mRNA of HUVEC. Also, immunocytochemistry demonstrated that endothelial AT1R expression was strongly increased by PIH sera and reduced by anti-TNF-alpha antibody. CONCLUSIONS: Endothelial AT1R expression is increased and AT2R expression is decreased in PIH. The TNF-alpha is related to the pathogenesis of PIH by reduced AT2R mRNA through an increase of AT1R mRNA.

Effect of estrogen and AT1 receptor blocker on neointima formation.

The present study explored the possibility that estrogen may enhance the inhibitory effect of an angiotensin (Ang) II type 1 (AT1) receptor blocker on neointima formation in vascular injury, and investigated the signaling mechanism involved in their actions. Polyethylene cuff placement around the femoral artery of mice induced neointima formation and increased bromodeoxyuridine (BrdU) incorporation into vascular smooth muscle cells. These changes were significantly smaller in female mice than in male mice. Ovariectomy enhanced neointima formation and BrdU incorporation in the injured artery, which were reversed by 17beta-estradiol (80 microg/kg per day) replacement. Treatment with a selective AT1 receptor blocker, olmesartan (3 mg/kg per day), significantly inhibited neointima formation and BrdU incorporation, whereas the inhibitory effects of olmesartan were more marked in intact female mice than in male or ovariectomized mice. Phosphorylation of extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription (STAT) 1, and STAT3 was increased in the injured artery. These increases were significantly smaller in intact female mice than in male or ovariectomized mice. Olmesartan or estrogen attenuated the phosphorylation of ERK and STAT in the injured artery, whereas these inhibitory effects were greater in intact female mice. Lower doses of olmesartan (0.5 mg/kg per day) or 17beta-estradiol (20 microg/kg per day) did not influence neointima formation, BrdU incorporation, and ERK and STAT phosphorylation in ovariectomized mice, whereas coadministration of olmesartan and 17beta-estradiol at these doses attenuated these parameters. These results indicate that estrogen and an AT1 receptor blocker synergistically attenuate vascular remodeling, which is at least partly via inhibition of ERK and STAT activity.

ACE inhibitor improves insulin resistance in diabetic mouse via bradykinin and NO.

Improvement of insulin resistance by ACE inhibitors has been suggested; however, this mechanism has not been proved. We postulated that activation of the bradykinin-nitric oxide (NO) system by an ACE inhibitor enhances glucose uptake in peripheral tissues by means of an increase in translocation of glucose transporter 4 (GLUT4), resulting in improvement of insulin resistance. Administration of an ACE inhibitor, temocapril, significantly decreased plasma glucose and insulin concentrations in type 2 diabetic mouse KK-Ay. Mice treated with temocapril showed a smaller plasma glucose increase after glucose load. We demonstrated that temocapril treatment significantly enhanced 2-[3H]-deoxy-D-glucose (2-DG) uptake in skeletal muscle but not in white adipose tissue. Administration of a bradykinin B2 receptor antagonist, Hoe140, or an NO synthase inhibitor, L-NAME, attenuated the enhanced glucose uptake by temocapril. Moreover, we observed that translocation of GLUT4 to the plasma membrane was significantly enhanced by temocapril treatment without influencing insulin receptor substrate-1 phosphorylation. In L6 skeletal muscle cells, 2-DG uptake was increased by temocaprilat, and Hoe140 inhibited this effect of temocaprilat but not that of insulin. These results suggest that temocapril would improve insulin resistance and glucose intolerance through increasing glucose uptake, especially in skeletal muscle at least in part through enhancement of the bradykinin-NO system and consequently GLUT4 translocation.

Angiotensin II subtype 2 receptor activation inhibits insulin-induced phosphoinositide 3-kinase and Akt and induces apoptosis in PC12W cells.

In the present study, we identified novel negative cross-talk between the angiotensin II subtype 2 (AT2) receptor and insulin receptor signaling in the regulation of phosphoinositide 3-kinase (PI3K), Akt, and apoptosis in rat pheochromocytoma cell line, PC12W cells, which exclusively express AT2 receptor. We demonstrated that insulin-mediated insulin receptor substrate (IRS)-2-associated PI3K activity was inhibited by AT2 receptor stimulation, whereas IRS-1-associated PI3K activity was not significantly influenced. AT2 receptor stimulation did not change insulin-induced tyrosine phosphorylation of IRS-2 or its association with the p85alpha subunit of PI3K, but led to a significant reduction of insulin-induced p85alpha phosphorylation. AT2 receptor stimulation increased the association of a protein tyrosine phosphatase, SHP-1, with IRS-2. Moreover, we demonstrated that AT2 receptor stimulation inhibited insulin-induced Akt phosphorylation and that insulin-mediated antiapoptotic effect was also blocked by AT2 receptor activation. Overexpression of a catalytically inactive dominant negative SHP-1 markedly attenuated the AT2 receptor- mediated inhibition of IRS-2-associated PI3K activity, Akt phosphorylation, and antiapoptotic effect induced by insulin. Taken together, these results indicate that AT2 receptor-mediated activation of SHP-1 and the consequent inhibition IRS-2-associated PI3K activity contributed at least partly to the inhibition of Akt phosphorylation, thereby inducing apoptosis.

Tumor necrosis factor-alpha inhibits growth factor-mediated cell proliferation through SHP-1 activation in endothelial cells.

Src homology 2-containing protein-tyrosine phosphatase 1 (SHP-1) is known to regulate signal transduction through the dephosphorylation of tyrosine kinases. In this study, we addressed the role of SHP-1 under tumor necrosis factor-alpha (TNF-alpha) stimulation in endothelial cells. The addition of recombinant vascular endothelial growth factor (50 ng/mL) or epidermal growth factor (50 ng/mL) significantly increased thymidine incorporation and c-fos promoter activity, whereas TNF-alpha (5 ng/mL) attenuated these effects in human or bovine aortic endothelial cells. In bovine aortic endothelial cells, we confirmed endogenous SHP-1 expression and that TNF-alpha activated SHP-1. Importantly, overexpression of dominant-negative SHP-1 attenuated the effect of TNF-alpha on thymidine incorporation and c-fos promoter activity. In addition, TNF-alpha attenuated vascular endothelial growth factor- and epidermal growth factor-induced extracellular signal-regulated kinase phosphorylation, whereas overexpression of dominant-negative SHP-1 prevented this inhibitory effect of TNF-alpha. Taken together, our results suggested that TNF-alpha inhibited growth factor-mediated cell proliferation through SHP-1 activation.

Estrogen activates phosphatases and antagonizes growth-promoting effect of angiotensin II.

Accumulating evidence suggests that estrogen exerts cardioprotective effects and protects against neointima formation in response to vascular injury in vivo, whereas angiotensin (Ang) II stimulation via the Ang II type 1 (AT(1)) receptor exaggerates vascular injury. We postulate that estrogen treatment antagonizes the AT(1) receptor-mediated growth-promoting effects in vascular smooth muscle cells (VSMCs). The present in vitro study was designed to explore this possibility and to establish the cellular mechanism whereby estrogen attenuates the growth of VSMCs. Primary cultures of VSMCs derived from male adult Sprague-Dawley rats express exclusively AT(1) receptors. Treatment with Ang II enhanced proliferation of VSMC and c-fos expression, whereas 17beta-estradiol (E2) attenuated these vasotrophic effects of Ang II. We also demonstrated that E2 attenuated AT(1) receptor-mediated extracellular signal-regulated kinase activation and that this effect of E2 was restored by pretreatment with vanadate or okadaic acid. Moreover, we demonstrated that E2 enhanced SHP-1 activity, rapidly reaching a peak after 3 minutes of E2 stimulation, whereas E2 transactivated mitogen-activated protein kinase phosphatase-1 expression, showing a peak after 60 minutes of E2 treatment. SHP-1 activation was not influenced by actinomycin D treatment, whereas E2-mediated mitogen-activated protein kinase phosphatase-1 expression was attenuated. Taken together, our results suggest a novel mechanism of vasoprotection by which estrogen antagonizes the effect of the AT(1) receptor via the activation and induction of phosphatases through nongenomic as well as genomic signaling.