K.B. Brosnihan

Angiotensin-(1–7) Augments Bradykinin-Induced Vasodilation by Competing With ACE and Releasing Nitric Oxide

Recent studies have shown that angiotensin-(1-7) [Ang-(1-7)] interacts with kinins and augments bradykinin (BK)-induced vasodilator responses by an unknown mechanism. In this study, we evaluated whether the potentiation of the BK-induced vasodilation by Ang-(1-7) may be attributable to inhibition of BK metabolism, release of nitric oxide, or both. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers. 125I-[Tyr0]-BK metabolism was determined in vascular rings by assessing the degradation of the peptide by high-performance liquid chromatography. Ang-(1-7) augmented the vasodilation induced by BK in a concentration-dependent manner in rings preconstricted with the thromboxane analog U46619. The EC50 of BK (2.45 +/- 0.51 nmol/L versus 0.37 +/- 0.08 nmol/L) was shifted leftward by 6.6-fold in the presence of 2 mumol/L concentration of Ang-(1-7). The response was specific for BK. since Ang-(1-7) did not augment the vasodilation induced by either acetylcholine (0.05 mumol/L) or sodium nitroprusside (0.1 mumol/L). Moreover, neither angiotensin I nor angiotensin II (Ang II) duplicated the augmented BK response of Ang-(1-7). Pretreatment of vascular rings with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NA; 100 mumol/L) completely abolished the effects of Ang-(1-7) on BK-induced vasodilation whereas pretreatment with indomethacin (10 mumol/L) was without effect. The potent specific BK B2 receptor antagonist, Hoe 140. nearly abolished the BK and the Ang-(1-7) potentiated responses at 2 mumol/L, whereas at a lower concentration (20 nmol/L) Hoe 140 shifted the response curve to the right for both Ang-(1-7) and vehicle; however, the augmented response to Ang-(1-7) persisted. Preincubation of vascular rings with 20 mumol/L of the AT1 (CV11974), AT2 (PD123319), or nonselective (Sar1 Thr8-Ang II) receptor antagonists had no significant effect on the Ang-(1-7)-enhanced vasodilator response to BK. Lisinopril (2 mumol/L) significantly enhanced the BK-induced vasodilator response while at the same time it abolished the synergistic action of Ang-(1-7) on BK. In addition, pretreatment with 2 mumol/L Ang-(1-7) significantly inhibited the degradation of 125I-[Tyr0]-BK and the appearance of the BK-(1-7) and BK-(1-5) metabolites in coronary vascular rings. Ang-(1-7) inhibited purified canine angiotensin converting enzyme activity with an IC50 of 0.65 mumol/L. In conclusion. Ang-(1-7) acts as a local synergistic modulator of kinin-induced vasodilation by inhibiting angiotensin converting enzyme and releasing nitric oxide.

Enhanced expression of Ang-(1-7) during pregnancy

Pregnancy is a physiological condition characterized by a progressive increase of the different components of the renin-angiotensin system (RAS). The physiological consequences of the stimulated RAS in normal pregnancy are incompletely understood, and even less understood is the question of how this system may be altered and contribute to the hypertensive disorders of pregnancy. Findings from our group have provided novel insights into how the RAS may contribute to the physiological condition of pregnancy by showing that pregnancy increases the expression of both the vasodilator heptapeptide of the RAS, angiotensin-(1-7) [Ang-(1-7)], and of a newly cloned angiotensin converting enzyme (ACE) homolog, ACE2, that shows high catalytic efficiency for Ang II metabolism to Ang-(1-7). The discovery of ACE2 adds a new dimension to the complexity of the RAS by providing a new arm that may counter-regulate the activity of the vasoconstrictor component, while amplifying the vasodilator component. The studies reviewed in this article demonstrate that Ang-(1-7) increases in plasma and urine of normal pregnant women. In preeclamptic subjects we showed that plasma Ang-(1-7) was suppressed as compared to the levels found in normal pregnancy. In addition, kidney and urinary levels of Ang-(1-7) were increased in pregnant rats coinciding with the enhanced detection and expression of ACE2. These findings support the concept that in normal pregnancy enhanced ACE2 may counteract the elevation in tissue and circulating Ang II by increasing the rate of conversion to Ang-(1-7). These findings provide a basis for the physiological role of Ang-(1-7) and ACE2 during pregnancy.

Bi-directional actions of estrogen on the renin-angiotensin system

Estrogen stimulates the renin-angiotensin system by augmenting both tissue and circulating levels of angiotensinogen and renin. We show, however, that angiotensin converting enzyme (ACE) activity in the circulation and in tissues is reduced in two animal models of postmenopausal chronic hormone replacement. We observed a reduction of ACE activity in association with a significant increase in plasma angiotensin I (Ang I) and hyperreninemia in ovariectomized monkeys treated with Premarin (conjugated equine estrogen) replacement for 30 months. Plasma angiotensin II (Ang II) levels were not increased in monkeys treated with estrogen, suggesting that the decrease in ACE curtailed the formation of the peptide. The Ang II/Ang I ratio, an in vivo index of ACE activity, was significantly reduced by estrogen treatment, further supporting the biochemical significance of estrogens inhibition of ACE. In ovariectomized transgenic hypertensive (mRen2)27 rats submitted to estrogen replacement treatment for 3 weeks, ACE activity in plasma and tissue (aorta and kidney) and circulating Ang II levels were reduced, whereas circulating levels of angiotensin-(1-7) (Ang-(1-7)) were increased. Ang-(1-7), the N-terminal fragment of Ang II, is a novel vasodilator and antihypertensive peptide. Thus, the net balance of these effects of estrogen on the reninangiotensin vasoconstrictor/vasodilator system is to promote the anti-hypertensive effect.

Therapeutic targets in liver transplantation: angiotensin II in nonsteatotic grafts and angiotensin-(1-7) in steatotic grafts.

Numerous steatotic livers are discarded as unsuitable for transplantation because of their poor tolerance of ischemia‐reperfusion(I/R). The injurious effects of angiotensin (Ang)‐II and the benefits of Ang‐(1–7) in various pathologies are well documented. We examined the generation of Ang II and Ang‐(1–7) in steatotic and nonsteatotic liver grafts from Zucker rats following transplantation. We also studied in both liver grafts the effects of Ang‐II receptors antagonists and Ang‐(1–7) receptor antagonists on hepatic I/R damage associated with transplantation. Nonsteatotic grafts showed higher Ang II levels than steatotic grafts, whereas steatotic grafts showed higher Ang‐(1–7) levels than nonsteatotic grafts. Ang II receptor antagonists protected only nonsteatotic grafts against damage, whereas Ang‐(1–7) receptor antagonists were effective only in steatotic grafts. The protection conferred by Ang II receptor antagonists in nonsteatotic grafts was associated with ERK 1/2 overexpression, whereas the beneficial effects of Ang‐(1–7) receptor antagonists in steatotic grafts may be mediated by NO inhibition. Our results show that Ang II receptor antagonists are effective only in nonsteatotic liver transplantation and point to a novel therapeutic target in liver transplantation based on Ang‐(1–7), which is specific for steatotic liver grafts.

Angiotensin-(1-7) serves as an aquaretic by increasing water intake and diuresis in association with downregulation of aquaporin-1 during pregnancy in rats

We previously demonstrated that kidney and urine levels of angiotensin-(1-7) [ANG-(1-7)] were increased in pregnancy. To explore the role of ANG-(1-7) on fluid and electrolyte homeostasis during pregnancy, we evaluated the effect of the ANG-(1-7) antagonist D-alanine-[ANG-(1-7)] (A-779) on kidney function. Virgin and pregnant rats received infusion of vehicle or A-779 (48 microg.kg(-1).h(-1)) for 8 days by osmotic minipumps. Metabolic studies were done on treatment day 7-8. Virgin and pregnant rats at day 15 and 19 were killed, and blood and kidneys were collected. Kidneys were prepared for Western blot analysis for aquaporin-1 (AQP1) and aquaporin-2. In virgin female rats, A-779 increased urine volume and decreased urinary osmolality and AQP1 with no change in water intake. In 19-day pregnant rats, A-779 significantly decreased water intake and urine volume and increased urinary osmolality and kidney AQP1 expression. Only in late gestation did A-779 treatment decrease the difference between intake and output (balance). A-779 treatment increased plasma vasopressin in late gestation but did not change vasopressin in virgins. In virgin and pregnant animals, A-779 administration had no effect on blood pressure, plasma volume, blood volume, or urinary electrolytes. These results suggest that ANG-(1-7) produces antidiuresis associated with upregulation of AQP1 in virgin rats, whereas ANG-(1-7) produces diuresis in late gestation with downregulation of AQP1. ANG-(1-7) contributes to the enhanced water intake during pregnancy, allowing maintenance of the normal volume-expanded state despite diuresis produced in part by decreased AVP and AQP1.

Effect of estrogen on neprilysin expression in uterus and kidney of Sprague–Dawley normotensive and heterozygous (mRen2)27-transgenic hypertensive rats

The present study was designed to determine whether estrogen modulates the angiotensin processing enzymes in membrane homogenates obtained from uterus and kidney cortex and medulla of Sprague-Dawley (SD) and heterozygous (mRen2)27-transgenic hypertensive (Tg(+)) female rats treated with or without 17beta-estradiol (E2). We evaluated estrogens influence on neprilysin (NEP), an endopeptidase that forms angiotensin-(1-7) [Ang-(1-7)] and on aminopeptidase (AMP), which degrades Ang-(1-7). Renal tissue from normotensive and hypertensive male rats was also evaluated. E2 up-regulated NEP mRNA in the uterus of both SD and Tg(+) and this was associated with increased NEP activity in the uterus of SD (0.31+/-0.03 nmol/min/mg versus 0.18+/-0.04 nmol/min/mg of protein, p<0.05) and Tg(+) (0.26+/-0.04 nmol/min/mg versus 0.13+/-0.02 nmol/min/mg of protein, p<0.05) female). E2 had no significant effect on NEP activity in cortex and medulla of hypertensive and normotensive female. In female animals, cortical NEP activity is two-fold higher than medullary; in males there is a four-fold higher cortical NEP activity as compared to medulla. In male animals, medullary NEP was significantly lower than females with or without E2 treatment; no gender specific effect was found in cortex. E2 treatment also caused a two-fold increase in AMP activity in the uterus and 1.6-fold decrease in kidney cortex of SD and Tg(+) female (p<0.05). Our studies indicate that NEP may be a primary candidate for increased Ang-(1-7) processing in the uterus with estrogen treatment; kidney NEP, on the other hand, showed no modulation by estrogen, suggesting that down regulation of other processing enzymes, like AMP and ACE, may come into play in the kidney with estrogen replacement. In addition, these studies showed that there is tissue-specific regulation of NEP with estrogen treatment that is strain independent.

Decidualized pseudopregnant rat uterus shows marked reduction in Ang II and Ang-(1-7) levels.

UNLABELLED Previous studies showed that angiotensin (Ang) II and Ang-(1-7) concentrations were reduced in the implantation site at day 7 of pregnancy in Sprague-Dawley rats as compared to the site immediately adjacent to it, which does not have the embryo attached, clearly showing the importance of the blastocyst in the regulation of renin-angiotensin system (RAS). OBJECTIVE The objective of this study was to evaluate the regulation of the RAS in the decidualized uterus in the pseudopregnant rat, a model without the presence of a conceptus. METHODS Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments; decidualization was induced by oil-injection of the right horn; the left horn served as a control. The uterine content of Ang I, Ang II, and Ang-(1-7) was examined in the decidualized and non-decidualized uteri. RESULTS Both Ang-(1-7) and Ang II and ACE and ACE2 mRNA were significantly reduced in the decidualized horn as compared to the non-decidualized horn. Immunocytochemical characterization of Ang II, Ang-(1-7), ACE and ACE2 demonstrated that Ang-(1-7), Ang II, and ACE2 polarize to the anti-mesometrial pole with decidualization. CONCLUSION The decidualization process elicits marked reduction in uterine Ang II and Ang-(1-7) content as compared to the non-decidualized horn. The differential immunocytochemical expression of Ang II and Ang-(1-7) with ACE2, but not ACE in the anti-mesometrial pole of the decidualized horn may favor the formation and action of Ang-(1-7) in the anti-mesometrial pole, an area which plays a role in triggering the decidualization process.

Characterization of vascular reactivity in dorsal hand veins after oral rosiglitazone treatment in healthy subjects

OBJECTIVE In clinical studies with diabetic patients thiazolidinediones have been shown to restore abnormal vascular function which might be attributed to improved blood sugar control or to restoration of vascular endothelium and smooth muscle responsiveness. The present study was undertaken to investigate whether rosiglitazone modulates vascular responsiveness to different vasoactive agents and exerts renin-angiotensin-system (RAS)-inhibiting properties in healthy subjects in vivo. METHODS 24 healthy male subjects were randomized to receive either rosiglitazone or placebo. Venoconstrictor responses to angiotensin II (Ang II) and phenylephrine, and endothelium-dependent response to histamine and insulin, and endothelium-independent response to glyceroltrinitrate were compared using the dorsal hand vein compliance method. Effects on the RAS were investigated by plasma level determinations of Ang II and angiotensin-(1-7). Treatment effects on the systemic arterial system were investigated by standardized pulse-wave-analysis. RESULTS Rosiglitazone significantly inhibited venoconstrictor responses to Ang II by 19% (-70% vs. -51% constriction, p = 0.034) and in the presence of rosiglitazone the ED80 for phenylephrine was increased (ED80: 317 A+/- 86 ng vs. 531 A+/- 102 ng; p = 0.010). Rosiglitazone treatment was without effect on endothelium-dependent dilation, blood pressure, pulse-wave-velocity and plasma angiotensin peptide levels. CONCLUSIONS The data of the present study in veins of healthy subjects are consistent with data from in vitro and animal studies supporting a direct effect of rosiglitazone on venous tone by modulation of the vascular smooth muscle response via AT1-receptor-downregulation.

Angiotensin-(1–7). Its Contribution to Arterial Pressure Control Mechanisms

A formidable body of evidence accumulated to date reveals the pleio-tropic actions of the renin-angiotensin system and its role in cardiovascular medicine. The characterization of the biological actions of angiotensin-(1-7) expanded knowledge of the endogenous control mechanisms regulating the actions of angiotensin II and revealed alternate enzymatic pathways for the processing of angiotensin I and angiotensin II. The following chapter reviews the tissue-specific pathways for formation and metabolism of this member of the renin-angiotensin system as well as its localization within brain, renal, and cardiac tissue. The unique actions of angiotensin-(1-7) in general oppose those of angiotensin II, and the preponderance of data identifies the regulation of the balance between the two peptides as important to overall cardiovascular health. We emphasize the physiological actions of the peptide in vascular, neural, and renal systems and provide a comprehensive review of its trophic and signaling mechanisms. The current status of the receptors mediating the actions of the peptide and the potential for non-receptor mechanisms are also presented. Finally, we discuss the significance of angiotensin-(1-7) in human disease, based on evidence emerging from recent studies suggesting that deficits in angiotensin-(1-7) are associated with hypertension in specific subpopulations of patients.