Angiotensin-(1-7): a peptide hormone with anti-cancer activity.

The development of peptides as therapeutic agents has progressed such that these small molecules of less than fifty amino acids are currently in use for the treatment of a variety of pathologies. This review focuses on the pre-clinical studies and clinical trials assessing the anti-cancer properties of angiotensin-(1-7) [Ang-(1-7)], an endogenous heptapeptide hormone of the renin-angiotensin system. Ang-(1-7) mediates biological responses by activating mas, a unique G protein- coupled receptor, thereby providing specific targeted actions when used as a therapeutic agent. Studies in in vitro as well as in vivo mouse models demonstrated that the heptapeptide hormone reduced proliferation of human cancer cells and xenograft tumors. This attenuation was concomitant with decreased angiogenesis, cancer associated fibrosis, osteoclastogenesis, tumor-induced inflammation and metastasis as well as altered regulation of growth promoting cellular signaling pathways. In three clinical trials, Ang-(1-7) was well tolerated with limited toxic or quality-of-life side effects and showed clinical benefit in cancer patients with solid tumors. Taken together, these studies suggest that Ang-(1-7) may serve as a first-in-class peptide chemotherapeutic agent, reducing cancer growth and metastases by pleiotrophic mechanisms as well as targeting the tumor microenvironment.

Downregulation of the AT1A receptor by pharmacologic concentrations of Angiotensin-(1-7).

Angiotensin (Ang)-(1-7), the amino terminal heptapeptide fragment of Ang II, is an endogenous Ang peptide with vasodilatory and antiproliferative actions. Because Ang II causes vasoconstriction and promotes growth through activation of Ang type 1 (AT1) receptors, we investigated whether the actions of Ang-(1-7) are due to its regulation of these receptors. Studies were performed in CHO cells stably transfected with the AT1A receptor. Ang-(1-7) competed poorly with [125I]-Ang II for the AT1A binding site and was ineffective at shifting the IC50 for Ang II competition with [125I]-Ang II for binding to the AT1A receptor. However, if CHO-AT1A cells were pretreated with Ang-(1-7) and then treated with acidic glycine to remove surface-bound ligand, the heptapeptide caused a concentration-dependent reduction in Ang II binding, with a maximal inhibition to 67.8 +/- 4.6% of total (p < 0.05) at 1 microM Ang-(1-7) compared with a reduction to 24% of total by 10 nM Ang II. Ang-(1-7) pretreatment caused a small but significant decrease in the affinity of [125I]-Ang II for the AT1A receptor and a significant reduction in the total number of binding sites. The Ang-(1-7)-induced reduction in binding was rapid (occurring as early as 5 min after exposure to the peptide), was maintained for 30 min during continued exposure of the cells to Ang-(1-7), and rapidly recovered after removal of the heptapeptide. The AT1 receptor antagonist L-158,809 reduced the Ang-(1-7)-induced downregulation of the AT1A receptor, suggesting that interactions with AT1A receptors mediate the regulatory events. Pretreatment with 1 microM or 10 microM Ang-(1-7) significantly reduced inositol phosphate production in response to 10 nM Ang II. The decrease in binding and responsiveness of the AT1A receptor after exposure to micromolar concentrations of Ang-(1-7) suggests that the heptapeptide downregulates the AT1A receptor to reduce responses to Ang II. Because downregulation of the receptor only occurred at micromolar concentrations of the heptapeptide, our findings suggest that Ang-(1-7) is not a potent antagonist at the AT1A receptor. However, when the balance between Ang II and Ang-(1-7) is shifted in favor of Ang-(1-7), such as during inhibition of Ang-converting enzyme, some contribution of this mechanism may come into play.

Angiotensin-(1-7) downregulates the angiotensin II type 1 receptor in vascular smooth muscle cells.

Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT(1)) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT(1) receptor. Ang-(1-7) competed poorly for [(125)I]Ang II binding to the AT(1) receptor on vascular smooth muscle cells, with an IC(50) of 2.0 micromol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [(125)I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 micromol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [B(max) 437.7+/-261.5 fmol/mg protein in Ang-(1-7)-pretreated cells compared with 607.5+/-301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT(1) receptor. Pretreatment with the AT(1) receptor antagonist L-158,809 blocked the reduction in [(125)I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II-stimulated phospholipase C activity; however, the decrease was significant (81.2+/-6.4%, P<0.01, n=5) only at 1 micromol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT(1) receptor on vascular cells and a reduction in Ang II-stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT(1) receptor or Ang-(1-7)-mediated downregulation of the vascular AT(1) receptor.

State-of-the-Art lecture. Antiproliferative actions of angiotensin-(1-7) in vascular smooth muscle.

Hemodynamic factors, circulating hormones, paracrine factors, and intracrine factors influence vascular smooth muscle growth and plasticity. The well-characterized role of angiotensin II in the modulation of vascular tone and cell function may be critically involved in the mechanisms by which vascular smooth muscle responds to signals associated with vascular endothelial dysfunction and increases in oxidative stress. Studies from this laboratory suggest that the trophic actions of angiotensin II may be intrinsically regulated by angiotensin-(1-7), a separate product of the angiotensin system derived from the common substrate, angiotensin I. Exposure of cultured vascular smooth muscle cells to angiotensin-(1-7) inhibited the trophic actions of angiotensin II and reduced the expression of the mitogenic effects of both normal serum and platelet-derived growth factor. The growth-inhibitory actions of angiotensin-(1-7) were blocked by the selective D-alanine(7)-angiotensin-(1-7) antagonist and the nonselective angiotensin receptor blocker sarcosine(1)-threonine(8)-angiotensin II. In contrast, subtype-selective antagonists for the AT(1) and AT(2) receptors had no effect on the inhibitory actions of angiotensin-(1-7), a finding that is consistent with the pharmacological characterization of a high-affinity (125)I-labeled angiotensin-(1-7) binding site in the vasculature by use of selective and nonselective angiotensin II receptor antagonists. The relevance of these findings to the proliferative response of vascular smooth muscle cells after endothelial injury was confirmed by assessment of the effect of a 12-day infusion of angiotensin-(1-7) on neointimal formation. In these experiments, the proliferative response produced by injuring the carotid artery was inhibited by angiotensin-(1-7) through a mechanism that could not be explained by changes in arterial pressure. Because plasma angiotensin-(1-7) increased to levels comparable to those found in animals and human subjects given therapeutic doses of angiotensin-converting enzyme inhibitors, angiotensin-(1-7) may be one factor participating in the reversal of vascular proliferation during inhibition of angiotensin II formation or activity.

Angiotensin II AT1-receptor blockade inhibits monocyte activation and adherence in transgenic (mRen2)27 rats.

This study investigated whether angiotensin II AT1-receptor blockade with losartan inhibits endothelium-monocyte interactions originating from long-term activation of the renin-angiotensin system in hypertensive transgenic rats [TGR(mRen2)27]. The number of circulating activated monocytes, monocytes adhered to thoracic aorta endothelium, and the extent of endothelial cell injury were compared in adult male transgenic (mRen2)27 and age-matched Hannover Sprague-Dawley (SD) rats after 12 days of continuous subcutaneous administration of saline (120 microl/24 h), losartan (10 mg/kg/24 h), or the vasodilator hydralazine (3 mg/kg/24 h). At the doses administered in this experiment, both losartan and hydralazine normalized mRen2 rat blood pressures equal to values in similarly treated SD rats. Compared with saline infusion, administration of either antihypertensive in mRen2 rats reduced (p<0.05) endothelial cell injury, but only losartan significantly (p<0.05) decreased the number of activated circulating and endothelium-adherent monocytes. Infusion of antihypertensives in SD rats had no effect on blood pressures, monocyte activity, or endothelial injury compared with saline administration. These findings suggest that the recruitment and infiltration of leukocytes into the subendothelium associated with renin-angiotensin system-induced hypertension is partly mediated by pressure-independent AT1-receptor pathways.

Angiotensin-(1-7) reduces smooth muscle growth after vascular injury.

Regulation of vascular smooth muscle cell growth is critical to the maintenance of normal blood flow and vessel patency. Angiotensin-(1-7) [Ang-(1-7)] inhibits proliferation of vascular smooth muscle cells in vitro and opposes the mitogenic effects of angiotensin II. The present study investigated whether Ang-(1-7) inhibits vascular smooth muscle cell growth in vivo by determining its effect on neointimal formation and medial remodeling in balloon-injured carotid arteries. The carotid arteries of adult male Sprague-Dawley rats were injured with a balloon embolectomy catheter. Ang-(1-7) in saline (24 microg/kg per hour) or saline alone was infused intravenously for 12 days after injury. Pumps containing bromodeoxyuridine were implanted at the same time to determine DNA synthesis. Intravenous infusion increased plasma Ang-(1-7) to 166. 0+/-41.2 fmol/mL (n=6) compared with 46.9+/-4.2 fmol/mL (n=8) in saline-infused rats. Plasma concentrations of Ang II were not changed by Ang-(1-7) infusion. Elevation in circulating Ang-(1-7) had no effect on either blood pressure or heart rate compared with saline controls. Histomorphometric analysis of carotid arteries indicated that Ang-(1-7) infusion significantly reduced neointimal area compared with rats infused with saline (0.063+/-0.011 versus 0. 100+/-0.009 mm2; P<0.05). In contrast, Ang-(1-7) infusion had no effect on medial area of the injured or the contralateral uninjured artery compared with saline controls. Ang-(1-7) infusion also reduced the rate of DNA synthesis in both the neointima and the media of the injured vessels. Therefore, exogenous Ang-(1-7) inhibited vascular smooth muscle cell proliferation associated with balloon-catheter injury. Similar increases in endogenous plasma Ang-(1-7) and inhibition of neointimal growth were observed in rats after angiotensin-converting enzyme inhibitor or angiotensin type 1 receptor antagonist administration, suggesting that Ang-(1-7) may contribute to the in vivo antiproliferative effects of these agents on vascular smooth muscle.

Angiotensin-(1-7): a novel vasodilator of the coronary circulation.

Angiotensin-(1-7) [Ang-(1-7)] possesses novel biological functions that are distinct from angiotensin II (Ang II). In coronary arteries, the octapeptide Ang II and the heptapeptide Ang-(1-7) exert opposing actions. Ang II elicits vasoconstriction and Ang-(1-7) is a vasodilator. Ang-(1-7) elicits vasodilation by an endothelium-dependent release of nitric oxide. Further, the vasorelaxant activity is markedly attenuated by the bradykinin (BK) B2 receptor antagonist icatibant and does not appear to be associated with the synthesis and release of prostaglandins. Ang-(1-7) vasodilation is mediated by a non-AT1/AT2 receptor, since [Sar1Thr8]-Ang II, but neither candesartan, an AT1 receptor antagonist, nor PD123319, an AT2 receptor antagonist, blocked the response. Specific and high affinity binding of 125I-Ang-(1-7) to the endothelial layer of canine coronary arteries was demonstrated using in vitro emulsion autoradiography. Binding was effectively competed for by either unlabeled Ang-(1-7) or the specific Ang-(1-7) antagonist [D-Ala7]-Ang-(1-7). Additionally, Ang-(1-7) potentiated synergistically BK-induced vasodilation. The EC50 of BK vasodilation (2.45 +/- 0.51 nmol/L vs 0.37 +/- 0.08 nmol/L) was shifted 6.6-fold left-ward in the presence of 2 mumol/L concentration of Ang-(1-7). The potentiated response was specific for BK, since Ang-(1-7) did not augment the vasodilation produced by either acetylcholine or sodium nitroprusside; further, it was specific for Ang-(1-7), since neither Ang I nor Ang II augmented the BK response. In contrast to the vasodilator actions of Ang-(1-7), the potentiated response was not blocked by candesartan, PD123319 or [Sar1Thr8]-Ang II. Novel studies from our group demonstrate that Ang-(1-7) is both a substrate and inhibitor for angiotensin converting enzyme (ACE). Ang-(1-7) was shown to retard the degradation of 125I-[Tyr0]-BK in coronary rings. These studies describe novel actions of Ang-(1-7) as a vasodilator and a local synergistic modulator of kinin-induced vasodilation in coronary arteries.

Counterregulatory actions of angiotensin-(1-7).

Angiotensin (Ang)-(1-7) is a bioactive component of the renin-angiotensin system that is formed endogenously from either Ang I or Ang II. The first actions described for Ang-(1-7) indicated that the peptide mimicked some of the effects of Ang II, including the release of prostanoids and vasopressin. However, Ang-(1-7) is devoid of vasoconstrictor, central pressor, or thirst-stimulating actions. In fact, new findings reveal depressor, vasodilator, and antihypertensive actions that may be more apparent in hypertensive animals or humans. Thus, the accumulating evidence suggests that Ang-(1-7) may oppose the actions of Ang II either directly or by stimulation of prostaglandins and nitric oxide. These observations are significant because they may explain the effective antihypertensive action of converting enzyme inhibitors in a variety of non-renin-dependent models of experimental and genetic hypertension as well as most forms of human hypertension. In this context, studies in humans and animals showed that the antihypertensive action of converting enzyme inhibitors correlated with increases in plasma levels of Ang-(1-7). In this review, we summarize our knowledge of the mechanisms accounting for the counterregulatory actions of Ang-(1-7) and elaborate on the emerging concept that Ang-(1-7) functions as an antihypertensive peptide within the cascade of the renin-angiotensin system.

Angiotensin II activates distinct signal transduction pathways in astrocytes isolated from neonatal rat brain.

In previous studies, we showed that angiotensin II (Ang II) and its congener peptides-angiotensin-(2-8) [Ang-(2-8)] and angiotensin-(1-7) [Ang-(1-7)]-activate 2 distinct signal transduction pathways in a mixed population of human cortical astrocytoma cells. This suggested that different populations of astrocytes could be heterogeneous with respect to their expression of Ang II receptors or the responses to which these receptors are coupled. To compare the responses which are activated by Ang II and its congener peptides in astrocytes from different brain regions, we measured phospholipase C (PLC) activity and prostaglandin release in isolated astrocytes from 4 different areas of neonatal rat brain. In medullary and cerebellar astrocytes, Ang II activated a phosphoinositide-specific PLC in a dose-dependent manner with EC50s of 1.74 and 1.86 nM, respectively. Ang-(2-8) also caused an increase in inositol phosphate release. PLC activity was coupled to an AT1 receptor in both medullary and cerebellar astrocytes, as demonstrated by the inhibition of Ang II-activation of inositol phosphate release by the AT1 antagonist losartan. The AT2 antagonist PD 123319 was ineffective. Ang II and Ang-(2-8) also released prostacyclin from medullary and cerebellar astrocytes, measured as the release of its stable metabolite 6-keto-PGF1 alpha. In contrast, Ang II did not activate PLC or release prostaglandins in astrocytes isolated from the cortex or hypothalamus. In addition, Ang-(1-7) did not stimulate the release of inositol phosphates or prostacyclin in astrocytes from any of the neonatal rat brain regions examined. However, bradykinin (1 microM) activated PLC or released prostacyclin in astrocytes isolated from all 4 brain regions. These results suggest that Ang II receptors on region-specific astrocytes activate distinct signal transduction mechanisms in response to different angiotensin peptides.

Bovine aortic endothelial cells contain an angiotensin-(1-7) receptor.

Angiotensin-(1-7) is a novel peptide of the renin-angiotensin system that counteracts the pressor and proliferative responses to angiotensin II. We now report that cultured bovine aortic endothelial cells contain a saturable, high-affinity [125I]angiotensin-(1-7) binding site with an affinity of 19.3 +/- 10.7 nmol/L and a density of 1351 +/- 710 fmol/mg protein. Angiotensin-(1-7) competed at a second lower-affinity site, with an IC50 of 2.9 mumol/L. The high-affinity angiotensin II receptor antagonist sarcosine1-isoleucine8-angiotensin II blocked [125I]angiotensin-(1-7) binding to bovine aortic endothelial cells at both a high- (IC50 = 1.3 nmol/L) and a low-affinity (IC50 = 6.2 mumol/L) binding site. In contrast, D-alanine7-angiotensin-(1-7) completely blocked [125I]angiotensin-(1-7) binding, with an IC50 of 19.8 nmol/L, suggesting that D-alanine7-angiotensin-(1-7) may selectively block responses to angiotensin-(1-7) in endothelial cells. Neither the AT1 antagonist losartan nor the AT2 antagonist PD 123319 exhibited significant competition for [125I]angiotensin-(1-7) binding to endothelial cells isolated from bovine aorta, in agreement with the absence of detectable mRNAs encoding typical angiotensin receptor subtypes 1 or 2 (AT1 or AT2). Angiotensin II also competed for [125I]angiotensin-(1-7) binding to bovine aortic endothelial cells; however, the relative affinity was 13-fold lower than angiotensin-(1-7), suggesting a preference for angiotensin-(1-7) over angiotensin II. These results demonstrate that bovine aortic endothelial cells contain a unique non-AT1, non-AT2 angiotensin receptor that preferentially binds angiotensin-(1-7).

Angiotensin-(1-7) inhibits vascular smooth muscle cell growth.

Although angiotensin II (Ang II) and the heptapeptide Ang-(1-7) differ by only one amino acid, the two peptides produce different responses in vascular smooth muscle cells. We previously showed that Ang II stimulated phosphoinositide hydrolysis, whereas Ang II and Ang-(1-7) released prostaglandins. We now report that Ang II and Ang-(1-7) differentially modulate rat aortic vascular smooth muscle cell growth. Ang-(1-7) inhibited [3H]thymidine incorporation in response to stimulation by fetal bovine serum, platelet-derived growth factor, or Ang II. The reduction in serum-stimulated thymidine incorporation by Ang-(1-7) depended on the concentration of the heptapeptide over the range of 1 nmol/L to 1 mumol/L, with a maximal inhibition of 60% by 1 mumol/L Ang-(1-7). Ang-(1-7) also inhibited the serum-stimulated increase in cell number to a maximum of 77% by 1 mumol/L Ang-(1-7). The attenuation of serum-stimulated thymidine incorporation by Ang-(1-7) was unaffected by antagonists selective for angiotensin type 1 (AT1) or type 2 (AT2) receptors; however, [Sar1,Ile1]Ang II and [Sar1,Thr2]Ang II were effective antagonists, indicating that growth inhibition by Ang-(1-7) was a result of angiotensin receptor activation. In contrast, Ang II stimulated [3H]thymidine incorporation in cultured vascular smooth muscle cells over the same concentration range, with a maximal stimulation of 314% at 1 mumol/L Ang II. Ang II also increased the total number of cells (to 145% of control), suggesting that enhanced thymidine incorporation was associated with vascular smooth muscle cell proliferation. The AT1 antagonist losartan or L-158,809 but not AT2 antagonists blocked [3H]thymidine incorporation by Ang II. These results suggest that Ang-(1-7) and Ang II exhibit opposite effects on the regulation of vascular smooth muscle cell growth. The inhibition of proliferation by Ang-(1-7) appears to be mediated by a novel angiotensin receptor that is not inhibited by AT1 or AT2 receptor antagonists.

Angiotensins differentially activate phospholipase D in vascular smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats.

We previously showed that angiotensin (Ang) II activates phospholipase D (PLD) through AT1 receptors in vascular smooth muscle cells (VSMC) isolated from Sprague-Dawley rats [Freeman and Tallant, Biochem J. 304:543-548, (1994)]. In the present study, we compared activation of PLD by angiotensin peptides in VSMC from spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats. Ang II caused a dose-dependent increase in PLD activity in VSMC from both rat strains. However, the response to Ang II in VSMC from hypertensive rats was approximately three times higher than that observed in VSMC from normotensive controls. Furthermore, Ang II-induced activation of PLD in VSMC from hypertensive rats was significant within 1 min, whereas significant increases in PLD activity in cells from normotensive rats were not seen until 10 min after exposure to Ang II. Ang-(2-8) caused a similar increase in PLD activity which was three times higher in SHR VSMC than in WKY controls. In contrast, Ang-(1-7) did not affect PLD activity in either smooth muscle cell population. The Ang II-mediated increases in PLD activity in VMSC from both rat strains were completely blocked by AT1 receptor antagonists (EXP 3174 or L-158,809). Conversely, the AT2 receptor antagonist PD 123177 (1 mumol/L) was ineffective. Thus Ang II stimulation of PLD in VSMC derived from both the hypertensive and normotensive rat aorta and the accumulation of its metabolites (e.g., phosphatidic acid and diacylglycerol) is coupled to activation of AT1 receptors predominantly and occurs in response to Ang II or Ang-(2-8) but not Ang-(1-7). Moreover, activation of PLD by angiotensins in VMSC from the SHR is significantly more robust than that observed in VSMC from the normotensive WKY rat. We conclude that increased activation of PLD by Ang II in genetically-induced hypertension may reflect an additional mechanism linking enhanced contractile responses to enhanced growth.

Opposing actions of angiotensin-(1-7) and angiotensin II in the brain of transgenic hypertensive rats.

Lack of specific antagonists to the amino-terminal heptapeptide angiotensin-(1-7) [Ang-(1-7)] prompted us to evaluate the central effects of delivering a specific affinity-purified Ang-(1-7) antibody on the blood pressure and heart rate of 12-week-old conscious homozygous female rats (n = 12) expressing the mouse submandibular Ren-2d gene [(mRen-2d)27] in their genome. Another group of transgenic hypertensive and strain-matched Sprague-Dawley controls were injected with a specific Ang II monoclonal antibody (KAA8). Cerebroventricular administration of the affinity-purified Ang-(1-7) antibody in conscious transgenic hypertensive rats caused significant dose-related elevations in blood pressure associated with tachycardia. The hypertensive response was augmented in transgenic rats studied 7 to 10 days after cessation of lisinopril therapy. Neutralization of Ang II with the Ang II antibody caused a hemodynamic response opposite to that obtained with the Ang-(1-7) antibody. All doses of the Ang II antibody produced hypotension and bradycardia. The magnitude of the depressor response was significantly augmented in transgenic rats weaned off lisinopril therapy. In contrast, central administration of either the Ang-(1-7) or Ang II antibodies had no effect on normotensive rats. Central injections of an affinity-purified IgG fraction were ineffective in both control and transgene-positive rats. These data suggest that in the brain of transgenic hypertensive rats, Ang-(1-7) opposes the action of Ang II on the central mechanism or mechanisms that contribute to the maintenance of this model of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of calcium and protein kinase C in the activation of phospholipase D by angiotensin II in vascular smooth muscle cells.

We previously showed that cultured rat aortic vascular smooth muscle cells (VSMC) possess an AT1 angiotensin (Ang) receptor coupled to the activation of a phospholipase D (PLD). AT1 receptors in VSMC are also coupled to the activation of a phosphoinositide-specific phospholipase C (PLC), mobilization of intracellular Ca2+, and activation of protein kinase C (PKC). To determine whether PLD stimulation by Ang II is the result of PLC activation and the subsequent elevation of cytosolic free Ca2+ and PKC activation, we investigated the role of Ca2+ and PKC in the activation of PLD. Chelation of extracellular Ca2+ by EGTA, blockade of voltage-sensitive Ca2+ channels, or chelation of intracellular Ca2+ with BAPTA partially attenuated PLD activation and Ca2+ mobilization in response to Ang II. However, the simultaneous chelation of extracellular Ca2+ with EGTA and intracellular Ca2+ with BAPTA completely attenuated both PLD activation and Ca2+ accumulation. Ca2+ ionophores mimicked Ang II and the combined effects of Ang II and ionophore resulted in no further stimulation of PLD activity above that observed in the presence of either agonist alone. Although the putative PLC inhibitor U73122 blocked the activation of PLD by Ang II, it also may inhibit PLD activation directly, since it attenuated both Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA)-mediated increases in PLD activity. PMA also activated PLD in VSMC in a dose-dependent manner; however, Ang II and PMA stimulation were additive. Down-regulation of PKC via exposure to phorbol dibutyrate almost completely blocked PMA-induced stimulation of PLD while it had no effect on Ang II- or Ca(2+)-ionophore-mediated increases in PLD activity. The PKC inhibitor staurosporine augmented basal PLD activity and partially inhibited PMA stimulation of PLD while it had little effect on Ang II-induced increases in PLD activity. Thus, optimal Ang II stimulation of PLD is dependent on the availability of both intracellular and extracellular Ca2+ and independent of PMA-mediated effects. Furthermore, these data suggest that Ang II stimulation of PLD may occur subsequent to activation of PLC, since Ang II activates PLC and PLC is shown to be responsible for increases in intracellular Ca2- in response to Ang II.

Characterization of angiotensin II receptor subtypes in pancreatic acinar AR42J cells.

The AR42J acinar cell line was characterized as a potential cellular model to assess the functional aspects of an exocrine pancreatic angiotensin system. Binding studies revealed that the AR42J cells express high affinity angiotensin II binding sites (Kd = 0.73 +/- 0.06 nM; Bmax = 292 +/- 15 fmol/mg protein, n = 3). Competition studies established that these cells, similar to the intact pancreas, express predominantly the AT2 receptor subtype. The AT2-selective antagonists CGP 42112A, PD 123177, and PD 123319 competed for the majority of angiotensin II binding. However, 10-15% of the angiotensin II binding sites were competed for by the AT1-selective antagonist DuP 753 (Losartan). Affinity labeling of these binding sites with [125I]angiotensin II followed by SDS gel electrophoresis under reducing conditions revealed a single band comprising a molecular mass of 108,000 Da. Competition with unlabeled angiotensin II or the AT2 antagonist, but not the AT1 antagonist, abolished the 108,000-Da band. In intact cells, angiotensin II caused a rapid increase in intracellular calcium (Ca2+) using Fura-2 as a Ca2+ indicator. Pretreatment of the cells with the AT1 antagonist DuP 753 completely inhibited the angiotensin II-induced rise in Ca2+; however, the AT2 antagonists CGP 42112A and PD 123177 were ineffective in blocking the Ca2+ increase. These results demonstrate that this pancreatic acinar cell line expresses both AT2 and AT1 angiotensin II receptor subtypes. The AT1 receptor is coupled to the mobilization of Ca(2+)--a characteristic shared by AT1 receptors in other tissues.

Vascular smooth-muscle cells contain AT1 angiotensin receptors coupled to phospholipase D activation.

We previously showed that angiotensin II (Ang II) and angiotensin-(2-8)-peptide [Ang-(2-8)] activate a phosphoinositide-specific phospholipase C (PLC) and cause calcium mobilization in rat aortic vascular smooth-muscle cells (VSMC), while Ang II and Ang-(1-7) produce prostaglandins. To define further the signal-transduction mechanisms activated by angiotensin peptides in smooth-muscle cells, we measured diacylglycerol (DAG) accumulation in response to different angiotensin peptides and its inhibition by subtype-selective receptor antagonists. Both an initial (10 s) and secondary (10 min) phase of DAG production in response to 100 nM Ang II were inhibited by 1 microM losartan (DuP 753), an AT1 antagonist, while 1 microM PD 123177, an AT2 antagonist, was ineffective. In contrast, the heptapeptide Ang-(1-7) did not produce DAG in VSMC. Ang II also caused the hydrolysis of phosphatidylinositol and phosphatidylcholine, the formation of phosphatidic acid and the formation of phosphatidylethanol (PEt) in the presence of ethanol, through activation of a PLD and a PLD-induced transphosphatidylation reaction. A similar concentration of Ang-(2-8) also activated PLD; in contrast, Ang-(1-7) was ineffective. PEt production by 100 nM Ang II was significantly attenuated by the AT1 antagonists losartan, its metabolite EXP 3174 or L-158,809 (all at 1 microM), whereas a similar concentration of the AT2 antagonists CGP 42112A or PD 123177 was ineffective. The production of PEt by Ang II was also partially attenuated by the removal of extracellular calcium and potentiated by increasing calcium concentrations, indicating that PLD activity is partially dependent on extracellular calcium. Thus VSMC PLD is coupled to an AT1 receptor and occurs in response to Ang II or Ang-(2-8), but not Ang-(1-7). Since AT1 receptors in VSMC are also coupled to activation of PLC, both PLC and PLD may be coupled to the same or a different AT1 receptor. Alternatively, PLD may be sequentially activated in response to Ang II activation of PLC and a subsequent increase in calcium concentration.

Alterations in prostaglandin production in spontaneously hypertensive rat smooth muscle cells.

We have characterized angiotensin binding sites in cultured smooth muscle cells obtained from the aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. In both strains of rats the binding of 125I-angiotensin II (125I-Ang II) in smooth muscle cells was time dependent and reached a maximum at 60 minutes. Scatchard analysis revealed a single binding site in both strains with equilibrium constants (KD) of 5.35 nmol/L in SHR and 3.47 nmol/L in WKY rats. Binding capacities (Bmax) in smooth muscle cells averaged 270 and 150 fmol/mg protein in SHR and WKY rats, respectively. Angiotensin peptides competed for 125I-Ang II binding with an order of potency of Ang II > angiotensin-(1-7) = angiotensin I. In smooth muscle cells of the SHR, basal prostaglandin E2 (PGE2) and prostacyclin (prostaglandin I2 [PGI2]) release were threefold and 15-fold lower than that found in WKY rat smooth muscle cells. Ang II as well as angiotensin-(1-7) stimulated PGE2 and PGI2 release in WKY rat smooth muscle cells. In smooth muscle cells from SHR, Ang II increased the production of both PGE2 and PGI2, whereas angiotensin-(1-7) enhanced only PGE2 but not PGI2 release. There was no significant difference between Ang II-stimulated PGE2 and PGI2 release or angiotensin-(1-7)-stimulated PGE2 production in SHR and WKY rat smooth muscle cells. However, angiotensin-(1-7)-stimulated PGI2 release was significantly lower (p < 0.0005) in SHR compared with WKY smooth muscle cells. Collectively, the data suggest that smooth muscle cells of SHR contain a higher number of angiotensin binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of prostaglandin synthesis by angiotensin peptides in porcine aortic smooth muscle cells: subtypes of angiotensin receptors involved.

We determined the role of AT1 and AT2 angiotensin receptors as mediators of prostaglandin (PG) release and mobilization of intracellular Ca++ in cultures of porcine vascular smooth muscle cells (VSMC) with subtype-selective angiotensin (Ang) II receptor antagonists. The binding of [125I]Ang II to porcine VSMC showed an equilibrium constant (KD) of 0.52 nM and a binding capacity (Bmax) of 14.8 fmol/mg protein. Using the AT1 antagonists DuP 753, its metabolite EXP 3174, and L-158,809, [125I]Ang II binding was displaced in a clearly biphasic manner, indicating the presence of two binding sites. Consistent with this, the AT2 antagonist CGP 42112A also displayed a biphasic curve, whereas another AT2 antagonist, PD 123177, showed a 20% reduction in binding. Ang I, Ang II and Ang-(1-7) stimulated PGE2 as well as PGI2 synthesis in a dose-dependent pattern. Ang II but not Ang I or Ang-(1-7) also caused an increase in the intracellular concentration of Ca++. Ca++ mobilization by Ang II was blocked by the AT1 antagonist DuP 753, but not by the AT2 antagonists. Ang II- and Ang I-stimulated (10 nM) PG production was attenuated by all three AT1 antagonists. However, both CGP 42112A (100 nM) and PD 123177 (100 nM) also attenuated PG release in response to Ang II. The enhancement in PG release by Ang I (10 nM) was significantly reduced by CGP 42112A (100 nM), but not by PD 123177 (1 microM). Of the AT1 antagonists, only high doses of DuP 753 or L-158,809 partially reduced the Ang-(1-7)-induced release of PG. CGP 42112A was ineffective for blocking Ang-(1-7)-stimulated PG release. Ang-(1-7)-stimulated PGE2 and PGI2 production was significantly reduced by PD 123177. Unlike DuP 753 or L-158,809, but similar to the sarcosine antagonists, EXP 3174 (10 nM) abolished the angiotensin peptide-induced PG production. These data show that Ang I and Ang II stimulate PGE2 and PGI2 release via activation of both AT1 and AT2 receptors in porcine VSMC. Ang II stimulates intracellular Ca++ mobilization via activation of AT1 receptors only. Because Ang-(1-7) enhanced PGE2 and PGI2 release via activation of angiotensin receptors having greater affinity for PD 123177 than CGP 42112A, although CGP 42112A showed a greater ability to block the Ang I response, these data further suggest differences in these two compounds at AT2 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiotensin-(1-7): a new hormone of the angiotensin system.

We provide a new foundation for an alternative interpretation of the biochemical physiology of the brain and other tissue angiotensin systems on the basis of research done in our laboratory. This perspective is prompted by the discovery that angiotensin-(1-7) has cellular functions that differ from those established for angiotensin II. Although angiotensin-(1-7) is not an agonist in terms of activating vasoconstriction, stimulating thirst, or promoting aldosterone release, the heptapeptide caused neuronal excitation and vasopressin release with a potency similar to that found with angiotensin II. Furthermore, angiotensin-(1-7) enhances the production of prostanoids by a receptor-mediated event that causes no associated rise in intracellular Ca2+. These actions of angiotensin-(1-7) provide a new understanding of the heterogeneous functions of angiotensin peptides as modulators of a wide range of regulatory functions in mammals.