Mahesh C. Khosla

Characterization of a new angiotensin antagonist selective for angiotensin-(1–7): Evidence that the actions of angiotensin-(1–7) are mediated by specific angiotensin receptors

In this study we describe a new angiotensin antagonist [Asp1-Arg2-Val3-Tyr4-Ile5-His6-D-Ala7, (A-779)] selective for the heptapeptide angiotensin-(1-7) [Ang-(1-7)]. A-779 blocked the antidiuretic effect of Ang-(1-7) in water-loaded rats and the changes in blood pressure produced by Ang-(1-7) microinjection into the dorsal-medial and ventrolateral medulla. In contrast, A-779 did not change the dipsogenic, pressor, or myotropic effects of angiotensin II (Ang II). Also, A-779 did not affect the antidiuretic effect of vasopressin or the contractile effects of angiotensin III, bradykinin, or substance P on the rat ileum. In the rostral ventrolateral medulla, the pressor effect produced by Ang-(1-7) microinjection was completely blocked by A-779 but not by AT1 or AT2 receptor antagonists (DUP 753 and CGP 42112A, respectively). Conversely, the pressor effect produced by Ang II was not changed by A-779 but was completely blocked by DUP 753. Binding studies substantiated these observations: A-779 did not compete significantly for 125I-Ang II binding to adrenocortical membranes at up to a 1 microM concentration. Low affinity binding was also observed in adrenomedullary membranes with an IC50 greater than 10 microM. Our results show that A-779 is a potent and selective antagonist for Ang-(1-7). More importantly, our data indicate that specific angiotensin receptors mediate the central and peripheral actions of Ang-(1-7).

Differential responses to angiotensin-(1–7) in the feline mesenteric and hindquarters vascular beds

Regional vascular responses to angiotensin (Ang)-(1-7), a heptapeptide derivative of Ang II were investigated in the feline hindquarters and mesenteric vascular beds under conditions of controlled flow. In the mesenteric vascular bed, injections of Ang-(1-7) in doses of 1, 3 and 10 micrograms produced dose-dependent decreases in mesenteric perfusion pressure whereas at doses of 30 and 100 micrograms, increases were observed. In contrast, in the hindquarters circulation, low doses produced increases while high doses produced decreases in perfusion pressure. In both vascular beds the degree of vasoconstriction was weak, being less than 1% of that elicited by Ang II. The vasoconstrictor effect of Ang-(1-7) in both the mesenteric and hindquarters vascular bed was blocked by DuP 753 (1 mg/kg i.v.), an Ang receptor subtype 1 (AT1) antagonist. The vasodilator responses in both vascular beds were partially blocked by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (100 mg/kg i.v.) but were unaffected by the cyclooxygenase inhibitor, meclofenamate (2.5 mg/kg i.v.). The present results show that in the peripheral vascular bed of the cat, Ang-(1-7) causes vasodilation or modest vasoconstriction, depending on the dose and the regional vascular bed studied. The present data also suggest that the vasodilator effect of the peptide may be mediated in part by the release of endothelium-derived relaxing factor and the vasoconstrictor effect by activation of the AT1 receptor subtype.

Evidence that angiotensin-(1-7) plays a role in the central control of blood pressure at the ventro-lateral medulla acting through specific receptors.

In this study we determined which angiotensin receptors may mediate the cardiovascular effects elicited by angiotensin-(1-7) [Ang-(1-7)] in the rostral ventrolateral medulla (RVLM) and caudal pressor area (CPA) of the ventrolateral medulla (VLM) of anesthetized rats. Furthermore the role of endogenous angiotensins in these areas was also investigated. The pressor effect produced by unilateral microinjection of Ang-(1-7) into the RVLM or CPA was not modified by either the AT1 receptor antagonist, DuP 753 or by the AT2 receptor antagonist, CGP 42112A, but was completely blocked by the Ang-(1-7) selective antagonist, A-779. In contrast, the pressor effect produced by microinjection of angiotensin II (Ang II) was completely blocked by DuP 753 but was not changed by CGP 42112A or A-779. Bilateral microinjection of A-779 into the RVLM or CPA produced a significant fall in mean arterial pressure and heart rate. Microinjection of DuP 753 produced a pressor effect comparable to bilateral injection of vehicle. These results indicate that, although Ang II acts in the VLM through an AT1 receptor subtype, the cardiovascular effects produced by microinjection of Ang-(1-7) into the RVLM and CPA are mediated by a specific angiotensin receptor (AT5?). Furthermore, our data provide evidence that endogenous Ang-(1-7) participates at the VLM in the neural control of arterial blood pressure.

Subtype 2 angiotensin receptors mediate prostaglandin synthesis in human astrocytes.

We have identified two distinct cellular responses that occur in human astrocytes in the presence of angiotensin (Ang) peptides and are linked to specific receptor subtypes. Ang II and the N-terminal heptapeptide Ang-(1–7) stimulated release of prostaglandin (PG) E2 and PGI2 (measured as the stable metabolite 6-keto-PGFlα). In contrast, only Ang II but not Ang-(1–7) activated phosphoinositide-specific phospholipase C, leading to mobilization of intracellular calcium. The Ang H-induced PGE2 and PGI2 syntheses were attenuated by [Sar1,Ile8] Ang II but not by [Sar1,Thr8]Ang II. Ang-(l–7)-induced PGE2 and PGI2 syntheses were not inhibited by either of these two classical antagonists. DuP 753, a subtype 1-selective Ang receptor antagonist, blocked the Ang II-induced release of PGE2 but not PGI2. In contrast, CGP 42112A, the subtype 2-selective antagonist, totally blocked the Ang II-induced PGI2 release and partially attenuated the PGE2 release. Ang-(l–7)-induced PGE2 and PGI2 release was not altered by DuP 753; however, CGP 42112A totally blocked the effects of Ang-(l–7) on PG stimulation. Calcium mobilization in response to Ang II was blocked by [Sar1,Thr8]Ang II, [Sar1,Ile8]Ang II, and DuP 753 but not by CGP 42112A. These data suggest that human astrocytes contain both Ang receptor subtypes. The subtype 1 Ang receptor participates both in the release of PGs and in the mobilization of calcium, whereas the subtype 2 receptor is coupled to the release of PGs only. In addition, PG release coupled to subtype 2 Ang II receptors occurs through a calcium-independent mechanism and responds uniquely to Ang-(1–7).

Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats

In this study we determined the cardiovascular effects produced by microinjection of angiotensin peptides [Angiotensin-(1-7) and Angiotensin II] and angiotensin antagonists (losartan, L-158,809, CGP 42112A. Sar1-Thr8-Ang II, A-779) into the rostral ventrolateral medulla of freely moving rats. Microinjection of angiotensins (12.5-50 pmol) produced pressor responses associated to variable changes in heart rate, usually tachycardia. Unexpectedly, microinjection of both AT1 and AT2 ligands produced pressor effects at doses that did not change blood pressure in anesthetized rats. Conversely, microinjection of Sar1-Thr8-Ang II and the selective Ang-(1-7) antagonist, A-779, produced a small but significant decrease in MAP an HR. These findings suggest that angiotensins can influence the tonic activity of vasomotor neurons at the RVLM. As previously observed in anesthetized rats, our results further suggest a role for endogenous Ang-(1-7) at the RVLM. The pressor activity of the ligands for AT1 and AT2 angiotensin receptor subtypes at the RVLM, remains to be clarified.

Evidence for a Physiological Role of Angiotensin-(1-7) in the Control of Hydroelectrolyte Balance

In this study we evaluated the possibility that angiotensin-(1-7) [Ang-(1-7)] acts as an endogenous osmoregulatory peptide by determining the effect of acute administration of its selective antagonist [D-Ala7]Ang-(1-7) (A-779) on renal function parameters in rats. In addition, we investigated the physiological mechanisms involved in the antidiuretic effect of Ang-(1-7). The antidiuretic effect of Ang-(1-7) (40 pmol/0.05 mL per 100 g BW) in water-loaded rats was completely blocked by A-779 (vehicle-treated, 3.34 +/- 0.43 mL/h; Ang-(1-7), 1.48 +/- 0.23; A-779, 2.72 +/- 0.35; Ang-(1-7) plus A-779, 3.26 +/- 0.49). In contrast, the antidiuretic effect of Ang-(1-7) was not significantly changed by a vasopressin V2 receptor antagonist in a dose that completely blocked the antidiuresis produced by an equipotent dose of vasopressin. In addition, Ang-(1-7) administration did not significantly change vasopressin plasma levels in water-loaded rats. The antidiuretic effect of Ang-(1-7) in water-loaded rats was associated with a reduction of creatinine clearance (0.68 +/- 0.04 versus 1.38 +/- 0.32 mL/min in vehicle-treated rats, P <.05) and an increase in urine osmolality (266.8 +/- 32.7 versus 182.8 +/- 14 mOsm/kg in vehicle-treated rats, P <.05). An effect of Ang-(1-7) in tubular water transport was demonstrated in vitro by a fourfold increase in the hydraulic conductivity of inner medullary collecting ducts in the presence of 1 nmol/L Ang-(1-7). Subcutaneous administration of A-779 (2.3 to 9.2 nmol/100 g) produced a significant increase in urine volume (4.6 nmol/100 g, 0.45 +/- 0.12 mL/h; vehicle-treated rats, 0.16 +/- 0.03 mL/h; P <.05) comparable to that of acute administration of a vasopressin V2 receptor antagonist. The diuretic effect of A-779 was associated with an increase in creatinine clearance and decrease in urine osmolality. In contrast, no significant effects on urine volume were observed after systemic administration of angiotensin subtype 1 or 2 receptor antagonists (DuP 753 and CGP 42112A, respectively). These findings suggest that endogenous Ang-(1-7), acting on specific receptors, participates in the control of hydroelectrolyte balance by influencing especially water excretion.

Angiotensin-(1-7). A member of circulating angiotensin peptides.

We measured the concentrations of three principal products of the renin-angiotensin system and seven of their metabolites in the plasma of anesthetized normal dogs and in dogs 24 hours after bilateral nephrectomy. The levels of the angiotensin peptides were measured by high-performance liquid chromatography combined with radioimmunoassay using three specific antibodies that recognized different epitotes in the sequences of angiotensin I, angiotensin II, and angiotensin-(1-7). The analysis revealed that angiotensin-(1-7) is present in the plasma of intact (4.9 +/- 2.2 fmol/ml) and nephrectomized (0.5 +/- 0.5 fmol/ml) dogs. An intravenous injection of purified hog renin (0.01 Goldblatt unit/kg) increased plasma levels of angiotensin I, angiotensin II, and angiotensin-(1-7) both before and after nephrectomy. These changes were associated with parallel increases in the concentrations of fragments of the three parent peptides. Administration of MK-422 led to the disappearance of circulating angiotensin II and its fragments both before and after a second injection of the same dose of renin. In contrast, MK-422 augmented the plasma levels of both angiotensin I and angiotensin-(1-7). The concentrations of these two peptides, but not the blood pressure, were again augmented by a second injection of renin given after blockade of converting enzyme. These effects were observed both before and after bilateral nephrectomy. These findings show that angiotensin-(1-7) circulates in the blood of normal and nephrectomized dogs. In addition, we found that angiotensin-(1-7) is generated in the blood from the cleavage of angiotensin I through a pathway independent of converting enzyme (EC 3.4.15.1).

Role of nitric oxide and angiotensin II in the regulation of sympathetic nerve activity in spontaneously hypertensive rats.

This study evaluated the actions of nitric oxide on the blood pressure and renal sympathetic nerve activity responses produced by angiotensin II (Ang II) blockade in conscious spontaneously hypertensive rats. Two days after implantation of electrodes, we measured mean arterial pressure, heart rate, and renal sympathetic nerve activity. Baroreceptor reflex function was assessed with a logistic function curve; the maximum slope of the curve estimated the baroreceptor reflex gain. Data were obtained in rats given acute intravenous administration of either vehicle, the Ang II type 1 receptor antagonist losartan, the type 2 antagonist CGP 42112A, or the converting enzyme inhibitor lisinopril. In comparison with vehicle (-1.1 +/- 0.2%/mm Hg), both losartan (-1.8 +/- 0.3%/mm Hg) and lisinopril (-2.4 +/- 0.2%/mm Hg) significantly increased the maximum gain of the baroreceptor reflex control of nerve activity (p < 0.05). In contrast, the type 2 receptor antagonist did not alter baroreceptor reflex function. Similar studies were performed in rats that received an intravenous injection of NG-monomethyl L-arginine (10 mg/kg). The nitric oxide synthase inhibitor increased baseline blood pressure and decreased renal sympathetic nerve activity. Subsequent administration of losartan or lisinopril returned blood pressure to initial hypertensive level, whereas sympathetic nerve activity was increased to a level above the initial control value. The maximum gain of the baroreceptor reflex control of renal nerve activity was increased after the nitric oxide inhibition. The present study demonstrates that blunted baroreceptor reflex function in conscious spontaneously hypertensive rats is mediated by an Ang II type 1 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones

In a previous study we observed that most neurones in the paraventricular nucleus are excited by angiotensin-(1-7). In comparison with angiotensin III this excitatory action was significantly delayed. The aim of the present microiontophoretic study of angiotensin II-sensitive rat paraventricular neurones was to compare the effect of the angiotensin-analogues angiotensin-(1-7), angiotensin-(2-7), angiotensin II and angiotensin III on the spontaneous activity of these neurones and to test angiotensin receptor subtype 1 antagonists (CGP 46027 or DuP 753) and subtype 2 selective antagonists (CGP 42112A and PD 123177) in order to acquire more evidence of the receptor subtype present. As previously observed angiotensin II, angiotensin III and angiotensin-(1-7) excited most neurones. The effect of angiotensin-(1-7) was usually weaker than that of angiotensin II, and in contrast to angiotensin III the latencies were not significantly different. Angiotensin-(1-7) seemed to be active by itself, because its effect was antagonised by angiotensin receptor antagonists. Angiotensin-(2-7) was mostly inactive, although a few cells were excited. Whereas the excitatory effects of angiotensin-(1-7), angiotensin II and angiotensin III could always be inhibited with both angiotensin receptor subtype antagonists 1 and 2, that produced by angiotensin-(2-7) was only weakly antagonised, if at all. Subtype 1 selective antagonists were effective at lower concentrations than selective subtype 2 antagonists.

Cardiovascular effects produced by micro-injection of angiotensin-(1–7) on vasopressor and vasodepressor sites of the ventrolateral medulla

In this study, we determined the cardiovascular effects produced by micro-injection of the heptapeptide Angiotensin-(1-7) [Ang-(1-7)] into the rat ventrolateral medulla (VLM). Micro-injection of Ang-(1-7) into the rostral VLM and the caudal pressor area of the VLM produced significant increases in arterial pressure, comparable to that observed with micro-injection of Ang II. The changes in arterial pressure were associated with more variable changes in heart rate (HR) (usually tachycardia). On the other hand, micro-injection of Ang-(1-7) into the caudal depressor area induced decreases in arterial pressure and HR. The results suggest that, besides Ang II, Ang-(1-7) is involved in the mediation of the cardiovascular actions of the renin-angiotensin system in the VLM.