Robert E. Widdop

Angiotensin AT2 receptors: cardiovascular hope or hype?

British Journal of Pharmacology (2003) 140, 809–824. doi:10.1038/sj.bjp.0705448

Nitroxyl (HNO): the Cinderella of the nitric oxide story

Until recently, most of the biological effects of nitric oxide (NO) have been attributed to its uncharged state (NO*), yet NO can also exist in the reduced state as nitroxyl (HNO or NO(-)). Putatively generated from both NO synthase (NOS)-dependent and -independent sources, HNO is rapidly emerging as a novel entity with distinct pharmacology and therapeutic advantages over its redox sibling, NO*. Thus, unlike NO*, HNO can target cardiac sarcoplasmic ryanodine receptors to increase myocardial contractility, can interact directly with thiols and is resistant to both scavenging by superoxide (*O2-) and tolerance development. HNO donors are protective in the setting of heart failure in which NO donors have minimal impact. Here, we discuss the unique pharmacology of HNO versus NO* and highlight the therapeutic potential of HNO donors in the treatment of cardiovascular disease.

AT2 receptors: Functional relevance in cardiovascular disease

Abstract The renin angiotensin system (RAS) is intricately involved in normal cardiovascular homeostasis. Excessive stimulation by the octapeptide angiotensin II contributes to a range of cardiovascular pathologies and diseases via angiotensin type 1 receptor (AT1R) activation. On the other hand, tElsevier Inc.he angiotensin type 2 receptor (AT2R) is thought to counter-regulate AT1R function. In this review, we describe the enhanced expression and function of AT2R in various cardiovascular disease settings. In addition, we illustrate that the RAS consists of a family of angiotensin peptides that exert cardiovascular effects that are often distinct from those of Ang II. During cardiovascular disease, there is likely to be an increased functional importance of AT2R, stimulated by Ang II, or even shorter angiotensin peptide fragments, to limit AT1R-mediated overactivity and cardiovascular pathologies.

Angiotensin-(1–7) Acts as a Vasodepressor Agent Via Angiotensin II Type 2 Receptors in Conscious Rats

Given that angiotensin-(1–7) (Ang-[1–7]) has been frequently reported to exert direct in vitro vascular effects but less often in vivo, we investigated whether a vasodepressor effect of Ang-(1–7) could be unmasked acutely in conscious spontaneously hypertensive rats (SHR) against a background of angiotensin II type 1 (AT1) receptor blockade. Mean arterial pressure (MAP) and heart rate were measured over a 5-day protocol in various groups of rats randomized to receive the following drug combinations: saline, AT1 receptor (AT1R) antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, Ang-(1–7) (5 pmol/min) alone, candesartan plus Ang-(1–7), and candesartan plus Ang-(1–7) and angiotensin II type 2 (AT2) receptor (AT2R) antagonist PD123319 (50 &mgr;g/kg per minute). In Wistar-Kyoto (WKY) rats, saline, Ang-(1–7), or candesartan alone caused no significant alteration in MAP, whereas Ang-(1–7) coadministered with candesartan caused a marked, sustained reduction in MAP. A similar unmasking of a vasodepressor response to Ang-(1–7) during AT1R blockade was observed in SHR. Moreover, the AT2R antagonist PD123319 markedly attenuated the enhanced depressor response evoked by the Ang-(1–7)/candesartan combination in SHR and WKY rats, whereas in other experiments, the putative Ang-(1–7) antagonist A-779 (5 and 50 pmol/min) did not attenuate this vasodepressor effect. In separate experiments, the bradykinin type 2 receptor antagonist HOE 140 (100 &mgr;g/kg IV) or the NO synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester (1 mg/kg IV) abolished the depressor effect of Ang-(1–7) in the presence of candesartan. Collectively, these results suggest that Ang-(1–7) evoked a depressor response during AT1R blockade via activation of AT2R, which involves the bradykinin–NO cascade.

Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors

AT1R (angiotensin type 1 receptor) and AT2R (angiotensin type 2 receptor) are well known to be involved in the complex cardiovascular actions of AngII (angiotensin II). However, shorter peptide fragments of AngII are thought to have biological activity in their own right and elicit effects that oppose those mediated by AngII. In the present study, we have used HEK (human embryonic kidney)-293 cells stably transfected with either AT1R or AT2R to perform a systematic analysis of binding affinities of all the major angiotensin peptides. Additionally, we tested the novel AT2R agonist Compound 21, as well as the MasR (Mas receptor) agonist and antagonist AVE0991 and A-779 respectively, for their ability to bind to AT1R or AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using 125I-[Sar1Ile8]AngII on the AT1R-transfected HEK-293 cells revealed only AngII, AngIII [angiotensin III; angiotensin-(2-8)] and candesartan to have high affinity for AT1R. In the AT2R-transfected HEK-293 cells, competition for 125I-[Sar1Ile8]AngII binding was observed for all ligands except candesartan, AVE0991 and A-779, the latter two compounds having negligible affinity at either AT1R or AT2R. The rank order of affinity of ligands at AT2R was CGP42112>AngII≥AngIII>Compound 21≥PD123319≫AngIV [angiotensin IV; angiotensin-(3-8)]>Ang-(1-7) [angiotensin-(1-7)]. Of note, although AngIV and Ang-(1-7) exhibited only modest affinity at AT2R compared with AngII, these two angiotensin peptides, together with AngIII, had substantial AT2R selectivity over AT1R. Collectively, our results suggest that shorter angiotensin peptides can act as endogenous ligands at AT2R.

AT2 Receptor Stimulation Enhances Antihypertensive Effect of AT1 Receptor Antagonist in Hypertensive Rats

In the present study, we investigated the role of the angiotensin type 2 (AT(2)) receptor in the regulation of blood pressure in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We tested the hypothesis that AT(2) receptor activation may contribute to the antihypertensive effects of angiotensin type 1 (AT(1)) receptor antagonists. Mean arterial pressure (MAP) and heart rate were measured over a 4-day protocol in various groups of rats that received the following drug combinations: the AT(1) receptor antagonist candesartan (0.01 or 0.1 mg/kg IV) alone, the AT(2) receptor agonist CGP42112 (1 microg/kg per minute) alone, and candesartan plus CGP42112. In both SHR and WKY, 4-hour infusions of saline and CGP42112 alone did not alter MAP. In WKY, both doses of candesartan alone caused small decreases in MAP, which were similar when combined with CGP42112. In SHR, candesartan (0.1 mg/kg) caused an immediate, marked decrease in MAP, which was unaffected when combined with CGP42112. By contrast, in separate SHR, a 10-fold lower dose of candesartan (0.01 mg/kg) caused a slower-onset depressor response, which was enhanced when combined with CGP42112. The involvement of AT(2) receptors was confirmed in another group of SHR, since this facilitation of the antihypertensive effect of candesartan by CGP42112 was abolished by the coinfusion of the AT(2) receptor antagonist PD123319 (50 microg/kg per minute) with the candesartan/CGP42112 combination. Collectively, these data suggest that in SHR, AT(2) receptor activation can facilitate the initial depressor response caused by an AT(1) receptor antagonist.

Stimulation of angiotensin AT2 receptors by the non‐peptide agonist, Compound 21, evokes vasodepressor effects in conscious spontaneously hypertensive rats

Background and purpose:  Angiotensin type 2 receptor (AT2 receptor) stimulation evokes vasodilator effects in vitro and in vivo that oppose the vasoconstrictor effects of angiotensin type 1 receptors (AT1 receptors). Recently, a novel non‐peptide AT2 receptor agonist, Compound 21, was described, which exhibited high AT2 receptor selectivity.

Vasoprotective and Atheroprotective Effects of Angiotensin (1-7) in Apolipoprotein E–Deficient Mice

Objective—To evaluate the effectiveness of long-term angiotensin (Ang) (1-7) treatment to inhibit the progression of atherosclerosis in apolipoprotein E-deficient (ApoE−/−) mice. Methods and Results—Ang (1-7) is a heptapeptide fragment that has been proposed to counterregulate the Ang II proatherogenic effects. The effect of long-term 4-week Ang (1-7) treatment on both inhibition of atherosclerotic lesion development and improvement of endothelial function was examined in apolipoprotein E−/− mice that had been fed an atherogenic high-fat (21%) diet for 16 weeks. Chronic Ang (1-7) treatment significantly improved endothelial function, an effect reversed with either angiotensin type 2 (AT2) or Mas receptor blockade. In these vessels, Ang (1-7) treatment significantly decreased superoxide production and increased endothelial nitric oxide synthase immunoreactivity when compared with vehicle treatment. These effects were blocked by both AT2 and Mas receptor antagonists. Lesion development, assessed as both fatty deposits (oil red O) and intima to media ratio, was also significantly decreased with Ang (1-7) treatment compared with respective controls. Cotreatment with either AT2 or Mas receptor antagonists reversed Ang (1-7)–mediated reduction in lesion development. Conclusion—Long-term Ang (1-7) treatment caused both vasoprotection, via improvement in endothelial function, and atheroprotection, with a reduction in lesion progression in a model of atherosclerosis. These effects appear to be mediated by the restoration of nitric oxide bioavailability and involve a complex interaction of both Mas and AT2 receptors.

Enhanced Angiotensin II Type 2 Receptor Mechanisms Mediate Decreases in Arterial Pressure Attributable to Chronic Low-Dose Angiotensin II in Female Rats

The renin-angiotensin system is a far more complex enzymatic cascade than realized previously. Mounting evidence suggests sex-specific differences in the regulation of the renin-angiotensin system and arterial pressure. We examined the hemodynamic responses, angiotensin II receptor subtypes, and angiotensin-converting enzyme 2 gene expression levels after graded doses of angiotensin II in males and females. Mean arterial pressure was measured via telemetry in male and female rats in response to a 2-week infusion of vehicle, low-dose (50 ng/kg per minute SC) or high-dose (400 ng/kg per minute SC) angiotensin II. The effect of concurrent infusion of the angiotensin II type 2 receptor (AT2R) blocker (PD123319) was also examined. The arterial pressure response to high-dose angiotensin II was attenuated in females compared with males (24±8 mm Hg versus 42±5 mm Hg; P for the interaction between sex and treatment <0.002). Remarkably, low-dose angiotensin II decreased arterial pressure (11±4 mm Hg; P for the interaction between sex and treatment <0.02) at a dose that did not have an effect in males. This decrease in arterial pressure in females was abolished by AT2R blockade. Renal AT2R, angiotensin-converting enzyme 2, and left ventricular AT2R mRNA gene expressions were markedly greater in females than in males with a renal angiotensin II type 1a receptor:AT2R ratio of ≈1 in females. Angiotensin II infusion did not affect renal AT2R mRNA expression but resulted in significantly less left ventricular mRNA expression. Renal angiotensin-converting enzyme 2 mRNA expression levels were greater in females than in males treated with high-dose angiotensin II (≈2.5 fold; P for the interaction between sex and treatment <0.05). In females, enhancement of the vasodilatory arm of the renin-angiotensin system, in particular, AT2R and angiotensin-converting enzyme 2 mRNA expression, may contribute to the sex-specific differences in response to renin-angiotensin system activation.

Nitroxyl Anion Donor, Angeli’s Salt, Does Not Develop Tolerance in Rat Isolated Aortae

The nitroxyl anion (HNO) is emerging as a novel regulator of cardiovascular function with therapeutic potential in the treatment of diseases such as heart failure. It remains unknown whether tolerance develops to HNO donors, a limitation of currently used nitrovasodilators. The susceptibility of the HNO donor, Angeli’s salt (AS), to the development of vascular tolerance was compared with the NO donors, glyceryl trinitrate (GTN) and diethylamine/NONOate (DEA/NO) in rat isolated aortae. Vasorelaxation to AS was attenuated (P<0.01) by the HNO scavenger l-cysteine, whereas the sensitivity to GTN and DEA/NO was decreased (P<0.01) by the NO· scavenger carboxy-[2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidozoline-1-oxy-3-oxide]. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one impaired responses to GTN≥AS≫DEA/NO. Pretreatment with 10, 30, and 100 &mgr;mol/L of GTN for 60 minutes induced a 4- (P<0.05), 13- (P<0.01), and 48-fold (P<0.01) decrease in sensitivity to GTN, demonstrating tolerance development. In contrast, pretreatment with AS or DEA/NO (10, 30, and 100 &mgr;mol/L) did not alter their subsequent vasorelaxation. All of the nitrovasodilators (30 &mgr;mol/L) displayed a similar time course of vasorelaxation and cGMP accumulation over a 60-minute period. Unlike vasorelaxation, the magnitude of peak cGMP accumulation differed substantially: DEA/NO≫AS>GTN. GTN did not induce cross-tolerance to either AS or DEA/NO. In contrast, pre-exposure to DEA/NO, but not AS, caused a concentration-dependent attenuation (P<0.01) of GTN-mediated relaxation, which was negated by the protein kinase G inhibitor guanosine 3′,5′-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-,Rp-isomer, triethylammonium salt. In conclusion, vascular tolerance does not develop to HNO, nor does cross-tolerance between HNO and GTN occur. Thus, HNO donors may have therapeutic advantages over traditional nitrovasodilators.