Patrick Vanderheyden

G-protein-coupled receptor mas is a physiological antagonist of the angiotensin II type 1 receptor

Background—We previously identified the G-protein–coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT1. Methods and Results—In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT1 receptor–mediated, angiotensin II–induced production of inositol phosphates and mobilization of intracellular Ca2+ was diminished by 50% after coexpression of Mas, despite a concomitant increase in angiotensin II binding capacity. Mas and the AT1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT1 receptor; mice lacking the Mas gene show enhanced angiotensin II–mediated vasoconstriction in mesenteric microvessels. Conclusions—These results demonstrate that Mas can hetero-oligomerize with the AT1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein–coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT1-Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.

Angiotensin IV Activates the Nuclear Transcription Factor-κB and Related Proinflammatory Genes in Vascular Smooth Muscle Cells

Inflammation is a key event in the development of atherosclerosis. Nuclear factor-&kgr;B (NF-&kgr;B) is important in the inflammatory response regulation. The effector peptide of the renin angiotensin system Angiotensin II (Ang II) activates NF-&kgr;B and upregulates some related proinflammatory genes. Our aim was to investigate whether other angiotensin-related peptides, as the N-terminal degradation peptide Ang IV, could regulate proinflammatory factors (activation of NF-&kgr;B and related genes) in cultured vascular smooth muscle cells (VSMCs). In these cells, Ang IV increased NF-&kgr;B DNA binding activity, caused nuclear translocation of p50/p65 subunits, cytosolic I&kgr;B degradation and induced NF-&kgr;B–dependent gene transcription. Ang II activates NF-&kgr;B via AT1 and AT2 receptors, but AT1 or AT2 antagonists did not inhibit NF-&kgr;B activation caused by Ang IV. In VSMC from AT1a receptor knockout mice, Ang IV also activated NF-&kgr;B pathway. In those cells, the AT4 antagonist divalinal diminished dose-dependently Ang IV–induced NF-&kgr;B activation and prevented I&kgr;B degradation, but had no effect on the Ang II response, indicating that Ang IV activates the NF-&kgr;B pathway via AT4 receptors. Ang IV also increased the expression of proinflammatory factors under NF-&kgr;B control, such as MCP-1, IL-6, TNF-&agr;, ICAM-1, and PAI-1, which were blocked by the AT4 antagonist. Our results reveal that Ang IV, via AT4 receptors, activates NF-&kgr;B pathway and increases proinflammatory genes. These data indicate that Ang IV possesses proinflammatory properties, suggesting that this Ang degradation peptide could participate in the pathogenesis of cardiovascular diseases.

Angiotensin IV is a potent agonist for constitutive active human AT1 receptors: distinct roles of the N- and C-terminal residues of angiotensin II during AT1 receptor activation*

The octapeptide hormone, angiotensin II (Ang II), exerts its major physiological effects by activating AT1 receptors. In vivo Ang II is degraded to bioactive peptides, including Ang III (angiotensin-(2–8)) and Ang IV (angiotensin-(3–8)). These peptides stimulate inositol phosphate generation in human AT1 receptor expressing CHO-K1 cells, but the potency of Ang IV is very low. Substitution of Asn111 with glycine, which is known to cause constitutive receptor activation by disrupting its interaction with the seventh transmembrane helix (TM VII), selectively increased the potency of Ang IV (900-fold) and angiotensin-(4–8), and leads to partial agonism of angiotensin-(5–8). Consistent with the need for the interaction between Arg2 of Ang II and Ang III with Asp281, substitution of this residue with alanine (D281A) decreased the peptides potency without affecting that of Ang IV. All effects of the D281A mutation were superseded by the N111G mutation. The increased affinity of Ang IV to the N111G mutant was also demonstrated by binding studies. A model is proposed in which the Arg2-Asp281 interaction causes a conformational change in TM VII of the receptor, which, similar to the N111G mutation, eliminates the constraining intramolecular interaction between Asn111 and TM VII. The receptor adopts a more relaxed conformation, allowing the binding of the C-terminal five residues of Ang II that switches this “preactivated” receptor into the fully active conformation.

Involvement of insulin-regulated aminopeptidase in the effects of the renin-angiotensin fragment angiotensin IV: a review.

For decades, angiotensin (Ang) II was considered as the end product and the only bioactive peptide of the renin–angiotensin system (RAS). However, later studies revealed biological activity for other Ang fragments. Amongst those, Ang IV has drawn a lot of attention since it exerts a wide range of central and peripheral effects including the ability to enhance learning and memory recall, anticonvulsant and anti-epileptogenic properties, protection against cerebral ischemia, activity at the vascular level and an involvement in atherogenesis. Some of these effects are AT1 receptor dependent but others most likely result from the binding of Ang IV to insulin-regulated aminopeptidase (IRAP) although the exact mechanism(s) of action that mediate the Ang IV-induced effects following this binding are until now not fully known. Nevertheless, three hypotheses have been put forward: since Ang IV is an inhibitor of the catalytic activity of IRAP, its in vivo effects might result from a build-up of IRAP’s neuropeptide substrates. Second, IRAP is co-localized with the glucose transporter GLUT4 in several tissue types and therefore, Ang IV might interact with the uptake of glucose. A final and more intriguing hypothesis ascribes a receptor function to IRAP and hence an agonist role to Ang IV. Taken together, it is clear that further work is required to clarify the mechanism of action of Ang IV. On the other hand, a wide range of studies have made it clear that IRAP might become an important target for drug development against different pathologies such as Alzheimer’s disease, epilepsy and ischemia.

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

The renin angiotensin system (RAS) produced hormone peptides regulate many vital body functions. Dysfunctional signaling by receptors for RAS peptides leads to pathologic states. Nearly half of humanity today would likely benefit from modern drugs targeting these receptors. The receptors for RAS peptides consist of three G-protein–coupled receptors—the angiotensin II type 1 receptor (AT1 receptor), the angiotensin II type 2 receptor (AT2 receptor), the MAS receptor—and a type II trans-membrane zinc protein—the candidate angiotensin IV receptor (AngIV binding site). The prorenin receptor is a relatively new contender for consideration, but is not included here because the role of prorenin receptor as an independent endocrine mediator is presently unclear. The full spectrum of biologic characteristics of these receptors is still evolving, but there is evidence establishing unique roles of each receptor in cardiovascular, hemodynamic, neurologic, renal, and endothelial functions, as well as in cell proliferation, survival, matrix-cell interaction, and inflammation. Therapeutic agents targeted to these receptors are either in active use in clinical intervention of major common diseases or under evaluation for repurposing in many other disorders. Broad-spectrum influence these receptors produce in complex pathophysiological context in our body highlights their role as precise interpreters of distinctive angiotensinergic peptide cues. This review article summarizes findings published in the last 15 years on the structure, pharmacology, signaling, physiology, and disease states related to angiotensin receptors. We also discuss the challenges the pharmacologist presently faces in formally accepting newer members as established angiotensin receptors and emphasize necessary future developments.

From angiotensin IV binding site to AT4 receptor

One of the fragments of the cardiovascular hormone Angiotensin II incited the interest of several research groups. This 3-8 fragment, denoted as Angiotensin IV (Ang IV) causes a number of distinct biological effects (see Introduction), unlikely to be explained by its weak binding to AT(1) and/or AT(2) receptors. Moreover the discovery of high affinity [(125)I]-Ang IV binding sites and their particular tissue distribution led to the concept of the AT(4) receptor. An important breakthrough was achieved by defining the AT(4) receptor as the membrane-bound insulin-regulated aminopeptidase (IRAP). Crucial for the definition as a receptor the binding of the endogenous ligand(s) should be linked to particular cellular and/or biochemical processes. With this respect, cultured cells offer the possibility to study the presence of binding sites in conjunction with ligand induced signaling. This link is discussed for the AT(4) receptor by providing an overview of the cellular effects by AT(4) ligands.

Metabolism of angiotensin II is required for its in vivo effect on dopamine release in the striatum of the rat

The effect of angiotensin (Ang) IV, an inhibitor of insulin‐regulated aminopeptidase (IRAP), on extracellular dopamine levels in the striatum of freely moving rats was examined using in vivo microdialysis. The Ang IV was administered locally in the striatum through the microdialysis probe. A concentration‐dependent (10–100 µm) increase in extracellular striatal dopamine was observed. The effect of Ang II (10–100 µm), which has only a weak affinity for IRAP, was similar to that observed for Ang IV. The effects of both peptides could not be blocked by the AT1 antagonist candesartan (10 nm and 1 µm) nor by the AT2 antagonist S‐(+)‐1‐([4‐(dimethylamino)‐3‐methylphenyl]methyl)‐5‐(diphenyl‐acetyl)‐4,5,6,7‐tetrahydro‐1H‐amidazo(4,5‐c) pyridine‐6‐carboxylic acid (1 µm), suggesting that the observed effects are both AT1 and AT2 independent. The effect of Ang II could be blocked by the aminopeptidase‐A inhibitor (S)‐3‐amino‐4‐mercaptobutylsulphonic acid as well as the aminopeptidase‐N inhibitor 2‐amino‐4‐methylsulphonylbutane thiol, indicating that the effect of Ang II is mediated via metabolism into Ang IV. Other IRAP inhibitors, such as Divalinal‐Ang IV and LVV‐haemorphin‐7, had similar effects on extracellular dopamine levels as compared with Ang IV. We propose a role for IRAP as mediator for the effects of Ang IV and related peptides on extracellular dopamine levels in the striatum of the rat.

The role of the central renin-angiotensin system in Parkinson’s disease

Since the discovery of a renin-angiotensin system (RAS) in the brain, several studies have linked this central RAS to neurological disorders such as ischaemia, Alzheimer’s disease and depression. In the last decade, evidence has accumulated that the central RAS might also play a role in Parkinson’s disease. Although the exact cause of this progressive neurodegenerative disorder of the basal ganglia remains unidentified, inflammation and oxidative stress have been suggested to be key factors in the pathogenesis and the progression of the disease. Since angiotensin II is a pro-inflammatory compound that can induce the production of reactive oxygen species due to activation of the NADPH-dependent oxidase complex, this peptide might contribute to dopaminergic cell death. In this review, three different strategies to interfere with the pathogenesis or the progression of Parkinson’s disease are discussed. They include inhibition of the angiotensin-converting enzyme, blockade of the angiotensin II type 1 receptor and stimulation of the angiotensin II type 2 receptor.

Agonist induction and conformational selection during activation of a G-protein-coupled receptor

Substitutions of Asn111 of the AT(1) angiotensin receptor and mutations of the corresponding amino acids in other G-protein-coupled receptors (GPCRs) cause constitutive receptor activation. Ligand binding and signalling of constitutively active mutant GPCRs are discussed and similarities and differences during the activation of amine and peptide GPCRs are identified. Studies using the AT(1) receptor suggest that conformational selection is not sufficient to explain the mechanism of receptor activation, and that agonist binding to the receptor provides energy to induce activation of the receptor. Because agonist binding also actively facilitates the conformational rearrangements leading to activation of other GPCRs we propose that agonist induction should be considered as a general mechanism of GPCR activation.

Clozapine, atypical antipsychotics, and the benefits of fast-off D2 dopamine receptor antagonism

Drug–receptor interactions are traditionally quantified in terms of affinity and efficacy, but there is increasing awareness that the drug-on-receptor residence time also affects clinical performance. While most interest has hitherto been focused on slow-dissociating drugs, D2 dopamine receptor antagonists show less extrapyramidal side effects but still have excellent antipsychotic activity when they dissociate swiftly. Fast dissociation of clozapine, the prototype of the “atypical antipsychotics”, has been evidenced by distinct radioligand binding approaches both on cell membranes and intact cells. The surmountable nature of clozapine in functional assays with fast-emerging responses like calcium transients is confirmatory. Potential advantages and pitfalls of the hitherto used techniques are discussed, and recommendations are given to obtain more precise dissociation rates for such drugs. Surmountable antagonism is necessary to allow sufficient D2 receptor stimulation by endogenous dopamine in the striatum. Simulations are presented to find out whether this can be achieved during sub-second bursts in dopamine concentration or rather during much slower, activity-related increases thereof. While the antagonist’s dissociation rate is important to distinguish between both mechanisms, this becomes much less so when contemplating time intervals between successive drug intakes, i.e., when pharmacokinetic considerations prevail. Attention is also drawn to the divergent residence times of hydrophobic antagonists like haloperidol when comparing radioligand binding data on cell membranes with those on intact cells and clinical data.