Serge P. Bottari

Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications

Thanks to the recent discovery of angiotensin II (ANG II) receptor subtypes linked to different signalling pathways, research in the different areas related to this peptide has regained a strong interest. In the following review, we first describe the biochemistry and actions of angiotensin peptides formed both in the circulation and locally at the tissue and organ level. Evidence for the existence and distribution of ANG II receptor subtypes in mammalian as well as in nonmammalian species and lower organisms is presented. The changes in receptor subtype expression during development and disease are described. The signal transduction mechanisms and biological actions of ANG II mediated by the recently cloned AT1 receptor are reviewed and the recent data concerning the signalling pathways linked to the AT2 receptor are discussed. Finally, based upon their molecular pharmacology, we present evidence and also speculate upon the physiological function of the ANG II receptor subtypes.

The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase

The signalling mechanism and cellular targets of the AT2 receptor are still unknown. We report that angiotensin II (Ang II) inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase (pGC) activity through AT2 receptors in rat adrenal glomerulosa and PC12W cells. This inhibition is blocked by the phosphotyrosine phosphatase (PTPase) inhibitor orthovanadate but not by the Ser/Thr phosphatase inhibitor okadaic acid, suggesting the involvement of a PTPase in this process. Moreover, Ang II induces a rapid, transient and orthovanadate sensitive dephosphorylation of phosphotyrosine containing proteins in PC12W cells. Our findings suggest that AT2 receptors signal through stimulation of a PTPase and that this mechanism is implicated in the regulation of pGC activity. This observation is also the first example of hormonal inhibition of basal pGC activity.

Angiotensin II AT2 receptors do not interact with guanine nucleotide binding proteins.

We have studied the effect of GTP gamma S on the affinity and binding kinetics of angiotensin II in plasma membrane particulate prepared from tissues expressing either only AT1 (human renal artery smooth muscle cells), only AT2 (human myometrium and bovine cerebellar cortex) or both angiotensin II receptor subtypes (rat adrenal glomerulosa). We also examined the ability of angiotensin II to stimulate GTP gamma[35S] incorporation in these membrane preparations. In contrast to its effects on angiotensin II binding to the AT1 receptor, GTP gamma S does not affect binding parameters to the AT2 receptor. Moreover, in tissues expressing solely AT2 receptors, angiotensin II was unable to induce GTP gamma[35S] incorporation. These findings indicate that AT2 receptors do not interact with G-proteins and that angiotensin II must therefore mediate some of its effects through G-protein-independent mechanisms.

The angiotensin AT2 receptor modulates T‐type calcium current in non‐differentiated NG108‐15 cells

We report here that angiotensin II (AII) and the AT2 receptor‐selective ligand, CGP 42112, modulate the T‐type calcium current in non‐differentiated NG108‐15 cells, which express only AT2 receptors. Both peptides decrease the T‐type calcium current at membrane potentials above −40 mV and shift the current—voltage curve at lower potentials with maximal effect between 5 and 10 min after application. These data describe a new cellular response to AII and suggest that the AT2 receptor mediates certain neurophysiological actions of this hormone.

Distribution of angiotensin II receptor subtypes in rat brain nuclei

Angiotensin II (ANG II) receptor subtypes in rat brain were characterized and quantified by competitive radioligand binding using [125I]Sar1 Ile8 angiotensin II ([125I]sarilesin) as a tracer and ANG II, sarilesin and the subtype selective ligands DuP 753 (AT1) and CGP 42112A (AT2) as competitors. The distribution of AT1 and AT2 receptors was determined in midbrain, brainstem, hypothalamus as well as in individual hypothalamic and periventricular nuclei. Whereas in midbrain and brainstem the AT1: AT2 ratio was 40%: 60% and 70%: 30% respectively, the AT1 receptors were by far predominant in hypothalamus and in the nuclei investigated. Interestingly, we found that approximately 25% of the ANG II receptors in hypothalamus did not bind DuP 753 even at 0.1 mM. These sites which bind CGP 42112A, ANG II and sarilesin may represent a third ANG II receptor subtype.

Agonistic and antagonistic properties of angiotensin analogs at the AT2 receptor in PC12W cells

Despite some recent reports describing the effects of AT2 receptor selective ligands in vitro and in vivo, the physiological function of this receptor is still a matter of debate. This problem stems amongst others from the difficulty in interpreting results from in vivo experiments with drugs of which it is not known whether they act as agonists or antagonists. We reported earlier that angiotensin II inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase activity through AT2 receptors in PC12W cells. We have used this parameter in intact PC12W cells in order to determine the pharmacological properties of different widely used angiotensin receptor ligands. We found CGP 42112 to behave as a full agonist in this system, whereas PD 123319 and Sar Ile angiotensin II act as antagonists. As expected, the AT1 antagonist losartan did not affect this response.

Radioiodinated CGP 42111A: A novel high affinity and highly selective ligand for the characterization of angiotensin AT2 receptors

CGP 42112A, a potent angiotensin AT2 receptor selective ligand, was radio-iodinated and its binding characteristics compared with those of [125I]angiotensin II. In human myometrium (only AT2 expressed), binding was saturable (Kd 1.03 x 10(-10) M; Bmax 807 fmol/mg) and reversible (K+1 1.89 x 10(8) M-1.min-1; K-1 3.77 x 10(-3) min-1). The order of potency of a number of peptides and non-peptides was the same as when [125I] angiotensin II was used as tracer. No specific binding could be detected on membranes from vascular smooth muscle cells (only AT1 expressed). In rat adrenal glomerulosa membranes (mixed AT1/AT2), [125I]CGP 42112A bound only to AT2. [125I]CGP 42112A can therefore be used as a specific probe for AT2 receptors and will be especially useful in tissues where other subtypes are also present.

Characterization and distribution of angiotensin II binding sites in fetal and neonatal astrocytes from different rat brain regions

Although angiotensin II (Ang II) binding sites have been extensively investigated in brain, revealing the presence of both AT1 and AT2 subtypes in various areas, the question as to which cells express AT1 and AT2 sites is still open. We report here that primary cultures of astrocytes obtained from various brain regions of fetal (F17) and one-day-old rats express Ang II binding sites belonging only to the AT1 subtype. The binding sites have the same binding profile in all regions tested; however, much less binding was observed in membranes of astrocytes derived from cortical than from subcortical regions and almost none were found in neonatal cortex. In addition, the dispersion method used at the onset of culture affects the number of binding sites present at the end of the culture period.

Labelling of vasopressin and oxytocin receptors from the human uterus

Four labelled ligands, [3H]arginine vasopressin ([3H]AVP), [3H]oxytocin ([3H]OT), [3H]d(CH2)5[Tyr(Me)2]AVP ([3H]VPA), and [125I]d(CH2)5[Tyr(Me)2-Thr4-Orn8-Tyr(NH2)9]OT([125I]OTA] and nine unlabelled analogues exhibiting enhanced selectivity for rat oxytocin (OT) and vasopressin (VP) receptors were used to characterize OT and VP receptors on myometrial membranes from non-pregnant and pregnant human uteri. On membranes from non-pregnant uteri, [3H]AVP, [3H]VPA, and [125I]OTA labelled with high affinity (Kd values: 3.2, 2 and 0.8 nM, respectively) a major and apparently homogeneous population of sites, the ligand selectivity of which resembled that of rat V1a VP receptors. On membranes from pregnant and non-pregnant uteri, [3H]OT labelled a single population of high-affinity sites that could be distinguished from VP receptors on the basis of ligand selectivity. Several analogues (in particular [125I]OTA) that are highly selective for rat OT receptors exhibited a much less pronounced selectivity for human OT receptors. Experiments with [3H]VPA allowed detection of VP receptors on myometrical membranes from pregnant uteri and confirmed that only OT but not VP receptors increase during pregnancy in humans.