Angiotensin II (Ang II) stimulates water and saline intakes when injected into the brain of rats. This arises from activation of the AT1 Ang II receptor subtype. Acute repeated injections, however, decrease the water intake response to Ang II without affecting saline intake. Previous studies provide evidence that Ang II-induced water intake is mediated via the classical G protein coupling pathway, whereas the saline intake caused by Ang II is mediated by an ERK 1/2 MAP kinase signaling pathway. Accordingly, the different behavioral response to repeated injections of Ang II may reflect a selective effect on G protein coupling. To test this hypothesis, we examined the binding of a radiolabeled agonist (125I-sarcosine1 Ang II) and a radiolabeled antagonist (125I-sarcosine1, isoleucine8 Ang II) in brain homogenates and tissue sections prepared from rats given repeated injections of Ang II or vehicle. Although no treatment-related differences were found in hypothalamic homogenates, a focus on specific brain structures using receptor autoradiography, found that the desensitization treatment reduced binding of both radioligands in the paraventricular nucleus of the hypothalamus (PVN) and median preoptic nucleus (MnPO), but not in the subfornical organ (SFO). Because G protein coupling is reported to have a selective effect on agonist binding without affecting antagonist binding, these findings do not support a G protein uncoupling treatment effect. This suggests that receptor number is more critical to the water intake response than the saline intake response, or that pathways downstream from the G protein mediate desensitization of the water intake response.

The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding <300 fmol/g initial wet weight was in the mediolateral medulla. 125I-SI Ang II binding was substantially higher by an average of 85% in wild-type mouse brains compared to neurolysin knockout brains, suggesting the presence of an additional non-AT1, non-AT2, non-neurolysin Ang II binding site in the mouse brain. Binding of 125I-SI Ang II to neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT1, non-AT2, non-neurolysin binding was also highest in the hypothalamus but had a different distribution than neurolysin. There was a significant reduction in AT2 receptor binding in the neurolysin knockout brain and a trend towards decreased AT1 receptor binding. In the neurolysin knockout brains, the size of the lateral ventricles was increased by 56% and the size of the mid forebrain (−2.72 to +1.48 relative to Bregma) was increased by 12%. These results confirm the identity of neurolysin as a novel Ang II binding site, suggesting that neurolysin may play a significant role in opposing the pathophysiological actions of the brain RAS and influencing brain morphology.

The discovery of a novel non-AT1, non-AT2 binding site for angiotensins in the rodent brain and testis that is unmasked by the organomercurial compound para-chloromercuribenzoic acid (PCMB) has catalyzed efforts to purify and characterize this protein. We recently reported that this protein is neurolysin and now report upon the specificity of this binding site for various neuropeptides. Competition binding assays in rat brain and testis used 125I-Sar1, Ile8 angiotensin II (Ang II) as the radioligand in the presence of saturating concentrations of AT1 and AT2 receptor antagonists and 100 µM parachloromercuribenzoate. Primary screening of 36 peptides and other compounds at 10 µM concentration revealed seven peptides that inhibited specific binding > 50%: ghrelin, Tyr1 S36057 (a melanin-concentrating hormone receptor ligand), orphanin FQ and its congeners (Tyr1 and Tyr14), Dynorphin A (1–8), and Ang (1–9). The selective neurolysin inhibitor Proline-Isoleucine dipeptide was inactive at 1 mM. These results suggest that the ability of PCMB to unmask high affinity binding of Ang II to neurolysin is a pharmacological effect and that neurolysin may significantly affect the activity of the reninangiotensin system.

Recently a novel, non‐AT1, non‐AT2 binding site for angiotensin II (Ang II) was identified as the metalloendopeptidase neurolysin. To assess the localization of this binding site in brain, 3 mice deficient in neurolysin and 3 wild‐type mouse brains were evaluated for radioligand binding with 125I‐Sar1, Ile8 Ang II (250 pM) in the presence of AT1 and AT2 receptor‐saturating concentrations of losartan and PD123319, and 150 μM p‐chloromercuribenzoate using in vitro autoradiography. Specific (10 μM Ang II displaceable) 125I‐Sar1, Ile8 Ang II binding in wild‐type mouse brains was abundant, with highest levels in the molecular layer of the cerebellum, cerebral cortex, hippocampus, amygdala, caudate‐putamen, hypothalamus, lateral septum, and external plexiform layer of the olfactory bulb. Specific 125I‐Sar1, Ile8 Ang II binding in the neurolysin‐deficient mouse brains was profoundly reduced, however, the extent of reduction was region‐specific. Large decreases were seen in the cerebral cortex, substantia nigra, hippocampus, paraventricular thalamus, lateral septum, nucleus accumbens. Cerebellar cortex and hypothalamus showed moderate reduction in binding. These results confirm neurolysin as the novel non‐AT1, non‐AT2 receptor binding protein, but may reveal additional non‐AT1 non‐AT2 binding sites in the mouse brain. Supported by NHLBI HL‐096357