Pharmacological characterization of a novel non-AT1, non-AT2 angiotensin binding site identified as neurolysin

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 (125)I-Sar(1), Ile(8) 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 \u3e50 %: ghrelin, Tyr(1) S36057 (a melanin-concentrating hormone receptor ligand), orphanin FQ and its congeners (Tyr(1) and Tyr(14)), 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 renin-angiotensin system

Loss of 125I-Sarcosine1, Isoleucine8 Angiotensin II Binding in the Brain of Neurolysin Knock-out Mice

Objective. To assess the distribution of a novel non-AT1, non-AT2 binding site in mouse brain and determine whether this binding site is the metalloendopeptidase neurolysin (E.C.3.4.24.16). Background.The brain renin-angiotensin system profoundly affects the cardiovascular system, often with pathological consequences. However, many unresolved questions about its functionality remain. Recently a novel, non-AT1, non-AT2 binding site for angiotensin II (AngII) was found in the mammalian brain. Methods. To assess the localization of this binding site in mouse brain, 3 mouse brains deficient in neurolysin as well as 3 wild-type mouse brains were evaluated for radioligand binding with 125ISarcosine1, Isoleucine8 AngII (250 pM) in the presence of receptor-saturating concentrations of losartan (an AT1 receptor antagonist), PD123319 (an AT2 receptor antagonist) and 150 mM parachloromercuribenzoate (to unmask the binding site) using in vitro autoradiography. Results. Specific (10 μM AngII displaceable) 125I-Sarcosine1,Isoleucine8 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 olfactory bulb. Specific 125I-Sar1,Ile8 AngII binding was profoundly reduced in neurolysin-deficient mouse brains. However, the extent of the reduction was region-specific. Greatest decreases were seen in the cerebral cortex, substantia nigra, hippocampus, paraventricular thalamus, lateral septum, and nucleus accumbens. The cerebellar cortex and hypothalamus showed the smallest reductions in 125I-Sarcosine1,Isoleucine8 AngII binding. Conclusions. These results verify that the previously-reported novel non-AT1, non-AT2 receptor binding protein, is neurolysin, but that there may be additional non-AT1 non-AT2 binding sites in the mouse brain. Grants. Supported by NHLBI HL-096357

Identification of Membrane-bound Variant of Endopeptidase 24.16 as the non-AT1, non-AT2Angiotensin II Binding Site

Objective. To determine the identity of the non-AT1, non-AT2 angiotensin II (AngII) binding site in the mammalian brain. Background. New discoveries about the renin-angiotensin system continue to abound in the 21st century, e.g., discovery of a renin receptor, identification of the mas oncogene protein as the angiotensin 1-7 receptor, discovery of an enzyme (angiotensin-converting enzyme-2, ACE-2) that converts AngII to angiotensin 1-7, use of AT2 receptor agonists as therapeutic agents, and discovery of a novel non-AT1, non-AT2 angiotensin binding site in the brain. Methods. An angiotensin analog, photoaffinity probe 125I-sarcosine1,benzoylphenylalanine8-AngII was used to specifically label the non-AT1, non-AT2 AngII binding site in membranes prepared from mouse forebrain and HEK293 cell overexpressing mouse neurolysin in the presence of AT1 receptor-saturating concentrations of losartan, AT2 receptor-saturating concentrations of PD123319, and 150 μM parachloromercuribenzoate. The 125Isarcosine1, benzoylphenylalanine8-AngII radiolabeled binding site was purified by 2-Dimensional electrophoresis and analyzed by mass spectrometry, or solubilized and immunoprecipitated. Saturation binding assays or in vitro autoradiography with 125I-sarcosine1,isoleucine8-AngII measured expression of the binding site in brain and HEK293 cell membranes. Results. The binding site is a ~75kDa membrane protein that fits a mass spectrometric profile of neurolysin. An antibody to neurolysin precipitated the 125Isarcosine1, benzoylphenylalanine8-AngII labeled protein. Overexpression of neurolysin in HEK293 cells increased non-AT1, non-AT2 125I-sarcosine1,isoleucine8-AngII binding. Binding of 125I-sarcosine1, isoleucine8-AngII to the non-AT1, non-AT2 binding site in neurolysin knock-out mouse brains was dramatically decreased compared to wild-type brains. Conclusion. A membrane-bound variant of the metalloendopeptidase neurolysin (E.C.3.4.24.16) is the novel AngII binding protein. Grants. Supported by NHLBI HL-096357

Acute repeated intracerebroventricular injections of angiotensin II reduce agonist and antagonist radioligand binding in the paraventricular nucleus of the hypothalamus and median preoptic nucleus in the rat brain

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 ((125)I-sarcosine(1) Ang II) and a radiolabeled antagonist ((125)I-sarcosine(1), isoleucine(8) 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

Autoradiographic film for WT1 KO1

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Autoradiographic film for WT3 KO3

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Autoradiographic film with KO1 and WT2

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Histology WT3 KO3

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

Histology WT1 WT2 KO1 KO2

<p>These files include autoradiographic films and histology files in .tiff format at 1200 dpi. These items were used to create the data shown in our manuscript. The nomenclature used for each animal (WT vs KO, -1, -2, -3, -4, 5) is described within the manuscript. We analyzed these files using MCID Image Analysis Software suite (http://www.mcid.co.uk/). Please warned that the files are large.</p

¹²⁵I-SI Ang II binding comparison.

<p>Comparison of ¹²⁵I-SI Ang II binding in the brains of neurolysin KO and WT mouse strains in the presence of PCMB, losartan, and PD123319. Bregma −7.2 mm for the KO and WT histological and autoradiogram sections.</p