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.