Association of the Novel Non-AT₁, Non-AT₂ Angiotensin Binding Site with Neuronal Cell Death

Abstract

We have discovered a non-AT 1 , non-AT 2 angiotensin binding site in rodent and human brain membranes, which, based on its pharmacological/biochemical properties and tissue distribution, is different from angiotensin receptors and key proteases processing angiotensins. In this study, the novel angiotensin binding site was localized to a specific brain cell type by using radioligand receptor binding assays. Our results indicate that the novel binding site is expressed in mouse primary cortical neuronal membranes but not in primary cortical astroglial and bEnd.3 brain capillary endothelial cell membranes. Whole-cell binding assays in neurons showed that the binding site faces the outer side of the plasma membrane. Consistent with our previous observations, the novel binding site was unmasked by the sulfhydryl reagent p-chloromercuribenzoate. This effect had a bell-shaped curve and was reversed by reduced glutathione, indicating that the function of the binding site might be regulated by the redox state of the environment. Density of the novel binding site measured by saturation binding assays was significantly increased in neuronal membranes of cells challenged in four in vitro models of cell death (oxygen-glucose deprivation, sodium azide-induced hypoxia, N-methyl-D-aspartate neurotoxicity, and hydrogen peroxide neurotoxicity). In addition, our in vivo data from developing mouse brains showed that the density of the novel angiotensin binding site changes similarly to the pattern of neuronal death in maturating brain. This is the first time that evidence is provided on the association of the novel angiotensin binding site with neuronal death, and future studies directed toward understanding of the functions of this protein are warranted.

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

“…As shown in Figure 1, optimal unmasking was observed at PCMB concentrations of 100–300 μM. The optimal PCMB concentration are slightly lower than our initial report [26], but is consistent with subsequent more recent studies [33,56]. Two-way ANOVA of the K D values indicated that the K D for testis was lower at 100 μM than at 300 μM (p<0.01) and that there was a main effect of higher affinity at 100 μM versus 300 μM (p=0.0033).…”

“…Among the sulfur-containing organic compounds, only cysteamine and amifostine, inhibited the unmasking by more than 50% in both brain and testis. Glutathione was not included in the primary screening based on a previous observation of its inhibitory effect [56]. Among the non-sulfur containing organic compounds (KCN was included here because it contains a carbon), only mersalyl acid inhibited the unmasking by more than 50% in both brain and testis.…”

In vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment

“…To confirm the neuroprotective effects of biphalin, we challenged neuronal cells with glutamate and hypoxia/aglycemia and used two independent approaches (LDH and MTT assays) to determine neuronal injury. LDH is an indicator of plasma membrane integrity and MTT is a widely used indicator of mitochondrial function, and both assays have been proved to be reliable indicators of neuronal injury (Lobner, 2000; Rashid et al, 2010). In our studies biphalin displayed a better ability to maintain integrity of plasma membrane (release of LDH), and mitochondrial function (conversion of MTT) compared to equimolar concentrations of selective OR agonists DPDPE, DAMGO and U 50 488 (Figures 2 and 3).…”

Distribution of Non-AT1, Non-AT2 Binding of 125I-Sarcosine1, Isoleucine8 Angiotensin II in Neurolysin Knockout Mouse Brains

“…Neurolysin has been shown to be present on the extracellular surface of cortical neurons and is therefore capable of metabolizing Ang I and Ang II in their extracellular environment [65], [66]. Moreover, formation of Ang (1–7) by neurolysin [45] diverts the conversion of Ang I into Ang II, directly counteracting the effects of the latter.…”