Further characterization of the novel, non‐AT1, non‐AT2 brain‐specific angiotensin binding site in the mouse brain

Abstract

To further characterize the non‐AT1, non‐AT2 binding site in mouse brain, differential centrifugation was used to assess its subcellular distribution. The fraction enriched in plasma membranes (P2) from both cerebral cortex (CTX) and hypothalamus (HT) showed a higher density of 125I‐Ang II binding relative to the nuclear/mitochondrial/debris (P1) fraction: CTX P1 Bmax= 21.9±3.6 fmol/mg protein, CTX P2 Bmax = 63.1±11.1 fmol/mg protein; HT P1 Bmax = 24.5±7.6 fmol/mg protein, HT P2 Bmax = 38.3±9.8 fmol/mg protein. The enriched binding in CTX and HT plasma membranes (71 and 66% of total binding, respectively) suggests that the non‐AT1, non‐AT2 angiotensin binding site is primarily localized to plasma membrane. Since p‐chloromercuribenzoate (PCMB), the agent that unmasks this binding site, may simulate pathophysiological conditions in the brain associated with an altered redox state, we examined the effect of reduced glutathione (GSH) on the ability of PCMB to unmask the binding site in P2 membranes. GSH at concentrations ranging from 0.3 to 10 mM inhibited 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site from 78 to 87% in a concentration‐related manner. A small residual amount of 125I‐Ang II binding to the non‐AT1, non‐AT2 binding site in the absence of PCMB was also inhibited by 0.3 to 10 mM GSH by 51 to 70%. Functionality of the non‐AT1, non‐AT2 binding site may thus depend on redox state of the brain.