Distribution of a novel binding site for angiotensins II and III in mouse tissues

A novel binding site for angiotensins II and III that is unmasked by parachloromercuribenzoate has been reported in rat, mouse and human brains. Initial studies of this binding site indicate that it is not expressed in the adrenal, liver or kidney of the rat and mouse. To determine if this binding site occurs in other mouse tissues, 8 tissues were assayed for expression of this binding site by radioligand binding assay and compared with the expression of this binding site in the forebrain. Particulate fractions of homogenates of testis, epididymis, seminal vesicles, heart, spleen, pancreas, lung, skeletal muscle, and forebrain were incubated with (125)I-sarcosine(1), isoleucine(8) angiotensin II in the presence or absence of 0.3mM parachloromercuribenzoate plus 10microM losartan and 10microM PD123319 (to saturate AT(1) and AT(2) receptors). Specific (3microM angiotensin II displaceable) high affinity binding occurred in the testis\u3eforebrain\u3eepididymis\u3espleen\u3epancreas\u3elung when parachloromercuribenzoate was present. Binding could not be reliably observed in heart, skeletal muscle and seminal vesicles. High affinity binding of (125)I-sarcosine(1), isoleucine(8) angiotensin II was observed in the absence of parachloromercuribenzoate in the pancreas on occasion. This suggests that this novel angiotensin binding site may have a functional role in these tissues

Characterization of the brain-specific non-AT(1), non-AT(2) angiotensin binding site in the mouse

In the present study the existence of a non-AT(1), non-AT(2) angiotensin (Ang) binding site unmasked by the organomercurial protease inhibitor p-chloromercuribenzoate (PCMB) was demonstrated in mouse brain membranes, consistent with observations previously reported in the rat (Karamyan and Speth, 2007b). The pharmacological specificity of the non-AT(1), non-AT(2) angiotensin binding site was similar to the rat brain: Sar(1)-Ile(8)-Ang II \u3e Ang III \u3eor= Ang II \u3e Ang I\u3e p-aminophenylalanine(6) Ang II\u3e CGP42112 \u3e\u3e Ang IV \u3e Ang 1-7 congruent with shorter angiotensin fragments. Neurotensin, bradykinin, and luteinizing hormone-releasing hormone showed K(i) values \u3e10 microM, while substance P and VIP had K(i) values of approximately 2 microM. The non-AT(1), non-AT(2) angiotensin binding site was not present in adrenal, liver or kidney. Subcellular fractionation showed a higher density of [(125)I]Ang II binding in plasma membrane (P2) fractions of cerebral cortex and hypothalamus relative to debris (P1) fractions. The binding site is present in the brains of mice in which the AT(1a), AT(1b), AT(2), Mas, and neprilysin (EC 3.4.24.11, neutral endopeptidase) was knocked out confirming that the binding site is not a heretofore described angiotensin receptor or neprilysin. These observations confirm that this novel Ang binding site is distinct from classical AT(1), AT(2), AT(4) and Ang 1-7 receptors while retaining a high specificity for angiotensins that act on the known angiotensin receptors. Whether this binding site functions as a novel receptor for angiotensins or a specific angiotensinase with variable functionality at different redox states will require further study