Immunocytochemical localization of angiotensin II receptor subtypes and angiotensin II with monoclonal antibodies in the rat adrenal gland

Angiotensin II (Ang II), a major regulator of cardiovascular function and b ody fluid homeostasis, mediates its biological actions via two subtypes of G protein-coupled receptors, termed AT(1) and AT(2). The primary goal of th is study was to raise monoclonal anti-peptide antibodies specific to angiot ensin AT(1)- and AT(2)-receptor subtypes and to Ang II itself and using the se monoclonal antibodies to determine the intraadrenal localization of AT(1 ) and AT(2) receptors and Ang II in male adult rats. Immunocytochemistry un ambiguously demonstrates a regional colocalization of Ang II and angiotensi n II receptors in the adrenal gland. The novel antibodies localized Ang II and the AT(1) receptors to the zona glomerulosa of the cortex and to the me dulla whereas AT(1) receptors were limited to the medulla. The specificity of immunostaining was documented by pre-adsorption of the antibody with the immunogenic peptide. Our data underscore that AT(1) appears to mediate mos t of the physiological actions of Ang II in adrenal. Western blot analysis of rat adrenal protein extracts using AT(1) antibody showed a predominant 7 3-kDa band and a weaker 97-kDa immunoreactive band corresponding to glycosy lated forms of the AT(1) receptor. Immunostaining with anti-AT(2) yielded o ne major immunoreactive band of 73-kDa size and one additional fainter band of 120 kDa. These antibodies may prove of value in unraveling the subcellu lar localization and intracellular effector pathways of AT(1) and AT(2). (C ) 2001 Elsevier Science B.V. All rights reserved.