Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect

Antibodies were produced against a synthetic peptide corresponding to amino acids (165-191) of the second extracellular loop of the human angiotensin II receptor subtype 1 (AT1) in rabbits. The purified antibodies had an appa rent affinity of about 1 nM and were monospecific for the AT1-receptor pept ide. Chemical modification of the carboxyl groups (glu at positions 173 and 185) and the sulfhydryl group (cys at position 180) of the AT1-receptor pe ptide did not alter the relative affinity of the coated AT1 -receptor pepti de to antibodies. The antibodies specifically stained CHO cells expressing the rat AT1(a) receptor. Immunoblots on rat kidney revealed that the antibo dy recognized a protein band of 59 +/- 3 kDa in a dose-dependent manner and this band was no longer detected after preincubating the antibodies with A T1-receptor peptide, Using electron microscopic and immunofluorescence immu nocytochemistry techniques, angiotensin II receptors were detected in (1) t he sarcolemma, T-tubules and nuclei of rat cardiomyocytes, (2) the translum inal side of endothelial cells and (3) fibroblast cells. These localization s are specific, as the immunostaining did not appear when preimmune rabbit serum was used and was blocked after preincubating antibodies with antigeni c peptide. Functionally, these antibodies did not affect the ligand binding properties of the receptors but displayed agonist-like activity as shown b y dose-dependent increases in beating frequency in cultured neonatal cardio myocytes, These results suggest that the antibodies against the second extr acellular loop of human AT1 receptors were able to specifically recognize A T1 receptors. In addition, they extend the observation that the second extr acellular loop of the G-protein coupled membrane receptors is a specific ta rget for antibodies with agonist-like activity.