Immunocytochemical localization of M2 muscarinic receptors in rat ventricles with anti-peptide antibodies.

We produced antibodies against a synthetic peptide corresponding to amino acids 168-192 of the second extracellular loop of the M2 human muscarinic receptor in rabbits. In immunoblot, affinity-purified antibodies specifically recognized a major band of rat ventricular muscarinic receptor protein with a molecular weight of about 80 KD. This recognition could be blocked by pre-incubation with peptide. Moreover, with both light (LM) and electron microscopic (EM) immunocytochemistry techniques, muscarinic receptors were detected on sarcolemma and T-tubules of rat cardiomyocytes. In addition, immunoreactions were localized in membranes of capillaries. Likewise, these reactivities were abolished by pre-incubation with peptide. These results suggest that the antibodies against the second extracellular loop of human M2 muscarinic receptor could specifically recognize rat ventricular muscarinic receptor protein and could be a powerful tool to study the fate of this receptor under different pathological or physiological conditions.