Stimulatory activity of anti-peptide antibodies against the second extracellular loop of human M-2 muscarinic receptors

Objective To study the activity of anti-peptide antibodies against the seco nd extracellular loop of human M-2 muscarinic receptors on cAMP production and inward calcium currents (I-Ca) in guinea pig ventricular myocytes. A co mparison was also made with those of a muscarinic receptor agonist. Methods cAMP content was determined by radioimmunoassay and the I-Ca in gui nea pig single ventricular cells were recorded by the whole-cell patch clam p technique. Results Both the muscarinic receptor agonist, carbachol (Carb 10 mu mol/L), and anti-peptide antibodies (Abs 100 nmol/L) could decrease basal cAMP lev els (by 46.9% +/- 4.2% and 60.2% +/- 4.6%, respectively) and basal I-Ca. Bo th Carb (10 mu mol/L) and Abs (100 nmol/L) could also inhibit the isoprenal ine-induced (Iso 0.8 mu mol/L) increases in cAMP production (from 108.2 +/- 7.0 to 88.4 +/- 7.2 pmol/mg.protein/min for Carb and 88.6 +/- 5.1 pmol/mg. protein/min for Abs, respectively) and the increases in I-Ca. The muscarini c receptor antagonist atropine (Atr) was able to prevent these effects of C arb and Abs. Conclusions Anti-peptide antibodies against an epitope located in the secon d extracellular loop of human M-2 muscarinic receptors, similar to muscarin ic receptor agonist, could decrease the basal I-Ca and beta-receptor agonis t stimulated increase of I-Ca by decreasing the basal and beta-receptor ago nist stimulated increase of cAMP production, and therefore could have an ef fect on their target receptor. These results further suggest that autoimmun ity may participate in the pathogenesis of human cardiomyopathy and the sec ond extracellular loop of human M-2 muscarinic receptor could be the main i mmunodominant region.