CHARACTERIZATION OF THE MUSCARINIC RECEPTOR SUBTYPES IN THE BOVINE CORNEAL EPITHELIAL-CELLS

Muscarinic receptor subtypes in the bovine corneal epithelial cells (B CE) were characterized on the basis of their: 1) ligand binding proper ties, 2) linkage to Ca2+ and cAMP cell signaling pathways, and 3) gene transcripts. Receptor subtypes, m1 and m2, are indicated by competiti on experiments using subtype-selective muscarinic receptor ligands. [H -3]N-methylscopolamine ([H-3]-MS) binding was displaced with IC(50)s o f: 1) 1 mu M for the ml antagonist, pirenzipine; 2) 51 mu M for the co mpetitive m2 antagonist, AFDX-116; 3) 100 mu M for the competitive m(3 ) antagonist, 4-DAMP. In fura2 loaded BCE, carbachol (0.001 - 100 mu M ) increased intracellular Ca2+ concentration ([Ca2+](i)), and these re sponses were significantly supressed if they were preincubated with ei ther atropine (1 mu M) or 1 mu M pirenzipine. In the absence of extrac ellular Ca2+, these carbachol-induced increases in [Ca2+](i) were depr essed. A considerable fall occurred with the presence of extracellular Ca2+ and 1 mu M verapamil, an L-type Ca2+ channel blocker. These resp onses suggest that carbachol increases Ca2+ influx through an L-type C a2+ channel in the plasma membrane, in addition to mobilizing Ca2+ fro m an intracellular store. BCE also possessed muscarinic receptors whic h were negatively linked to cAMP production insofar as: 1) preincubati on with 10 mu M carbachol significantly suppressed the increases in cA MP accumulation induced by isoproterenol (1 - 25 mu M); 2) this blunti ng effect of carbachol on cAMP production was eliminated when the BCE were preincubated with either 1 mu M AFDX-116, or 100 ng/ml pertussis toxin. The results of probing for muscarinic receptor gene expression are partially consistent with the ligand binding and functional assays . Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis re vealed the presence of m2 but nor m1, m3 or m4 gene transcripts. In su mmary, we obtained pharmacological and functional evidence for mi and m2 receptors in BCE. However, only the m2 gene transcript could be det ected.