Characterization of the Muscarinic Receptor Subtypes in the Bovine Corneal Epithelial Cells

Abstract

ABSTRACT

Muscarinic receptor subtypes in the bovine corneal epithelial cells (BCE) were characterized on the basis of their: 1) ligand binding properties, 2) linkage to Ca²⁺ and cAMP cell signaling pathways, and 3) gene transcripts. Receptor subtypes, m1 and m2, are indicated by competition experiments using subtype-selective muscarinic receptor ligands. [³H]N-methylscopolamine([³H]-MS) binding was displaced with IC₅₀s of: 1) 1 μM for the m1 antagonist, pirenzipine; 2) 51 μM for the competitive m2 antagonist, AFDX-116; 3) 100 μM for the competitive m3 antagonist, 4-DAMP. In fura2 loaded BCE, carbachol (0.001 - 100 μM) increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and these responses were significantly supressed if they were preincubated with either atropine (1 μM) or 1 μM pirenzipine. In the absence of extracellular Ca²⁺, these carbachol-induced increases in [Ca²⁺]_i were depressed. A considerable fall occurred with the presence of extracellular Ca²⁺ and 1 μM verapamil, an L-type Ca²⁺ channel blocker. These responses suggest that carbachol increases Ca²⁺ influx through an L-type Ca²⁺ channel in the plasma membrane, in addition to mobilizing Ca²⁺ from an intracellular store. BCE also possessed muscarinic receptors which were negatively linked to cAMP production insofar as: 1) preincubation with 10 μM carbachol significantly suppressed the increases in cAMP accumulation induced by isoproterenol (1 - 25 μM); 2) this blunting effect of carbachol on cAMP production was eliminated when the BCE were preincubated with either 1 μM 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 revealed the presence of m2 but not m1, m3 or m4 gene transcripts. In summary, we obtained pharmacological and functional evidence for m1 and m2 receptors in BCE. However, only the m2 gene transcript could be detected.