Effect of continuous administration of a cholinergic agonist on [H-3]4-DAMP binding and m3 mRNA expression in cultured human ciliary muscle cells

It is well known that chronic topical administration of cholinergic agonist s results in a subsensitization in ciliary muscle-mediated increases in out flow facility and accommodation in monkey eyes in vivo. These physiologic c hanges are apparently mediated by the M3 subtype receptor. However, the nat ure of this subsensitization remains unclear. This study investigated the e ffect of the continuous presence of carbachol, a muscarinic agonist, on the expression of the muscarinic receptor subtype m3 and the binding of [H-3]4 -DAMP in cultured human ciliary muscle cells (H7CM). The H7CM cell line, derived from the ciliary muscle of a one-day-old human infant, was used in this study. Confluent monolayers were treated individua lly with 1 mM carbachol for 2, 6, 24 and 48 hours. The level of mRNA encodi ng muscarinic receptor subtype m3 was measured by RNase protection. For con firmation, a receptor binding assay was done using [H-3]4-DAMP, a radioliga nd selective for M3 subtype receptors. At each timepoint, results were comp ared with untreated controls. Treatment with carbachol resulted in a down regulation ranging from 23.4% t o 34.8% of m3 mRNA expression at all time points. All [H-3]4-DAMP binding a ssay results also decreased, ranging from 24.5% to 31.0%.