Effect of Continuous Administration of a Cholinergic Agonist on [ 3 H]4-DAMP Binding and m3 mRNA Expression in Cultured Human Ciliary Muscle Cells

Abstract

ABSTRACT

It is well known that chronic topical administration of cholinergic agonists results in a subsensitization in ciliary muscle-mediated increases in outflow facility and accommodation in monkey eyes in vivo. These physiologic changes are apparently mediated by the M3 subtype receptor. However, the nature of this subsensitization remains unclear. This study investigated the effe[unk]t of the continuous presence of carbachol, a muscarinic agonist, on the expression of the muscarinic receptor subtype m3 and the binding of [³H]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 individually with 1 mM carbachol for 2,6,24 and 48 hours. The level of mRNA encoding muscarinic receptor subtype m3 was measured by RNase protection. For confirmation, a receptor binding assay was done using [³H]4-DAMP, a radioligand selective for M3 subtype receptors. At each timepoint, results were compared with untreated controls.

Treatment with carbachol resulted in a down regulation ranging from 23.4% to 34.8% of m3 mRNA expression at all time points. All [³H]4-DAMP binding assay results also decreased, ranging from 24.5% to 31.0%.