Muscarinic effects on cellular functions in cultured human ciliary muscle cells

Purpose. To characterize the pharmacology of the carbachol-induced changes of phospholipase C (PLC) activity and intracellular calcium concentration ([Ca²⁺](i)) in cultured human ciliary muscle cells. Methods. Changes in PLC activity of cultured human ciliary muscle cells were determined by production of inositol phosphates. Single-cell dynamic fluorescence ratio imaging was used to determine [Ca²⁺](i). Results. Carbachol, oxotremorine-M, aceclidine, and pilocarpine stimulated PLC with mean EC₅₀s of 20, 8, 17, and 2 μM, respectively. The effect of carbachol on PLC was partially suppressed by extracellular Ca²⁺ depletion. This muscarinic effect was blocked by muscarinic antagonists, such as atropine (apparent pK(i) = 9.12, nonselective for muscarinic receptor subtypes), pirenzepine (pK(i) = 6.76, selective for the M₁ receptor subtype), 4DAMP (pK(i) = 9.25, selective for the M₁ and M₃ subtypes), and fHHSiD (pK(i) = 7.77, selective for the M₃ subtype). In [Ca²⁺](i) experiments, carbachol increased [Ca²⁺](i) transients in human ciliary muscle cells in a dose-dependent manner with a mean EC₅₀ of 7 μM. 4DAMP was approximately 100 times more potent than pirenzepine in the inhibition of the carbachol-induced [Ca²⁺](i) increase. [Ca²⁺](i) oscillations were observed after carbachol stimulation and persisted after extracellular Ca²⁺ depletion. Conclusions. Muscarinic agonists activate PLC and increase [Ca²⁺](i) in cultured human ciliary muscle cells through an M₃-like muscarinic receptor subtype.