Mechanisms of Pilocarpine-Induced Nitric Oxide and Prostaglandin Production in Porcine Ciliary Muscle

Pilocarpine, a muscarinic receptor agonist, is clinically used to reduce intraocular pressure via ciliary muscle contraction. However, the intracellular signaling pathways mediating pilocarpine-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) synthesis in the ciliary muscle are not fully understood. This study aimed to characterize the muscarinic receptor subtypes and intracellular mechanisms involved in pilocarpine-induced NO and PGE₂ production in porcine ciliary muscle and to explore the potential implications of these pathways in age-related ocular changes, including presbyopia. Ciliary muscle strips from adult porcine eyes were incubated in Krebs-Ringer buffer with pharmacological agents. Nitrate levels were measured using a Griess reaction-based colorimetric assay, and PGE₂ concentrations were quantified via ELISA. Selective receptor antagonists and enzyme inhibitors were used to investigate receptor involvement and signaling cascades. Statistical significance was determined using unpaired t-tests (p < 0.05). Pilocarpine elicited a concentration-dependent increase in nitrate and PGE₂ production, peaking at 1 × 10^-7 M. This effect was inhibited by atropine, pirenzepine (M₁ antagonist), and J104129 (M₃ antagonist), but not by the M₂ antagonist AFDX 116. Inhibitors of nitric oxide synthase (L-NMMA, L-NIO), phospholipase C (U-73122), cyclooxygenase (diclofenac), and calcium signaling (verapamil, TFP) all reduced mediator production, while calcium ionophore A23187 enhanced it. Pilocarpine activates M₁ and M₃ muscarinic receptors in the porcine ciliary muscle, stimulating NO and PGE ₂ production via calcium-dependent PLC-NOS-COX signaling. These pathways may influence ciliary muscle function and lens physiology, offering potential therapeutic targets for glaucoma and presbyopia.