Effects of muscarinic agents on cultured human trabecular meshwork cells

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the nonselective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M₁-selective), p-fHHSiD (M₃-selective), and 4-DAMP (M₁, M₃ subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M₃ receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to ³H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M₃-like receptor subtype. Therefore, the muscarinic M₃ receptor may play a role in trabecular meshwork cell function(s).