Novel Insights Into Muscarinic and Purinergic Responses in Primary Cultures of Rat Lacrimal Gland Myoepithelial Cells

PURPOSE: The functional characteristics of receptors that regulate lacrimal gland myoepithelial cells are still somewhat unclear. To date, mainly muscarinic receptors have been of interest; however, further knowledge is needed regarding their expression and functional roles. For this purpose, primary cultures of rat lacrimal gland myoepithelial cells were established and examined functionally. METHODS: Rat lacrimal glands were excised, minced, and further digested, yielding mixtures of cells that were seeded in culturing flasks. After 4-6 weeks, primary monocultures of myoepithelial cells were established, verified by immunocytochemistry. The cells were stained for all muscarinic receptor subtypes (M1–M5) and examined functionally regarding intracellular [Ca(2+)] responses upon activation of muscarinic receptors. For methodological verification, purinergic functional responses were also studied. RESULTS: Expression of muscarinic receptor subtypes M2-M5 was detected, whereas expression of muscarinic M1 receptors could not be shown. Activation of muscarinic receptors by the non-selective muscarinic agonist methacholine (3 × 10(−11)–10(−3) M) did not cause a significant increase in intracellular [Ca(2+)]. However, activation of purinergic receptors by the non-selective purinergic agonist ATP (10(−8)–10(−3) M) caused a concentration-dependent increase in intracellular [Ca(2+)] that could be blocked by the P2 antagonists PPADS and suramin. CONCLUSIONS: Primary cultures of rat lacrimal gland myoepithelial cells were established that displayed a heterogeneous expression of muscarinic receptors. Purinergic functional responses demonstrated a viable cell population. Upon treatment with methacholine, no significant increase in intracellular [Ca(2+)] could be detected, indicating that cholinergic activation of myoepithelial cells occurs via other intracellular messengers or is dependent on interaction with other cell types.