Cholinergic agonists activate P2X7 receptors to stimulate protein secretion by the rat lacrimal gland.

PURPOSE To determine the interaction of M3 muscarinic receptors (M3AChR) and P2X(7) receptors to increase intracellular [Ca2+] ([Ca2+]i) and stimulate protein secretion in rat lacrimal gland cells. METHODS Exorbital lacrimal glands from male Sprague-Dawley rats were divided into pieces or digested with collagenase to form acinar clumps. [Ca2+]i was measured using an imaging system in acini incubated with fura-2/AM. Adenosine triphosphate (ATP) release was determined using the luciferin-luciferase reaction. Peroxidase secretion, our index for protein secretion, was measured spectrophotometrically. Acini were stimulated with the P2X7 receptor agonist, (benzoylbenzoyl)adenosine 5' triphosphate (BzATP), cholinergic agonist carbachol, or the activator of conventional and novel PKC isoforms, phorbol 12-myristate 13-acetate (PMA). RESULTS The increase in [Ca2+]i caused by carbachol and BzATP used simultaneously was less than additive, but the increase in protein secretion was additive. The M3AChR antagonist atropine blocked the BzATP-stimulated increase in [Ca2+]i and peroxidase secretion. The P2X7 antagonist did not alter the carbachol-stimulated increase in [Ca2+]i or peroxidase. PMA- and BzATP-stimulated increases in [Ca2+]i were additive. Neither constitutively active PKCα, dominant-negative PKCα, nor PKCε altered BzATP-stimulated increases in [Ca2+]i. Carbachol increased ATP release from lacrimal gland pieces but not from acini. CONCLUSIONS In lacrimal gland cells, the activation of M3AChRs stimulates P2X7 receptors to increase [Ca2+]i and protein secretion. The underlying mechanisms are unknown but could include the release of ATP or intracellular interactions not mediated by PKC isoforms. In addition, M3AChRs use signaling pathways that overlap with those used by P2X7 receptors to increase [Ca2+]i, but they also use signaling pathways not used by P2X7 receptors to stimulate protein secretion.