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

Lacrimal glands secrete protein, electrolytes, and water into the tear film that overspreads the cornea and conjunctiva.1 Lacrimal gland protein secretion is stimulated by multiple neurotransmitters, including the parasympathetic neurotransmitters acetylcholine (activates muscarinic type 3 acetylcholine receptors [M3AChR]) and vasoactive intestinal peptide (VIP) (stimulates VIPAC1 receptors) and the sympathetic neurotransmitter norepinephrine (interacts with α1D-adrenergic receptors [α1D-AR]).1 Each of these neurotransmitters activates a separate distinct signaling pathway. Thus lacrimal gland protein secretion can be induced by increasing the intracellular [Ca2+] ([Ca2+]i) and activating protein kinase C (PKC), raising the cellular level of cAMP, or elevating cellular cGMP levels.2–6 Cholinergic agonists stimulate lacrimal gland protein secretion by activating M3AChR coupled to Gαq G proteins, which activate phospholipase Cβ (PLCβ).7,8 PLCβ activation cleaves phosphatidylinositol 1,4-bisphosphate to produce the PKC activator diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). InsP3 activates Ca2+-selective InsP3 receptors located in the endoplasmic reticulum of lacrimal gland acinar cells that increase the [Ca2+]i.2 Depletion of the endoplasmic reticulum Ca2+ pool triggers extracellular Ca2+ influx and a sustained elevation of [Ca2+]i.9 The increase in [Ca2+]i along with activation of the PKC isoforms PKCα, PKCδ, and PKCe stimulate the secretion of protein stored in preformed secretory granules.3 Protein secretion occurs across the apical membrane and along with isotonic electrolyte and water secretion, also induced by cholinergic agonists, forms lacrimal gland fluid.10 After modification by ductal cell secretion, lacrimal gland fluid is secreted onto the cornea and conjunctiva. In addition to M3AChR, VIPAC1, and α1D-AR, lacrimal gland acinar cells express purinergic P2 receptors that are coupled to an increase in [Ca2+]i and stimulate protein secretion.11 The P2 receptor family consists of P2Y receptors that are G-protein–coupled (metabotropic) and P2X receptors that are ion channels (ionotropic).12 Both types of P2 receptors are activated by extracellular di- and tri-nucleotides. P2Y receptors cause an increase in [Ca2+]i by InsP3-induced Ca2+ mobilization from intracellular stores similar to muscarinic receptors, whereas P2X receptors act as ligand-gated, nonselective ion channels that allow the influx of extracellular Ca2+.12 P2X7 receptors are a major functional P2 receptor in the lacrimal gland.11 Activation of P2X7 receptors with (benzoylbenzoyl)adenosine 5′ triphosphate (BzATP) causes an increase in [Ca2+]i and the stimulation of lacrimal gland protein secretion.11 The BzATP-stimulated increase in [Ca2+]i in lacrimal acinar cells was increased in the absence of Mg2+ and was blocked by two P2X7 antagonists, brilliant blue G and A438979.11 Similarly, protein secretion induced by BzATP was prevented by brilliant blue G.11 Thus the activation of P2X7 receptors is a Ca2+-dependent stimulus of lacrimal gland protein secretion. Neurotransmitters often work together, thus changing the secretory response. Simultaneous activation of two different receptors and their signaling pathways can cause three different outcomes: a less than additive response, an additive response, or potentiation of the response. A less than additive response can occur if two receptors activate the same or overlapping signaling pathways or if activation of one receptor inhibits the second receptor. In the lacrimal gland, a less than additive secretory response occurs when the cholinergic agonist M3AChR is activated with carbachol and PKC isoforms are activated by the phorbol ester 4β-phorbol 12,13 dibutyrate (PdBU) because M3AChR and PdBU both activate PKC isoforms.13 An additive response results if two receptors use different signaling pathways. No interaction of two separate signaling pathways occurs in the lacrimal gland in the presence of activation of the M3AChR (Ca2+- and PKC-dependent) and the α1D-AR (cGMP- and Ca2+-dependent), leading to additivity of secretion.13 Finally, potentiation is produced if the two pathways interact synergistically and cause a response that is greater than that of the two pathways activated together. In the lacrimal gland interaction of M3AChR (Ca2+- and PKC-dependent) and VIPAC1 (cAMP and Ca2+-dependent) receptors stimulated by cholinergic agonists and VIP, respectively, leads to potentiation.13,14 In the present study the interaction of M3AChR and P2X7 receptors is explored. Our results demonstrate that the activation of M3AChR stimulates P2X7 receptors to increase [Ca2+]i and induce protein secretion. M3AChR does not activate PKC to alter P2X7 receptors but can release adenosine triphosphate (ATP) from nonacinar cells. Activation of both receptors increases [Ca2+]i and stimulates lacrimal gland protein secretion because both receptors are at least partially Ca2+-dependent. Muscarinic agonists, however, activate an additional pathway, probably a PKC-dependent pathway, that causes protein secretion independently of P2X7 receptors because secretion stimulated by muscarinic and P2X7 agonists is additive.