Identification of the Raf-1 Signaling Pathway Used by cAMP to Inhibit p42/p44 MAPK in Rat Lacrimal Gland Acini: Role in Potentiation of Protein Secretion

The lacrimal gland (LG) is a tubuloacinar gland that is composed of acinar cells arranged in a pyramidal shape surrounding a central lumen. Acinar cells synthesize and secrete into the lumen proteins in the tear film as well as electrolytes and water. The lumen is lined with ductal cells that modify the primary secretory product by the secretion of electrolytes and water. The lumen coalesces with other ducts to form a main excretory duct that empties onto the ocular surface. The LG also contains a third major cell type, the myoepithelial cell. Myoepithelial cells are stellate-shaped cells that surround the acinar cells. Because these cells contain α-smooth muscle actin, it is possible that their contractions aid in expulsion of the secretory products from the acinar cells.1 The LG secretes a major portion of the aqueous portion of the tear film and includes water, electrolytes, and protein. Given that tears are necessary to provide a smooth refractive surface to ensure clear vision, secretion from the LG is tightly controlled because either an excess or a deficiency in tears can be harmful to vision. Sensory nerves in the cornea provide an efferent pathway by which stimuli to the ocular surface activate an afferent pathway by way of the parasympathetic and sympathetic nerves that innervate the LG to stimulate secretion.1 Parasympathetic nerves release the neurotransmitters acetylcholine (Ach) and vasoactive intestinal peptide (VIP). Ach, in the LG, binds to M3 muscarinic receptors on the basolateral membranes of acinar cells.2 Through a Gq/11α G-protein, M3 muscarinic receptors activate phospholipase Cβ to cleave phosphatidylinositol bisphosphate into inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG).3 IP3 diffuses to the endoplasmic reticulum, where it binds to its receptors to release Ca2+ into the cytosol from intracellular stores. The released Ca2+ is involved in the activation of kinases that are involved in secretion.3 The DAG that is released activates a family of proteins called protein kinase C (PKC). There are at least 10 PKC isoforms, of which four are present in the LG: PKCα, -δ, -e, and -λ.4 Cholinergic agonists activate PKCα, -δ, and -e to induce protein secretion.5 VIP binds to its receptors, VPAC1 and VPAC2, present on LG acinar cells to stimulate the Gsα subunit of G-proteins to activate adenylyl cyclase (AC).6 The LG contains at least three types of ACs, namely II, III, and IV.6 Activation of AC increases the intracellular levels of cAMP, which in turn activates protein kinase A (PKA). Activation of PKA stimulates protein secretion as the endogenous inhibitor of PKA—PKI—inhibits VIP-stimulated protein secretion.6 Growth factors are also stimuli of protein secretion from the LG. Epidermal growth factor (EGF) stimulates secretion through dimerization of the EGF receptor, leading to the activation of phospholipase Cγ.7 This leads to an increase in intracellular Ca2+ and the activation of PKCα and -δ to stimulate protein secretion.7 The Ca2+/PKC- and cAMP-dependent pathways are not separate, independent pathways but, in fact, can interact in many different tissues.8–10 In the LG, this was demonstrated by the observation that the addition of VIP and cholinergic agonists11 or VIP and EGF12 caused a potentiation of secretion, that is, a response greater than the sum of the individual responses. It was established that a potentiation of secretion was not a result of the potentiation of the increase in intracellular Ca2+, cAMP concentration, or PKC activity.13,14 One possible point at which these two pathways can interact is the activation of p42/p44 mitogen-activated protein kinase (MAPK, also known as ERK 1/2). Cholinergic agonists activate MAPK to attenuate stimulated protein secretion.15 Cholinergic agonists and EGF activate MAPK in different ways. Cholinergic agonists activate the nonreceptor tyrosine kinases Pyk2 and c-Src to activate the signaling cascade of Ras, Raf-1, MEK, and ultimately MAPK.15,16 EGF activates Shc, Grb2, and Sos to activate MAPK through Ras, Raf-1, and MEK.15 An additional pathway by which Raf-1, MEK, and MAPK can be activated is through the stimulation of Rap-1. Rap-1 is a small GTPase and a member of the Ras family. Rap-1 can be activated by a variety of stimuli, including compounds that increase intracellular cAMP concentrations, nerve growth factor, and epinephrine.17 In renal tubule epithelial cells, PKA has been shown to activate MAPK mediated by Rap-1.17–19 Activation of Rap-1 plays a role in cellular processes such as cell adhesion and proliferation.19 In support of the hypothesis that MAPK plays a role in the potentiation of secretion, we previously found that VIP through cAMP inhibits the activation of MAPK, preventing MAPK from attenuating secretion.12 Increasing cAMP concentrations within a cell through either the activation of adenylyl cyclase (using VIP or forskolin) or the inhibition of the phosphodiesterases, which degrade cAMP, inhibits the basal and stimulated activation of MAPK and causes the potentiation of protein secretion by blocking the MAPK-dependent inhibitory pathway.12 In the present study, we identify the components of the MAPK signal transduction pathway by which cAMP inhibits both basal- and agonist-stimulated MAPK activity.