The broad long term objectives of this proposal are to understand on a molecular level intracellular signals that are involved in receptor-mediated alterations of salivary cells, particularly those signals that are initiated by secretagogues involved in saliva formation (fluid secretion) by parotid acinar cells. Fluid secretion is controlled by neurotransmitters that act on muscarinic, Substance P, and alpha-adrenergic receptors, which are linked to phospholipase C (PLC) by heterotrimeric GTP- binding (G) proteins. These receptors promote the PLC-mediated generation of DAG, which activates Protein Kinase C (PKC), and IP3, which mobilizes Ca2+. Parotid acinar cells also have multiple P2X ATP-binding receptors, including P2X7 receptors, that are nonselective cation channels, and these also elevate [Ca2+]i. ATP may be a neurotransmitter or act as an autocrine signal on salivary cells. P2X receptors are not coupled to G proteins, yet activation of the P2X7 receptor appears to stimulate PKC-dependent pathways in common with the muscarinic receptor in parotid acinar cells. These pathways may require the activation of PKCdelta, the one PKC family member that is activated by phosphorylation on tyrosine residues. PKCs and MAPKs play important roles in salivary and epithelial functions, and our data indicates that diverse receptor types, including the P2X7 receptor/ion channel and the muscarinic receptor, activate both of these proteins, which may be differentially regulated by these receptors.
Aim 1 is to determine the effects of signaling molecules/events, including tyrosine phosphorylation, MAPK, and Src, on indices of fluid/electrolyte/protein secretion.
Aim 2 is to define and compare the signal transduction cascades that activate MAPKs in secretagogue-treated and Na pump-inhibited rat parotid acinar cells, and to use MAPK activation as a paradigm to study receptor crosstalk and other aspects of cellular control. Na pump inhibition appears to activate a distinct signaling cascade that links pump activity to MAPK.
Aim 3 is to study the activation of PKC by P2X7 and other receptors.
Aim 4 is to determine downstream targets of PKCdelta and other proteins by identifying binding partners of these proteins in parotid acinar cells. PKCs and MAPKs have important functions in salivary cells and epithelia, although most studies have been performed using cell lines. Tyrosine phosphorylation, Src, PKCs, and MAPK may affect ion transport proteins, tight junction permeability, exocytosis, and other salivary functions. Understanding the signaling events that control their activation, as well as the effects of these proteins on secretion, is critical to providing a basis for understanding the regulation of salivary cells in health and in disease states.
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