Salivary gland hypofunction negatively impacts normal speech, mastication and general oral health. A significant proportion of these cases are of unknown origin (idiopathic). Our central hypothesis is that many of these cases reflect a disruption of the normal signaling pathways regulating the stimulation of salivary flow. The major mechanisms of stimulation of salivary gland fluid secretion involves muscarinic receptor- induced increases in intracellular Ca2+ concentration ([Ca2+]i), via activation of the phospholipase C/inositol triphosphate (PLC/InsP3) pathway, the details of which are fairly well-established. However, a substantial body evidence indicates the importance of a concurrent activation of cyclic AMP/protein kinase A (cAMP/PKA) signaling pathways along with the PLC/InsP3 pathway. This simultaneous activation of the two pathways profoundly potentiates overall fluid secretion via a mechanism that is, as yet, unclear. It is our hypothesis that proteins involved in this interaction are likely loci fort the underlying disruption seen in the diseased state. A major goal of this project is therefore to identify the key points of convergence of the two pathways in parotid acinar cells. Mouse parotid acinar cells will be utilized as a model system, however, the validity of the model will be confirmed in human tissue when available. We will examine the effects of PKA linked agonists on the generation and turnover of Insp3, and on the specific temporal and spatial features of the [Ca2+]i signals evoked by PLC- coupled agonists, both within and between acinar cells. The underlying mechanisms will be analyzed by identifying the molecular species and sub-cellular location of the individual signaling components involved, and by determining the properties of their regulation by PKA. In addition, potential interactions between PKA- and PLC-linked agonists at the level of the C1- and K+ conductances and the basolateral Na-2Cl-K co- transporter will be considered. The effects of potentiating combinations of agonists on the activation and modulation of individual Cl- and K+ conductances will be investigated, specifically focusing on those conductances demonstrated to be the most relevant in Subproject 2, and the nature of the effects determined. The activity of the co-transporter will also be assessed to determine if this is a potential site for potentiation by PKA-linked agonists. It is envisioned that these studies will identify loci which underlie the physiologically important regulation of salivary secretion, thereby increasing our understanding of the etiology of salivary gland dysfunction with a long term goal of designing effective therapies.
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