In the exocrine pancreas agonist-evoked elevations of [Ca2+]c play a key role underlying both digestive enzyme secretion and longer term adaptive responses such as gene transcription and growth. Inappropriate [Ca2+]c signaling is also strongly implicated in pancreatic disease. Agonist-stimulated Ca2+ oscillations in acinar cells carry distinct spatial and temporal information, as defined by differences in Ca2+ release, Ca2+ influx and Ca2+ clearance. These characteristics are unique to individual agonists. In this proposal we will test the hypothesis that cross-talk between the cAMP and Ca2+ signaling pathways plays a major role in """"""""shaping"""""""" the Ca2+ signals for cholecystokinin (CCK) and bombesin but not muscarinic receptor stimulation. An additional, related question is whether the specific spatio-temporal characteristics of the Ca2+ signal stimulated by individual agonists are important for the specific activation of down-stream effectors. Mouse pancreatic acinar cells from native and protein kinase A (PKA)-regulatory subunit null transgenic animals will be used as a model system. Using digital imaging of Ca2+ indicator dyes and flash photolysis of caged compounds together with biochemical assays the effects of raising cAMP on the processes of Ca2+ release, influx and removal will be evaluated for their contribution to the signaling pattern stimulated by CCK and bombesin. We will determine which inositol 1,4,5 trisphosphate receptor subtypes (InsP3R) are phosphorylated and if ryanodine receptor phosphorylation occurs. In genetically engineered cell-lines we will study each receptor in isolation to determine the functional effects of phosphorylation of individual InsP3R subtypes in order to predict the subtypes important in acinar cells. Using site directed mutagenesis and expression of constructs on a novel null background we will determine the sites of phosphorylation of InsP3R sub types. The dominant Ca2+ influx pathway occurring during oscillations (store-operated or non-capacitative) will be defined and the effects of raising cAMP determined. We will also consider that any effects of cAMP may be independent of PKA. The effects of raising cAMP will be evaluated on Ca2+ removal from the cytoplasm and the loci of the effect determined. Finally, we will determine if the specific characteristics of the Ca2+ signal are important in efficiently activating distract physiological effectors in acinar cells by studying the characteristics of the Ca2+ signal necessary and sufficient to efficiently stimulate gene transcription. This proposal is designed to provide a detailed, molecular understanding of the processes underlying [Ca2+]c signaling in the exocrine pancreas. This information is fundamentally important to appreciating the normal functioning of the gland and to the subsequent development of effective strategies for the treatment of pancreatic dysfunction.
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