The long-term objective of this project is to elucidate how the various functional elements of stimulus-response coupling become established in rat salivary acinar cells during postnatal development. Our findings during the current grant period have shown age-related and dose-related differences in the intermediate (IP3 formation, Ca2+ mobilization) steps of the signal transduction pathway after cholinergic stimulation. They have also shown an apparent dissociation between IP3/Ca2+ responses and more distal events (K and Cl efflux) of the secretory pathway in the early stages of postnatal development. Based on these observations, our goal for the new grant period is to further investigate aspects of the signal transduction process that may help explain these functional differences during Postnatal gland development. As in previous studies, we will use submandibular cells of 1, 7, 14 and 21-day old and of adult rats to compare the following: 1) Proximal events in signalling, including receptor affinity for acetylcholine; the presence and receptor coupling of G proteins; the activity and isoforms of phospholipase C; membrane phosphoinositides;c activities of lP3 Kinase, IP3 phosphatase, PI Kinase and PIP Kinase; the functional coupling of phospholipase C to muscarinic, substance P and alpha-adrenergic receptors. 2) Ca 2+ storage sites and the localization of the changes in intracellular Ca2+ that occur upon stimulation. A combination of methods will be used for this purpose, including x-ray microprobe analysis, quantitation of 35S-thio-IP3 binding, imaging with fluorescent probes and 45 Ca or ruthenium-red labeling of proteins in subcellular fractions. 3) The altered coupling between Ca2+ and monovalent ion transport. Cell Ca 2+ will be 'clamped' with ionomycin and the efflux of 86 Rb and 36Cl will be measured at different external Ca2+. Cell volume will be measured under these conditions using isotopic, imaging and light scattering methods and K channels will be evaluated with 125I charybdotoxin. Ion channels will also be studied by patch clamping. The x-ray microprobe analysis and the patch clamping will be done in collaboration with Profs. G. Roomans (University of Uppsala) and O.H. Petersen (University of Liverpool). Our working hypothesis is that immature salivary cells have differences in: a) the phospholipid/phospholipase complex or in their coupling to the acetylcholine receptor; and b) intracellular Ca 2+ pools, their localization or compartmentalization or Ca2+-regulated channels. The proposed studies should contribute to our understanding of the functional development of salivary glands and of how the signalling mechanism evolves during this process.
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