The overall objective of this project is to characterize the functional development of the salivary glands. Using the rat submandibular gland as a model of postnatal development, the specific goal for the new grant period is to investigate various elements of a model of the secretory pathway involved in the response to parasynmpathomimetic stimulation and the temporal sequence of their appearance in the early postnatal period. This model involves a stimulation-induced increase in cellular Ca++, which increases membrane permeability to Na+ and activates the Na, K ATPase. The Na+ gradient created by this pump favors a further influx of Na+ and Cl- by a carrier-mediated, furosemide sensitive cotransport system. Cl- diffuses out through the luminal cell membrane, creating an electrochemical gradient which favors the extrusion of Na+. This causes an osmotic gradient for the transfer of water into the lumen. Submandibular glands of 1, 7, 14 and 21 day old rats will be used to study the following aspects of this model: (1) Gland slices will be used to study the effects of parasympathomimetic stimuli onion (Na, Cl, Ca) content, ion (22Na, 36Cl, 45Ca) fluxes, Na+, K+ ATPase activity, cGMP levels and guanylate cyclase and phosphodiesterase activities. The effects of antagonists and transport inhibitors on these parameters will also be investigated, as will the effect of cation ionophores (A23187, monensin). Effects of exogenous cGMP on Na+, K+ ATPase will also be evaluated. Release and uptake of K+ will be monitored as an index of in vitro secretory responses in all these experiments. (2) In situ glands will be used to study the effects of furosemide on acetylcholine-induced secretion of saliva, glandular Na+, K+ ATPase activity and cGMP metabolism. The results will be compared with those obtained with the in vitro slice system. A morphological study and an electrophoretic analysis of changes in glandular proteins will be carried out at the various postnatal ages to support the view that the observed changes represent primarily the contribution of the rapidly proliferating acinar cells. The proposed studies will determine whether the model indicated above applies to the cholinergic secretory pathway of developing rat submandibular glands and the time of its appearance as an integrated mechanism during postnatal gland differentiation.
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