Stimulation-induced fluid secretion requires an inwardly directed Na chemical gradient. In sublingual mucous acinar cells, this approximately 10-fold gradient drives both the extrusion of protons via a Na/H exchange mechanism and the net re-uptake of C1 mediated by the Na/K/2C1 cotransporter. These two Na transport pathways are thus important for regulating intracellular pH and for driving anion-dependent fluid secretion. Muscarinic receptor stimulation causes the intracellular pH of sublingual acinar cells to acidify due to HCO3 efflux. Injury to the cell by this agonist-induced drop in the intracellular pH is prevented by the activation of a Na/H exchanger. Consistent with the hypothesis that Na/H exchange is a functionally significant transport mechanism in salivary acini, fluid secretion is reduced markedly when the Na/H exchanger is inhibited. We have recently observed that following the agonist-induced loss of K and C1, the Na/K/2Cl cotransporter is the primary, if not the only, C1 re-uptake pathway present in sublingual acinar cells. Emphasis will be placed on combining two complementary approaches, molecular biological and physiological, in order to gain a better understanding of the structure-function relationships of sublingual gland sodium transport proteins in the fluid secretion process. (1) Inhibitor susceptibility and kinetic properties of sodium transport proteins will be examined in sublingual acini and functional expression systems. The regulation of these proteins by pH, Mg2+, and Ca2+ will be examined using ion-sensitive fluorescent dyes and radioisotope studies. These studies will reveal possible similarities to Na transport proteins in other tissues and provide critical information about transport mechanism and function. (2) Defining the primary sequence of the Na/H exchanger protein in sublingual salivary glands is an important first step in determining the structural basis for the uptake of Na and the regulation of intracellular pH by the Na/H exchanger. We will screen cDNA libraries from the rat sublingual gland with specific cDNA probes for NHE1, NHE3 and NHE4, isoforms of the Na/H exchange gene family (generously provided by Dr. Gary Shull, University of Cincinnati). This will permit us to isolate positive clones an to deduce the primary sequence by conceptual translation of the sublingual gland Na/H exchange protein. Functional expression of the sublingual gland Na/H exchanger in a Na/H exchange (-) cell line will permit functional verification and detailed kinetic analyses of the regulation of this important Na transport mechanism. Insight gained from these studies may therefore aid in developing rationales for preventing and/or treating salivary gland dysfunctions caused by a myriad of perturbations.