Upregulation of Na+-K+-2Cl(-) cotransporter activity in rat parotid acinar cells by muscarinic stimulation

1. The effects of fluid secretory stimuli on the Na+-K+-2Cl(-) cotransporter in rat parotid acini were investigated. Cotransporter activity was measured using NH4+ as a K+ surrogate and following cotransporter-mediated NH4+ fluxes by monitoring intracellular pH. 2. A dramatic upregulation (15- to 20-fold) of acinar Na+-K+-2Cl(-) cotransporter activity was induced by muscarinic, alpha(1)-adrenergic and peptidergic stimuli. A half-maximal effect of the muscarinic agonist carbachol was observed at similar to 0.5 mu M. 3. Our results indicate that the rise in intracellular calcium concentration ([Ca2+](i)) which accompanies these stimuli is both a necessary and a sufficient condition for this effect; but it is not a consequence of the KCl loss and concomitant isotonic shrinkage caused by increased [Ca2+](i) as it persists when these effects are prevented. 4. The effect of muscarinic stimulation on the cotransporter can, however, be blocked by inhibitors of phospholipase A, (4-bromophenacylbromide and manoalide), by a general inhibitor of arachidonic acid metabolism (5,8,11,14-eicosatetraynoic acid) and by specific inhibitors of the cytochrome P450 pathway (methoxsalen and ketoconazole). 5. These latter results argue strongly for the involvement of a product of the cytochrome P450 pathway of arachidonic acid metabolism in upregulation of the salivary Na+-K+-2Cl(-) cotransporter. 6. Owing to the complexity of the arachidonic acid cascade a wide variety of agents could potentially interfere with this upregulation of the cotransporter, and thereby result in decreased salivary fluid production. We suggest that such an effect could underlie the dry mouth (xerostomia) that occurs as an unexplained side-effect of many commonly prescribed medications.