MUSCARINIC AGONISTS INDUCE PHOSPHORYLATION-INDEPENDENT ACTIVATION OF THE NHE-1 ISOFORM OF THE NA+ H+ ANTIPORTER IN SALIVARY ACINAR-CELLS/

Cholinergic agonists stimulate isotonic fluid secretion in the parotid gland. This process is driven by the apical exit of Cl-, which enters the cells partly via Cl-/HCO3- exchange across the basolateral membra ne. Acidification of the cytosol by the extrusion of HCO3- is prevente d by the concomitant activation of the Na+/H+ exchanger (NHE), which i s directly activated by cholinergic stimulation. Multiple isoforms of the NHE have been described in mammalian cells, but the particular iso form(s) present in salivary glands and their mechanism of activation h ave not been defined. Reverse transcriptase-polymerase chain reaction with isoform-specific primers was used to establish that NHE-1 and NHE -2, but not NHE-3 or NHE-4, are expressed in parotid glands. The prese nce of NHE-1 was confirmed by immunoblotting and immunofluorescence, w hich additionally demonstrated that this isoform is abundant in the ba solateral membrane of acinar cells. The predominant role of NHE-1 in c arbachol-induced Na+/H+ exchange was established pharmacologically usi ng HOE694, an inhibitor with differential potency toward the individua l isoforms. Because muscarinic agonists induce stimulation of protein kinases in acinar cells, we assessed the role of phosphorylation in th e activation of the antiport. Immunoprecipitation experiments revealed that, although NHE-1 was phosphorylated in the resting state, no furt her phosphorylation occurred upon treatment with carbachol. Similar ph osphopeptide patterns were observed in control and carbachol-treated s amples. Together, these findings indicate that NHE-1, the predominant isoform of the antiporter in the basolateral membrane of acinar cells, is activated during muscarinic stimulation by a phosphorylation-indep endent event. Other processes, such as association of Ca2+-calmodulin complexes to the cytosolic domain of the antiporter, may be responsibl e for the activation of Na+/H+ exchange.