Gramicidin-perforated patch recording revealed the oscillatory nature of secretory Cl- movements in salivary acinar cells

Elevations of cytoplasmic free calcium concentrations ([Ca2+](i)) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl- through Ca2+-activated Cl- channels, while Cl- enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na+-K+-2Cl(-) cotransporter (NKCC) and/or parallel operations of Cl--HCO3- and Na+-H+ exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net Cl- secretion, we analyzed carbachol (CCh)-activated Cl- currents in submandibular acinar cells using the gramicidin-perforated patch recording configuration. Since the linear polypeptide antibiotic gramicidin creates monovalent cation-selective pores, CCh-activated Cl- currents in the gramicidin-perforated patch recording were carried by Cl- efflux via Cl- channels, dependent upon Cl- entry through Cl- transporters expressed in the acinar cells. CCh-evoked oscillatory Cl- currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated Cl- currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitor, nor elimination of external HCO3- had significant effects, suggesting that the cotransporter rather than parallel operations of Cl--HCO3- and Na+-H+ exchangers is the primary Cl- uptake pathway. Pharmacological manipulation of the activities of the Ca2+-activated Cl- channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory Cl- currents, while adjusting to the rate imposed by the Ca2+-activated Cl- channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory Cl- movements may effectively provide a driving force for fluid secretion in intact acinar cells.