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⁻-HCO⁻₃ 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 HCO⁻₃ had significant effects, suggesting that the cotransporter rather than parallel operations of Cl⁻-HCO⁻₃ 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.