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

Elevations of cytoplasmic free calcium concentrations ([[[Ca.sup.2+]].sub.i]) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary, acinar cells. This process is driven by the apical exit of [Cl.sup.-] through [Ca.sup.2+]-activated [Cl.sup.-] channels, while [Cl.sup.-] enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive [Na.sup.2+] -[K.sup.+] -2[Cl.sup.-]-2[Cl.sup-] cotransporter (NKCC) and/or parallel operations of [Cl.sup.-] -HC[O.sup.-.sub.3] and [Na.sup.+][-H.sup.+] exchangers, located in the basolateral membrane. To characterize the contributions of those activities to net [Cl.sup.-] secretion, we analyzed carbachol (CCh)-activated [Cl.sup.-] 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 CI currents in the gramicidin-perforated patch recording were carried by [Cl.sup.-] efflux via [Cl.sup.-] channels, dependent upon [Cl.sup.-] entry through [Cl.sup.-] transporters expressed in the acinar cells. CCh-evoked oscillatory CI currents were associated with oscillations of membrane potential. Bumetanide, a loop diuretic, decreased the CCh-activated C1 currents and hyperpolarized the membrane potential. In contrast, neither methazolamide, a carbonic anhydrase inhibitox; nor elimination of external HC[O.sup.-.sub.3] had significant effects, suggesting that the cotransporter rather than parallel operations of [Cl.sup.-] -HC[O.sup.-.sub.3] and [Na.sup.+][-H.sup.+] exchangers is the primary [Cl.sup.-] uptake pathway. Pharmacological manipulation of the activities of the [Ca.sup.2+]-activated [Cl.sup.-] channel and the NKCC revealed that the NKCC plays a substantial role in determining the amplitude of oscillatory [Cl.sup.-] currents, while adjusting to the rate imposed by the [Ca.sup.2+]-activated [Cl.sup.-] channel, in the gramicidin-perforated patch configuration. By concerting with and being controlled by the cation steps, the oscillatory form of secretory [Cl.sup.-] movements may effectively provide a driving force for fluid secretion in intact acinar cells. KEY WORDS: chloride secretion * exocrine glands * chloride channels * cation-chloride cotransporter * gramicidin