Initial activation processes of the Na⁺-K⁺-2Cl⁻ cotransporter and the Cl⁻ channel in carbachol-stimulated acinar cells of rat parotid glands

Abstract

To clarify the role of the intracellular Ca²⁺ and Cl⁻ in the activation process of the Na⁺-K⁺-2Cl⁻ cotransporter in rat parotid acinar cells, we made simultaneous recording of the membrane conductance and the reversal potential of the Cl⁻ current at each peak of the oscillatory Cl⁻ current by using the gramicidin-perforated patch recording method. The conductance reflects the intracellular Ca²⁺ level. The intracellular Cl⁻ concentration was calculated from the reversal potential. We blocked the K⁺ channel by charybdotoxin and obtained the data in the second carbachol (CCh, 0.25 μM) application, changing the membrane potential from -80 to -85 mV. The intracellular Cl⁻ concentration was 30-60 mM just after the start of CCh application. It decreased to 10-30 mM in 2 min and then increased to a steady-state level of 20-40 mM. The increase was blocked by the Na⁺-K⁺-2Cl⁻ cotransporter blocker, bumetanide, suggesting that it reflects the cotransporter activity. The membrane conductance began to increase just after the start of CCh application and became almost steady-state in 0.5 min. These suggest that the Cl⁻ channel is activated by the intracellular Ca²⁺ increase before the Na⁺-K⁺-2Cl⁻ cotransporter activation, and that the decrease in the intracellular Cl⁻ concentration may play an important role in the activation of the cotransporter under the condition of the high intracellular Ca²⁺ level. [Jpn J Physiol 54 Suppl:S68 (2004)]