Amplification of the thapsigargin-evoked increase in the cytosolic free Ca2+ concentration by acetylcholine in acutely isolated mouse submandibular acinar cells

摘要

pThe intracellular Casup2+/sup concentration was measured in single, acutely isolated, mouse submandibular acinar cells loaded with fura-2 AM. All experiments were performed in the absence of extracellular Casup2+/sup in order to eliminate Casup2+/sup influx. The microsomal ATPase inhibitor, thapsigargin, was used to release Casup2+/sup from intracellular stores and simultaneously prevent re-uptake into the stores. Sequential application of thapsigargin (2 μM) and the Casup2+/sup ionophore ionomycin (500 nM) indicated that thapsigargin was able to mobilize practically all intracellular Casup2+/sup. Furthermore, in comparison with results obtained following inhibition of the plasma membrane Casup2+/sup-ATPase by Lasup3+/sup (2 mM), it may be shown that slowly unloading the intracellular Casup2+/sup stores using thapsigargin does not normally cause a massive, cytotoxic, increase in the cytosolic Casup2+/sup concentration, because Casup2+/sup is rapidly extruded from the cell across the plasma membrane. Application of a submaximal dose of acetylcholine (500 nM) during the rising phase of the response to thapsigargin caused a 3–4-fold increase in the amplitude of the rise in the cytosolic Casup2+/sup concentration without any significant alteration of the time course of the response. As thapsigargin alone is capable of mobilizing all releasable Casup2+/sup, this increase in amplitude is most likely the result of inhibition of the Casup2+/sup extrusion process by acetylcholine./p