Intracellular Ca2+ levels and amylase secretion in Quin 2-loaded mouse pancreatic acini

Abstract

Dispersed mouse pancreatic acini were loaded with the Ca2+-sensitive fluorescence probe Quin 2. Stimulation with carbamylcholine or cholecystokinin-octapeptide (CCK-OP) resulted in a rapid increase in Quin 2 fluorescence, which returned to a lower and sustained plateau level within 2 min of secretagogue stimulation. The magnitude of the initial rise in fluorescence intensity and of amylase secretion were closely related to the concentration of agonist used. Maximal fluorescence changes and amylase secretion were noted with 1 X 10(-5) M carbamylcholine and 1 X 10(-9) M CCK-OP, whereas both responses were half maximal in the presence of 5 X 10(-7) M carbamylcholine and approximately 1 X 10(-10) M CCK-OP. The resting cytosolic free Ca2+ concentration, as measured by the intensity of Quin 2 fluorescence, was calculated to be 1.03 +/- 0.12 X 10(-7) M. Cytosolic free Ca2+ rose to 1.3 +/- 0.3 X 10(-6) M after addition of 1 X 10(-5) M carbamylcholine and 1.25 +/- 0.21 X 10(-6) M following 1 X 10(-9) M CCK-OP. Amylase secretion, but not the Quin 2 fluorescence response, was attenuated at higher secretagogue concentrations. Both secretagogue-induced Quin 2 fluorescence and amylase secretion were inhibited by secretagogue antagonists. Removal of Ca2+ from the extracellular medium resulted in a 48.8 +/- 2.0% reduction of carbamylcholine-induced Quin 2 fluorescence. Following addition of carbamylcholine, CCK-OP was unable to stimulate a second increase in Quin 2 fluorescence without the intervening addition of a cholinergic antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)