Relation between free cytosolic calcium and amylase release by pancreatic acini

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Abstract
Pancreatic acini were loaded with the Ca-selective fluorescent indicator quin-2 by incubation with its acetyoxymethyl ester. Loading acini with 844 .+-. 133 .mu.M quin-2 altered neither their ultrastructure nor their viability. The rate of amylase release from quin-2-loaded acini in response to the secretagogue carbachol, however, was significantly smaller than that of control acini. Studies in which acini were loaded with both quin-2 and a similar Ca-chelating compound, BAPTA, indicated that this reduced amylase release was related to the Ca buffering properies of quin-2. The concentration of free intracellular Ca calculated from the fluorescence of quin-2 was 90 .+-. 18 nM. Stimulation by carbachol of acini suspended in media containing 1.25 mM Ca caused a rapid, transient enhancement of this value. After stimulation with 10-6 M carbachol, an optimal concentration for stimulating amylase release, the onset of the rise in free cytosolic Ca levels was observed in 1.1 .+-. 0.1 s following the addition of agonist, and peak Ca levels(545 .+-. 112 nM) were obtained within 5.3 .+-. 0.3 s. For concentrations of carbachol .ltoreq. 10-6 M, a stoichiometric relation was found between stimulated amylase release and the peak concentration of free cytosolic Ca achieved. At higher concentrations of carbachol, however, the peak free cytosolic Ca remained constant while amylase release declined. The latency of the rise in intracellular Ca following stimulation of acini suspended in normal media, but the rise time was significantly prolonged. In the presence of extracellular Ca, the intracellular level of Ca remained elevated 2.8-fold above basal levels for at least 15 min following stimulation with 10-6 M carbachol, whereas it had returned to near resting levels by 15 min when either 3 .times. 10-7 or 3 .times. 10-5 M carbachol was the stimulus. The Ca ionophore ionomycin (10-6 M) induced changes in the level of free cytsolic Ca similar to those caused by 10-6 M carbachol. Ionomycin, however, stimulated only approximately one-third as much amylase release. These data suggest that factors in addition to changes in free cytosolic Ca may be important in regulating enzyme secretion by pancreatic acinar cells.

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