The effect of calcium and cyclic AMP on amylase release in digitonin-permeabilized parotid gland cells

Abstract

Rat parotid cells were permeabilized with digitonin to examine their secretory dynamics. Cells were isolated by a modification of the method previously described by Hootman ((1985). J. Biol. Chem. 260, 4186–4194) in which α-chymotrypsin was included. The final preparation consisted of approx. 40–60% single cells. The cells were 85–90% viable by trypan blue exclusion and secreted amylase when stimulated with isoproterenol. Digitonin (2 or 5 μM) was sufficient for permeabilization while 2 μM digitonin was somewhat more effective in maintaining cell integrity as indicated by lactate dehydrogenase release. Digitonin had minimal effects on intracellular granules in the whole cell and was, thus, relatively selective. The response of digitonin-permeabilized cells to calcium (without secretagogues) in the incubation medium was monitored by amylase release. For a wide range of applied free calcium concentrations (1·10 −7 M to 10 −4 M) a statistically significant increase in amylase secretion was observed. Control cells did not release amylase to a similar extent without secretagogue. Cyclic AMP (50 μM) significantly enhanced amylase secretion from digitonin-treated cells at all concentrations of free calcium tested. Neither calcium nor cyclic AMP alone was sufficient to stimulate maximal amylase release. Our results provide direct evidence for a model in which calcium and cyclic AMP work on separate pathways as interacting regulators of exocytosis.