Ca(2+)-dependent amylase secretion from SLO-permeabilized rat pancreatic acini requires diffusible cytosolic proteins

Abstract

Streptolysin O (SLO)-permeabilized pancreatic acini are now frequently used to study regulated exocytosis in the exocrine pancreas. In this paper we introduce alpha-toxin as a possible alternative permeabilization agent to SLO. Both alpha-toxin and SLO are bacterial cytolysins, but the membrane pores generated by SLO are approximately 5-10 times larger than those formed by alpha-toxin. The Ca2+ requirements for amylase secretion from both types of permeabilized acini were identical, maximal amylase secretion being obtained at 30 microM Ca2+ with an effective concentration of approximately 3-4 microM Ca2+ producing 50% of the maximal response. However, Ca(2+)-stimulated amylase secretion from the SLO-permeabilized acini stopped after 10-15 min, unlike secretion from the alpha-toxin-permeabilized cells, which continued for at least 50 min. The rapid cessation of secretion from the SLO-treated acini reflects the rapid decline in the responsiveness of the cells observed after permeabilization. This decline in Ca(2+)-dependent secretion appears to be due to the loss of cytosol, since addition of purified rat brain cytosol to nonresponsive SLO-permeabilized acini reconstituted regulated secretion. Because alpha-toxin-permeabilized acini maintained their responsiveness, the cytosolic factors lost from the SLO-permeabilized cells must be retained within the toxin-treated cells. The reconstitutive activity of the brain cytosol was nondialyzable but heat and trypsin sensitive, suggesting that the factors responsible are proteins. Of the cytosols screened (brain, liver, spleen, muscle, and lacrimal) only those prepared from brain or lacrimal gland reconstituted Ca(2+)-dependent amylase secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

CRHSP-28 Regulates Ca2+-stimulated Secretion in Permeabilized Acinar Cells

“…Secretion of the digestive enzyme, amylase, into the medium was measured at various times over 30 min. Consistent with previous studies utilizing SLO-permeabilized acini (30,34), the highest rate of Ca 2ϩ -stimulated secretion occurred during the first 10 min of incubation when 6.1% of total cellular amylase was released into the medium (filled squares). A considerably slower rate of secretion occurred during the following 10-min intervals adding up to 9.5 and 11.9% of total cellular amylase at 20 and 30 min, respectively.…”

“…Again, CRHSP-28 slowly diffused from the cells over the 15-min incubation, and its appearance in the medium was clearly enhanced at each time point when Ca 2ϩ was elevated. Characterization of CRHSP-28-enhanced Secretion-Corresponding with previous studies utilizing SLO-permeabilized acini (30,34), amylase secretion in these preparations was stimulated by physiological levels of free Ca 2ϩ with maximal release occurring between 3 and 10 M Ca 2ϩ (Fig. 3).…”

“…2ϩ -stimulated Secretion in Permeabilized Acini-A role for CRHSP-28 in pancreatic secretion was examined utilizing a well characterized SLO-permeabilized acinar cell preparation (11,18,30,34) (Fig. 1).…”

“…CRHSP-28 Leakage from SLO-permeabilized Acini-Previous studies indicate that SLO permeabilization of acini is complete within 2-5 min at 37°C and is not altered by micromolar concentrations of free Ca 2ϩ (11,30,34). To ensure complete permeabilization of cells in our preparations, diffusion of the 12-kDa cytosolic protein cyclophilin A from acini was analyzed by immunoblotting following SLO treatment ( Fig.…”

Multiple Fatty Acid Sensing Mechanisms Operate in Enteroendocrine Cells

“…This technique has been previously applied to study intracellular Ca 2ϩ stores or exocytosis in several cell types including gastric epithelial cells (24), pancreatic acinar cells (13,25), and platelets (26). We optimized and validated this method for use in STC-1 cells.…”