A role for Ca 2+ in mediating hormone-induced biphasic pepsinogen secretion from the chief cell determined by luminescent and fluorescent probes and X-ray microprobe

Abstract

In isolated chief cells from the guinea pig, cholecystokinin (10 nM) and a high concentration of ionomycin each caused a biphasic pattern of pepsinogen secretion. The initial fast response to cholecystokinin was not dependent on medium Ca 2+ and was mimicked by low concentration of ionomycin (100 nM). Inositol 1,4,5-trisphosphate caused a similar fast release from permeabilized cells. The slow component of release was dependent on medium Ca 2+, however, and was mimicked by the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (TPA) (100 nM) or the diacylglycerol analogue 1-oleoyl-2-acetylglycerol (OAG) (100 μM). Ionomycin (100 nM) and TPA (and/or OAG), when applied together, reproduced the biphasic pattern of pepsinogen secretion, suggesting that the signalling pathways utilized by both types of agonist contribute to the response evoked by cholecystokinin-hormone stimulation. Both fura-2 and aequorin were used to monitor changes of intracellular Ca 2+. Three pathways were found to contribute to the Ca 2+ transient. A rapid release of Ca 2+ from intracellular store(s), a rapid Ca 2+ entry from the extracellular space, and a more sustained Ca 2+ entry from the extracellular space. Cholecystokinin induced a rapid increase in cytoplasmid Ca 2+ ([Ca 2+] i) as estimated with fura-2 and aequorin. This rise was reduced but not abolished upon removal of extracellular Ca 2+, suggesting that both Ca 2+ entry from the extracellular space and Ca 2+ mobilization from the intracellular store(s) contribute to the initial, fast component of the Ca 2+ transient. A second, more sustained component of the Ca 2+ transient induced by cholecystokinin was abolished by lanthanum. TPA and OAG induced a biphasic Ca 2+ transient that could be detected only with aequorin. The late, sustained component of this response was again abolished by lanthanum as well as by removal of extracellular Ca 2+. It appears that the late component of the Ca 2+ transient is dependent on Ca 2+ influx from the extracellular space and is too localized to be detected by fura-2. Prestimulation of cells with TPA or OAG preventes the aequorin transient caused by cholecystokinin and vice versa, suggesting that TPA, OAG and cholecystokinin activate the same pathways of Ca 2+ entry into the cytosol from the intracellular store(s) or the extracellular space. The stimulation-sensitive Ca 2+ pool was examined with electron probe X-ray microanalysis. It appears to be restricted to an area enriched in secretory granules or peripheral endoplasmic reticulum just beneath the apical plasma membrane and in close association with the microtubular-microfilamentous system. Ionomycin stimulation decreased the Ca level in secretory granules and reciprocally increased the cytoplasmic level of Ca. These changes were accompanied by entry Na + and Cl - from the extracellular space and by K + efflux from the cell. Our data suggest that the initial fast component of pepsinogen secretion is associated with a high, fast and more global rise of cytoplasmic Ca 2+, and that the sustained component is associated with a late, prolonged and more localized elevation of cytoplasmic Ca 2+ that is dependent on influx of Ca 2+ from the extracellular space.