THAPSIGARGIN DEFINES ROLES OF CA2+ IN INITIAL, SUSTAINED, AND POTENTIATED STIMULATION OF PEPSINOGEN SECRETION

The roles of Ca2+ in agonist-induced pepsinogen secretion from guinea pig chief cells remain unclear. We used cholecystokinin octapeptide (C CK-8) or secretin alone or with thapsigargin (TG) to clarify these rol es. TG releases Ca2+ from intracellular stores by inhibiting microsoma l Ca2+-adenosinetriphosphatase (ATPase), thereby depleting intracellul ar Ca2+ (Ca-i(2+)) stores. In most cells TG also causes Ca2+ influx. I n the present study, with an extracellular Ca2+ concentration ([Ca2+]( o)) of 1.5 mM, CCK-8 (0.1 mu M) caused a rapid increase in pepsinogen secretion; however, the rate decreased with time. With [Ca2+](o) = 0, the initial increase was similar but later secretion was abolished, su ggesting that Ca2+ influx was important for sustained secretion. With [Ca2+](o) = 1.5 mM, TG (0.1 mu M) caused a 2.7-fold sustained increase in in Ca-i(2+) concentration ([Ca2+](i)) and a ninefold sustained inc rease in pepsinogen secretion. With [Ca2+](o) = 0, TG caused a transie nt 66% increase in [Ca2+](i) and a 50% increase in pepsinogen secretio n. The time course of TG-induced pepsinogen secretion correlated with the time course of TG-induced increases in [Ca2+](i). These data demon strated that Ca2+ influx itself was a potent stimulant of pepsinogen s ecretion. We further focused on the roles of increasing [Ca2+](i) from Ca-i(2+) stores. With or without extracellular Ca2+ (Ca-o(2+)) presen t, addition of CCK-8 (0.1 mu M) 10 min after TG caused no further incr ease in [Ca2+](i), demonstrating depletion of the inositol 1,4,5-trisp hosphate-sensitive pool. The Ca2+-mobilizing agent CCK-8 caused no pep sinogen secretion 10 min after TG preincubation, demonstrating that mo bilization of Ca2+ from intracellular stores was important in the rapi d initial phase stimulation of pepsinogen secretion caused by CCK-8. I n contrast, preincubation with TG had no effect on pepsinogen secretio n by secretin, an agent that increases adenosine 3',5'-cyclic monophos phate. A 6-min preincubation with TG potentiated the subsequent stimul ation of pepsinogen secretion caused by secretin in the presence of Ca 2+ where [Ca2+](i) remained elevated. However, TG-induced potentiation of secretin-stimulated pepsinogen secretion was abolished once [Ca2+] (i) had returned to the basal level in the absence of Ca2+. These resu lts suggest that 1) increases in [Ca2+](i) due to Ca2+ influx caused b y TG or CCK-8 can result in pepsinogen secretion, 2) mobilization of C a2+ from intracellular stores is important in the rapid initial phase stimulation of pepsinogen secretion caused by CCK-8, and 3) the increa se in [Ca2+](i) per se, whether caused by mobilization from Ca-i(2+) s tores or Ca2+ influx, is responsible for CCK-8 or TG potentiating secr etin-stimulated pepsinogen secretion.