Proceedings of The Physiological Society
University of Central Lancashire (2002) J Physiol 543P, S239
Effect of hydrogen peroxide on cholecystokinin-induced secretory activity in mouse pancreatic acinar cells monitored by the membrane fluorescent dye FM1-43
A.I. Lajas, G.M. Salido and J.A. Pariente
Department of Physiology, Faculty of Veterinary Science, University of Extremadura, 10071-Caceres, Spain
Stimulation of pancreatic acinar cells induces the release of digestive enzyme via exocytotic fusion of zymogen granules. Recently, optical measurements of membrane turnover have been developed that use membrane-sensitive fluorescent probes that can provide real-time measurements of secretory activity (Smith & Betz, 1996). The most extensively used of these probes is the non-permeant membrane indicator FM1-43, which is non-fluorescent in aqueous solution but becomes fluorescent when incorporated into the plasma membrane. Previous studies in pancreatic acinar cells have reported that reactive oxygen species, such as hydrogen peroxide (H2O2), are able to mobilize calcium from intracellular stores but inhibit CCK-induced calcium mobilization (Pariente et al. 2001). In the present study we have studied the effect of H2O2 on CCK-8-induced secretory activity by monitoring FM1-43 fluorescence using confocal microscopy. FM1-43 was excited by the 488 nm laser light line from the krypton-argon laser, and emitted light passed through a 515 nm light long-pass filter before measurement of fluorescence intensity. The optical thickness was typically 1 mm and taken through an equatorial region of an acinus. In addition, amylase release was also determined using the Phadebas blue starch method (Ceska et al. 1969). Cells were incubated with secretagogue for 30 min and amylase activities were expressed as a percentage of the total content of amylase at the beginning of the incubation. The donor mice were killed by rapid cervical dislocation. First of all, spontaneous exocytosis was measured by perifusion of cells with FM1-43 (1 mM) in the absence of secretagogue for 10 min. In these conditions the FM1-43 fluorescence remained constant or increased slowly over minutes but never increased by more than 5.2 ± 3.1 % (mean ± S.E.M.) with respect to the fluorescence intensity at time zero. Removal of FM1-43 rapidly reduced the cell fluorescence to a residual level. Perifusion of pancreatic acinar cells with 1 nM CCK-8 caused an increase in FM1-43 fluorescence by an additional 80.9 ± 10.7 %. This increase was observed in all 49 cells recorded, and usually consisted of a rapid initial rise, followed by a sustained plateau in fluorescence. The application of 1 mM H2O2 to acinar cells evoked a slight increase in FM1-43 fluorescence (9.3 ± 5.6 %, n = 28 cells), which was significantly smaller (P < 0.05, paired t test) than that evoked by CCK-8. Additionally, simultaneous addition of CCK-8 and H2O2 clearly abolished the CCK-8-induced fluorescence increase. FM1-43 fluorescence change was only 3.1 ± 9 % (n = 15 cells). Similar results have been obtained in amylase experiments, where 1 mM H2O2 attenuated the secretory effect of 1 nM CCK-8 (9.2 ± 0.8 %, n = 15) compared with the response to CCK-8 in the absence of H2O2 (14.5 ± 0.9 %, n = 15). Our results show that the reactive oxygen species H2O2 inhibits the exocytotic activity induced by the secretagogue CCK. Further investigation should be carried out to clearly identify the mechanism of action of H2O2 on CCK-induced secretory activity in the exocrine pancreas.
This work was supported by the Junta de Extremadura (Consejeria de Sanidad y Consumo) grant 01/32.
All procedures accord with current national guidelines.
Where applicable, experiments conform with Society ethical requirements