Mitochondria play a critical role in shaping the exocytotic response of rat pancreatic acinar cells.
We have previously demonstrated [M. Campos-Toimil, T. Bagrij, J.M. Edwardson, P. Thomas, Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry, Curr. Biol. 12 (2002) 211-215] that in rat pancreatic acinar cells, Gd(3+)-sensitive Ca(2+) entry is instrumental in governing which second messenger pathways control secretory activity. However, in those studies, we were unable to demonstrate a significant increase in cytoplasmic [Ca(2+)] during agonist application as a result of this entry pathway. In the present study, we combined pharmacology with ratiometric imaging of fura-2 fluorescence to resolve this issue. We found that 2 microM Gd(3+) significantly inhibits store-mediated Ca(2+) entry. Furthermore, both the protonophore, CCCP (5 microM) and the mitochondrial Ca(2+)-uptake blocker, RU360 (10 microM), led to an enhancement of the plateau phase of the biphasic Ca(2+) response induced by acetylcholine (1 microM). This enhancement was completely abolished by Gd(3+); and as has been previously shown for Gd(3+), RU360 led to a switch to a wortmannin-sensitive form of exocytosis. Using MitoTracker Red staining we found a close association of mitochondria with the lateral plasma membrane. We propose that in rat pancreatic acinar cells, capacitative Ca(2+) entry is targeted directly to mitochondria; and that as a result of Ca(2+) uptake, these mitochondria release "third" messengers which both enhance exocytosis and suppress phosphatidylinositol 3-kinase-dependent secretion.
Thomas
Paul
P
Henry Wellcome Laboratory for Cell Imaging, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. p.thomas@uea.ac.uk
Bagrij
Tanya
T
Campos-Toimil
Manuel
M
Edwardson
J Michael
JM
D000109:Acetylcholine / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D005682:Gadolinium / Q000494:pharmacology; D008297:Male; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D009930:Organic Chemicals; D010179:Pancreas / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D011976:Receptors, Muscarinic / Q000378:metabolism; D017975:Ruthenium Compounds / Q000494:pharmacology; D015398:Signal Transduction; D000077191:Wortmannin
D000730:Androstadienes; D009930:Organic Chemicals; D011976:Receptors, Muscarinic; C112020:Ru 360; D017975:Ruthenium Compounds; C107472:red dye CMXRos; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D005682:Gadolinium; D019869:Phosphatidylinositol 3-Kinases; D000109:Acetylcholine; D002118:Calcium; D000077191:Wortmannin