Induction of Ca2+ oscillations by selective, U73122-mediated, depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells.
The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca2+ sequestering and releasing properties of internal Ca2+ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca2+ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P3]-sensitive, but not the Ins(1,4,5)P3-insensitive, Ca2+ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca2+ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca2+ concentration ([Ca2+]i,av), which was largely independent of external Ca2+. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonist-evoked [Ca2+]i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca(2+)-ATPase activity resulted in a [Ca2+]i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P3- casu quo agonist-sensitive internal Ca2+ store, whereas thapsigargin affects both the Ins(1,4,5)P3-sensitive and -insensitive Ca2+ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca2+]i. The U73122-evoked oscillations were abolished in the absence of external Ca2+. The ability of U73122 to generate external Ca(2+)-dependent Ca2+ oscillations suggests that depletion of the agonist-sensitive store leads to an increase in Ca2+ permeability of the plasma membrane and that the Ins(1,4,5)P3-insensitive Ca2+ pool is necessary for the Ca2+ oscillations.
Willems
P H
PH
Department of Biochemistry, University of Nijmegen, The Netherlands.
Van de Put
F H
FH
Engbersen
R
R
Bosch
R R
RR
Van Hoof
H J
HJ
de Pont
J J
JJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004963:Estrenes / Q000494:pharmacology*; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology*; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology*; D011817:Rabbits; D012844:Sincalide / Q000031:analogs & derivatives / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D004963:Estrenes; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C058451:JMV 180; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D012844:Sincalide; D002118:Calcium