hide
Free keywords:
fusion; ruthenium red; heparin; GTP
Abstract:
Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP3) sensitive and IP3-insensitive nonmitochondrial intracellular Ca2+ pools in acinar cells of the exocrine pancreas. For further characterization of Ca2+ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designated P1 to P5 from the top to the bottom of the gradient. Measuring Ca2+ uptake and Ca2+ release with a Ca2+ electrode, we could differentiate three nonmitochondrial intracellular Ca2+ pools: (i) an IP3-sensitive Ca2+ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca2+ pool (CasCaP), vanadate- and IP3-insensitive, and (iii) a vanadate-sensitive Ca2+ pool (VasCaP), neither IP3- nor caffeine-sensitive, into which Ca2+ uptake is mediated via a Ca2+ ATPase sensitive to vanadate at 10-4 mol/liter. A fourth Ca2+ pool is neither IP3- nor caffeine- or vanadate-sensitive. Percoll fraction P1 contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca2+ uptake and Ca2+ release. When membrane vesicles were incubated in the presence of caffeine (2 x 10-2) mol/liter), Ca2+ uptake up to the steady state [Ca2+] did not appear to be altered as compared to the control Ca2+ uptake. However, in control vesicles spontaneous Ca2+ release occurred after the steady state had been reached, whereas caffeine-pretreated vesicles did not spontaneously release Ca2+. Addition of IP3 at steady state [Ca2+] induced similar Ca2+ release followed by Ca2+ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca2+ was released from all Ca2+ pools including the IsCaP. Following Ca2+ reuptake after IP3 had been added, a second addition of IP3 to control vesicles induced further but smaller Ca2+ release, and a third addition resulted in a steady Ca2+ efflux by which all Ca2+ that had been taken up was released. This steady Ca2+ release started at a Ca2+ concentration between 5.5-8 x 10-7 mol/liter and could also be induced by the IP3 analogue inositol 1,4,5-trisphosphorothioate (IPS3) or by addition of Ca2+ itself. Ruthenium red (10-5 mol/liter) inhibited both caffeine-induced as well as Ca2+-induced but not IP3-induced Ca2+ release. Heparin (100 micrograms/ml) inhibited IP3- but not caffeine-induced Ca2+ release. The data indicate the presence of at least three separate Ca2+ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca2+ uptake these Ca2+ pools appear to be separate
