Interaction of caffeine-, IP3- and vanadate-sensitive Ca2+ pools in acinar cells of the exocrine pancreas

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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 designatedP 1 toP 5 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 fractionP 1 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 (2×10−2 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 cfffeine-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 ×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 μg/m) 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. However, when steady state is reached, we assume that these Ca2+ pools come into contact and total Ca2+ release from all three pools can occur. The mechanism of this “contact” of Ca2+ pools is not clear but seems to be different from that induced by GTP in the presence of polyethylene glycol, which probably involves fusion of membranes.