| Summary: | Since the discovery that inositol 1,4,5-trisphosphate (IP₃) can release Ca²⁺ from intracellular stores, the phosphatidylinositol signal transduction system has been recognized as a major transduction pathway in most cell types. However, IP₃ is not the only intracellular Ca²⁺ mediator. In some cells, guanosine 5'-triphosphate (GTP) and arachidonic acid are known to mobilize intracellular Ca²⁺ independent of the action of IP₃ and probably play important roles in Ca²⁺ signalling. In pancreatic ductal cells the precise mechanisms of intracellular Ca²⁺ movement however are not well known. The aims of the present study are to identify and characterize intracellular Ca²⁺ pools and to investigate regulation of intracellular Ca²⁺ movement in PANC-1 cells (ductal cells of human pancreatic carcinoma) origin).
Endoplasmic reticulum (ER)-enriched microsomal membrane fractions were prepared from PANC-1 cells. Ca²⁺ could be actively accumulated into the microsomes driven by an ATP-dependent Ca²⁺ -ATPase. Addition of IP₃ maximally caused a 20% release of actively accumulated Ca²⁺ which was completely blocked by heparin, an antagonist of the IP₃ receptor. Extravesicular Ca²⁺ produced an inhibition of IP₃-activated Ca²⁺ release. GTP alone stimulated a 5% Ca²⁺ release. In the presence of 3% polyethylene glycol (PEG), GTP maximally discharged 60-65% of the accumulated Ca²⁺ from the microsomal membrane fractions. The combination of GTP and IP₃ resulted in a greater release than either agent alone. The GTP effect was independent of IP₃ and not inhibited by heparin, indicating that the IP₃-activated Ca²⁺ channel is probably not involved in GTP-induced Ca²⁺ release. The release in response to GTP appeared to be mediated by an enzymatic GTP hydrolytic process since GTP7S, a nonhydrolyzable GTP analogue, had no effect on the release of Ca²⁺ . Arachidonic acid mobilized intracellular Calf in a concentration-dependent manner and maximally released 80% of the Ca²⁺ from the microsomes. Addition of indomethacin or nordihydroguaiaretic acid (NDGA), inhibitors of cyclooxygenase and lipoxygenase products, failed to block the action of arachidonic acid. These results indicate that arachidonic acid mediates therelease of Ca²⁺ from the microsomes by a direct effect, not by its metabolites. 1P3was ineffective in releasing any further Ca²⁺ from the microsomes following maximal Ca²⁺ release by arachidonic acid, indicating that a proportion of IP₃- and arachidonic acid-sensitive Ca²⁺ pools overlap. Other fatty acids also induced similar effects on Ca²⁺ release, suggesting that arachidonic acid-mediated Ca²⁺ release appeared to be nonspecific. Thapsigargin, an inhibitor of the intracellularCa²⁺ -ATPase, was shown to inhibit Ca²⁺ accumulation into and induce Ca²⁺ release from PANC-1 microsomes. The thapsigargin-releasable Ca²⁺ pool included the 1P3- or arachidonic acid-sensitive pool. Studies carried out using phospholipid vesicles showed that arachidonic acid and thapsigargin did not alter membrane permeability, indicating that the arachidonic acid- and the thapsigargin-inducedCa²⁺ release was not due to either a Ca²⁺ ionophore or a membrane detergent effect. The present experiments have provided evidence for the existence of multiple non-mitochondrial Ca²⁺ pools in PANC-1 cells. These Ca²⁺ pools could be released via distinct mechanisms, in response to a variety of cellular second messengers. === Pharmaceutical Sciences, Faculty of === Graduate
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