Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells.
In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.
Glitsch
Maike D
MD
Laboratory of Molecular and Cellular Signalling, Department of Physiology, University Of Oxford, Parks Road, Oxford OX1 3PT, UK.
Bakowski
Daniel
D
Parekh
Anant B
AB
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D001491:Basophils / Q000187:drug effects / Q000378:metabolism*; D002109:Caffeic Acids / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism; D015289:Leukotrienes / Q000494:pharmacology; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D009637:Masoprocol / Q000494:pharmacology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D010539:Permeability; D051381:Rats; D012964:Sodium / Q000037:antagonists & inhibitors / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002109:Caffeic Acids; D004791:Enzyme Inhibitors; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; C099255:cinnamyl-3,4-dihydroxycyanocinnamate; D019284:Thapsigargin; C025348:arachidonic acid 5-hydroperoxide; D009637:Masoprocol; D000255:Adenosine Triphosphate; D012964:Sodium; D019311:Staurosporine; D002118:Calcium