Arachidonate and other fatty acids mobilize Ca2+ ions and stimulate beta-glucuronidase release in a Ca(2+)-dependent fashion from undifferentiated HL-60 cells.
Arachidonate (1-300 microM) mobilized Ca2+ ions from an intracellular store and stimulated the entry of Ca2+ ions from the extracellular fluid in undifferentiated HL-60 cells that had been loaded with Fura-2. The integrated response was biphasic in form: arachidonate liberated Ca2+ ions from the intracellular store first, resulting in a transient increase in cytosolic free Ca2+ concentration ([Ca2+]i). Ca2+ entry from the extracellular fluid was not evident for a further 1-2 min. At baseline, [Ca2+]i was 48.1 +/- 14.0 nM (SEM, n = 5). Upon addition of arachidonate (100 microM), [Ca2+]i rose to a transient peak level of 217 +/- 38.6 nM (SEM, n = 5) and a later plateau level of 427 +/- 118 nM (SEM, n = 5). Removal of added Ca2+ ions from the extracellular fluid in the presence of EGTA (1.0 mM) had no effect on the initial transient response but abolished the second phase of the response. In HL-60 cells that had been loaded with BAPTA/AM, the initial transient phase of the response was abolished but the elevation in [Ca2+]i due to Ca2+ entry from the extracellular fluid was unaffected. Undifferentiated HL-60 cells also responded to arachidonate (100 microM) with an increase in the release of the lysosomal enzyme beta-glucuronidase. Arachidonate-induced beta-glucuronidase release from BAPTA-loaded cells or in control cells exposed to Ca(2+)-free solutions was inhibited by about 50%. In BAPTA-loaded cells that were incubated with Ca(2+)-free solutions, arachidonate-induced beta-glucuronidase release was inhibited by about 90%. Leukotriene B4 failed to elevate [Ca2+]i in the concentration range 0.01-1 microM and failed to activate beta-glucuronidase release in concentrations up to 10 microM. Furthermore, the cyclo-oxygenase/lipoxygenase inhibitor ETYA (100 microM) was without effect on secretion. Consistent with this finding, we found that a large number of unsaturated fatty acids could reproduce the effect of arachidonate on [Ca2+]i and beta-glucuronidase release. Fatty acids belonging to the omega-3, omega-6 and omega-7 unsaturated fatty acid families were effective in elevating [Ca2+]i and stimulating beta-glucuronidase release. However, three unsaturated fatty acids, all belonging to the omega-9 fatty acid family, were ineffective.
Packham
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DE
Department of Biochemistry, University of Sydney, New South Wales, Australia.
Jiang
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Conigrave
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AD
D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002412:Cations; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism / Q000494:pharmacology*; D016257:Fura-2; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000378:metabolism / Q000473:pathology; D014407:Tumor Cells, Cultured
D002412:Cations; D005227:Fatty Acids; D016718:Arachidonic Acid; D004533:Egtazic Acid; D005966:Glucuronidase; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D016257:Fura-2