Dissociation of platelet-activating factor production and arachidonate release by the endomembrane Ca(2+)-ATPase inhibitor thapsigargin. Evidence for the involvement of a Ca(2+)-dependent route of priming in the production of lipid mediators by human polymorphonuclear leukocytes.
The production of platelet-activating factor (PAF) and the release of [3H]arachidonate were studied in human polymorphonuclear leukocytes (PMN) stimulated with thapsigargin, an inhibitor of endomembrane Ca(2+)-ATPase. Concentrations of thapsigargin as low as 10-25 nM primed PMN for both PAF production and [3H]arachidonate release in response to the chemotactic peptide (fMLP), whereas concentrations in the range 25-200 nM induced a time- and dose-dependent production of PAF, which occurred in the absence of both [3H]arachidonate release and [3H]phosphatidylethanol formation. Studies in fura-2/AM-loaded cells showed that concentrations of thapsigargin that elicited PAF production induced a protracted and long lasting elevation of cytosolic free calcium concentration ([Ca2+]i) between 200 and 700 nM. The lower concentrations primed the cells for a late [Ca2+]i elevation in response to fMLP similar to that elicited by cytochalasin B or ionomycin. PAF production showed a good correlation with the increase of [Ca2+]i (r = 0.91) irrespective of the procedure used to grade [Ca2+]i. In contrast, phorbol 12,13-dibutyrate failed to induce both PAF production and elevation of [Ca2+]i, but it was a very effective stimulator of [3H]arachidonate release and [3H]phosphatidylethanol production. These data indicate that PAF production and [3H]arachidonate release in PMN differ in both biochemical pathway and modulatory mechanisms. Whereas PAF production seems extremely sensitive to changes in [Ca2+]i, which seems to exert its modulatory effect at the lyso-PAF:acetyl-CoA acetyltransferase step, [3H]arachidonate release seems tightly modulated by protein kinase C-dependent mechanisms and is coincidental with activation of phospholipase D.
Garcia Rodriguez
C
C
Departamento de Bioquímica y Biología Molecular y Fisiología, Consejo Superior de Investigaciones Científicas, Facultad de Medicina, Valladolid, Spain.
Montero
M
M
Alvarez
J
J
García-Sancho
J
J
Sánchez Crespo
M
M
D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D020404:Glycerophospholipids; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin
D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010972:Platelet Activating Factor; D013729:Terpenes; C051521:phosphatidylethanol; D016718:Arachidonic Acid; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium