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Leukotriene synthesis in calcium-depleted human neutrophils: arachidonic acid release correlates with calcium influx.

ABSTRACT: The relationship between intracellular calcium concentration ([Ca2+]i), the release of arachidonic acid and the synthesis of leukotriene B4 (LTB4) was investigated using Ca(2+)-depleted human polymorphonuclear leucocytes (PMNs) in which [Ca2+]i can be manipulated by varying the concentration of exogenous Ca2+ added with agonists. In this model, Ca2+, platelet-activating factor (PAF) and N-formyl-Met-Leu-Phe (FMLP), added alone, were unable to induce arachidonic acid release or LTB4 synthesis, as assessed by measurements of the products by MS and HPLC, respectively. However, the simultaneous addition of Ca2+ and either PAF or FMLP to these Ca(2+)-depleted PMNs resulted in an influx of Ca2+ proportional to the extracellular concentration of Ca2+ and caused a substantial release of arachidonic acid and synthesis of LTB4. The [Ca2+]i values for threshold and maximal arachidonic acid release were found to be 150 nM and 350 nM respectively, suggesting the involvement of cytosolic phospholipase A2 (cPLA2). Under stimulatory conditions resulting in similar [Ca2+]i, Ca(2+)-depleted PMNs released significant amounts of arachidonic acid but normal (Ca(2+)-repleted) PMNs did not, indicating that Ca2+ depletion of PMNs altered the normal regulation of arachidonic acid release and facilitated the release of the fatty acid upon stimulation with agonists. cPLA2 and mitogen-activated protein kinase (MAP kinase) phosphorylation, as assessed by changes of electrophoretic mobility, occurred in both Ca(2+)-depleted and Ca(2+)-depleted PMNs upon addition of agonist. These data demonstrate that in Ca(2+)-depleted PMNs stimulated with agonists, arachidonic acid release and LTB4 synthesis correlated with extracellular Ca2+ influx.

SUBMITTER: Krump E

PROVIDER: S-EPMC1135949 | biostudies-other | 1995 Sep

REPOSITORIES: biostudies-other

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Project description:Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H(2)O(2) stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H(2)O(2) and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H(2)O(2), sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H(2)O(2) and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H(2)O(2) and sorbitol increased phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA(2) by H(2)O(2) occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA(2) activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.

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Leukotriene synthesis in calcium-depleted human neutrophils: arachidonic acid release correlates with calcium influx.

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