EFFECTS OF DILAZEP ON CYTOPLASMIC CALCIUM ION CONCENTRATION AND ARACHIDONIC ACID METABOLISM IN HUMAN PLATELETS AND NEUTROPHILS.

Abstract

We studied effects of dilazep(DZ), an antiplatelet drug, on the cytoplasmic Ca2+ concentration ([Ca2+]-i) and arachidonic acid (AA) metabolism in human platelets and neutrophils. [Ca2+]i of aequor-in-loaded platelets was estimated by using the platelet ionized calcium aggregometer. AA metabolism was studied by the determination of AA metabolites including hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE) and leukotriene B4 (LTB4) by reversed-phase high-performance liquid chromatography. When aequorin-loaded platelets were preincubated with DZ (0−0.5 mM) for 1 min at 37°C, both platelet aggregation and [Ca2+]i elevation induced by thrombin, AA and collagen were inhibited by DZ in a concentration dependent manner, while only aggregation was inhibited after stimulation by the calcium ionophore A23187 (1 yM). Both influx and release of Ca2+ into platelet cytoplasm induced by thrombin or AA were inhibited by DZ, while neither of them was affected when induced by A23187. The production of HHT and 12-HETE by the reaction of 100 yM AA with platelets preincubated with DZ (0−1 mM) was not inhibited, whereas their production by throirfbin (10 u/ml) was remarkably inhibited by DZ in a concentration dependent manner. When DZ (0−0.3 mM)-treated neutrophils were incubated with 5 μM A23187 in the presence or absence of 20 μM AA, the production of LTB4 and 5-HETE was increased by DZ in the presence of AA, whereas their production was inhibited by DZ in its absence. When platelet/neutrophil mixtures preincubated with DZ (0−0.3 mM) and cytochalasin B were stimulated by thrombin (5 u/ml) in the presence of FMLP, DZ inhibited LTB4 production in a concentration dependent manner, while 5S,12S-diHETE synthesis was enhanced by lower concentrations of DZ and inhibited by its higher concentrations (> 0.1 mM).Thus, DZ inhibits platelet aggregation induced by any agonist including A23183, while [Ca2+]i elevation is inhibited by the drug only when it is induced by the receptor-mediated agonist. Furthermore, it was suggested that AA liberation from phospholipids in platelets and neutrophils was inhibited by DZ, leading to reduced production of all endogenous AA metabolites by these cells after appropriate stimulation, although lipoxygenase metabolites of liberated or exogenous AA could be increased.