Vol. 180, No. 2, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1036-1040

October 31, 1991

RECEPTOR-MEDIATED ACTIVATION OF ARACHIDONIC ACID RELEASE IN MOUSE PERITONEAL MACROPHAGES IS LINKED TO EXTRACELLULAR CALCIUM INFLUX Bel6n Fern~indez I and Jesfis Balsinde 2"

1Centro de Investigaci6n del Hospital Doce de Octubre, Carretera Andalucia s/n, E-28041 Madrid, Spain ~Centro de Investigaciones Biol6gicas, C.S.I.C., Vel6zquez 144, E-28006 Madrid, Spain Received September 16, 1991

The role of external calcium in platelet-activating factor- and zymosan-stimulated arachidonic acid release from mouse macrophages was investigated. Deprivation of external Ca2÷ led to strong inhibition of receptor-mediated arachidonic acid release, which was completely restored when Caz÷was added to the incubation medium. When arachidonic acid release was examined in CaZ*-depleted ceils, the response took place only in presence of external Ca2÷. Verapamil, a voltage-dependent Ca2÷ channel blocker, nearly abolished arachidonic acid release in response to both platelet-activating factor and zymosan. These results suggest that extracellular Ca2. influx is functionally linked to arachidonic acid release and hence to phospholipase Az activation in mouse peritoneal macrophages. ® 199~Ao~d~mio Press,

Inc.

Phagocytic cells, as well as other cell types, release arachidonic acid (AA) when challenged by receptor-directed stimuli. Although diverse pathways could potentially contribute to AA release upon receptor stimulation [1], the key enzyme for mobilizing AA in macrophages is phospholipase A2 [2-6; Fernandez et aL, manuscript in preparation]. Increased Ca2÷ availability is thought to play a key role in regulating AA release. In this regard, AA release from macrophages responding to both receptor-directed and soluble stimuli is highly dependent on the presence of extracellular Ca2. [7-10]. Our previous work has suggested that AA release from zymosan-stimulated macrophages is parallel and independent of polyphosphoinositide hydrolysis [9]. Pretreatment of macrophages with phorbol ester substantially inhibited zymosan-stimulated inositol phosphates production but potentiated AA release [9]. Since phorbol ester did not block stimulated 45Ca2+uptake, it was suggested that extracellular Ca2÷ influx, and not discharge of internal Ca2÷ stores, was responsible of

"To whom correspondence should be sent. Abbreviations used: AA, arachidonic acid; PAF, platelet-activating factor. o006-291X/91 $1.5o Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Vol. 180, No. 2, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sustained AA release [9]. Indeed, a direct correlation was found between stimulated AA release and extracellular Ca 2÷ concentration [9,10]. The present work was designed to further assess the role of extracellular Ca 2÷ in receptor-mediated AA release from mouse peritoneal macrophages. The results herein reported show that elevated levels of Ca2. inside the cell are necessary for receptor activation of AA release, and that sustained AA release is the consequence of receptor-promoted Ca2÷ entry. Since the same results were obtained with two different receptor-directed stimuli, namely platelet-activating factor (PAF) and zymosan, the data suggest that a common mechanism may exist for the Ca2÷-dependent, receptor-mediated activation of AA release in macrophages.

MATERIALS AND METHODS

Cells. Peritoneal macrophages from Swiss mice were harvested and purified as described [11,12]. Cell monolayers were incubated at 37°C overnight in a humidified atmosphere at 5% CO2 and 95% air in RPMI 1640 medium (Flow Labs., Lrvine, Scotland, U.K.) supplemented with 10% (v/v) heat-inactivated fetal calf serum, penicillin (100 units/ml), streptomycin (100 fxg/ml), 2 mM L-glutamine, and 0.35 ~tCi/ml of [3H]AA (New England Nuclear, Boston, MA, U.S.A.; sp. act. 76.0 Ci/mmol). Macrophage stimulation. Ceils were placed in serum-free medium for 30 min before addition of stimulants and/or inhibitors. The reactions were initiated by adding either vehicle or stimulus for the time indicated. For preparation of Ca2÷-depleted macrophages, cells were incubated with 1 mM EGTA and 40 ~tM quin2/AM in a CaZ÷-free medium for 60 min at 37°C [13,14], washed twice and stimulated as appropriate. When verapamil was used, it was added 30 rain before cell stimulation [15]. Assay of.4,4 release. For measurements of [3H]AArelease, the supernatants were poured off, cleared of cells by centrifugation, and assayed for radioactivity by liquid scintillation counting [9].

RESULTS In this study, different pharmacological approaches have been carried out to assess the role of external Ca2~ in AA release. In order to obtain a more general view of the effect of calcium, two different receptor-directed stimuli were used, namely PAF and zymosan. Although zymosan can bind to several surface receptors in mononuclear phagocytes, it seems that binding occurs preferentially to a 180 kDa surface glycoprotein [16,17]. When experiments were conducted in the absence of calcium in the incubation medium, PAF- and zymosan-stimulated [3H]AA release was strongly inhibited (Fig. 1). However, replacing the Ca2÷-free medium with the original medium containing 1.3 mM CaCI2 restored both PAFand zymosan-stimulated AA release (Fig. 1). Receptor-mediated [3H]AA release under these conditions was very close to that observed for control stimulated cells (Fig. 1). 1037

Vol. 180, No. 2, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Receptor-mediated activation of arachidonic acid release in mouse peritoneal macrophages is linked to extracellular calcium influx.

The role of external calcium in platelet-activating factor- and zymosan-stimulated arachidonic acid release from mouse macrophages was investigated. D...
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