Characterization of Pardaxin-Induced Dopamine Release from Pheochromocytoma Cells: Role of Calcium and Eicosanoids SALEH ABU-RAYA, EUGENIA BLOCH-SHILDERMAN, PETER I. LELKES, VICTORIA TREMBOVLER, ESTHER SHOHAMI, YEHUDA GUTMAN and PHILIP LAZAROVICI 1 Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel (S.A.-R., E.B.-S., V.T., E.S., Y.G., P.L.); and Laboratory of Cell Biology, University of Wisconsin, Medical School, Milwaukee Clinical Campus, Milwaukee, Wisconsin (P.I.L) Accepted for publication August 5, 1998 This paper is available online at http://www.jpet.org ABSTRACT Pardaxin, an excitatory neurotoxin, induced dopamine release from pheochromocytoma (PC12) cells both in the presence and absence of extracellular calcium ([Ca] o ). In the presence of extracellular calcium, nifedipine, an L-type calcium channel blocker, did not affect dopamine release, whereas 1,2-bis (2- aminophenoxy) ethane N,N, N'N'-tetra-acetic acid (BAPTA), a chelator of cytosolic calcium, and dantrolene, a blocker of calcium release from intracellular stores, inhibited only partially (30 – 40%) pardaxin-induced dopamine release. In the absence of [Ca] o , BAPTA and dantrolene were ineffective. Pardaxin stim- ulated the arachidonic acid (AA) cascade in PC12 cells inde- pendently of [Ca] o . The phospholipase inhibitors mepacrine and bromophenacyl bromide inhibited both pardaxin-induced AA release and pardaxin-induced dopamine release. Dopamine release induced by pardaxin also was blocked by the lipoxy- genase inhibitors nordihydroguaiaretic acid, esculetin, and 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzo- quinone. Under these conditions, a parallel reduction in 5-hy- droxyeicosatetranoic acid release also was observed. Suppres- sion of pardaxin-induced dopamine release by inhibitors of phospholipase A 2 and lipoxygenase was more pronounced in calcium-free medium. These results indicate the involvement of the lipoxygenase pathway in pardaxin-induced dopamine re- lease and suggest the use of this toxin as a novel pharmaco- logical tool for investigating the mechanism of calcium-inde- pendent neurotransmitter release. Neurotransmitter release from the synaptic terminal oc- curs via regulated secretion (exocytosis) as synaptic vesicles fuse with the neuronal plasma membrane and release their contents into the synaptic cleft. Regulated exocytosis of cat- echolamines has been largely investigated in bovine adrenal chromaffin cells (Burgoyne, 1991) and in rat pheochromocy- toma (PC12) cells (Ahnert-Hilger et al., 1985). The secretory vesicles of these cells, the chromaffin granules, store dopa- mine, norepinephrine, ATP, and various proteins. The essen- tial role of calcium in catecholamine secretion from chromaf- fin cells has been well established (Burgoyne, 1991). The concentration of cytosolic calcium ([Ca] i ) is strictly regulated, and it is thought that the increase in calcium concentration within the microdomain of the active exocytotic zone allows vesicles to fuse and release their catecholamines content (Burgoyne and Morgan, 1995). [Ca] i can be increased because of membrane depolarization, opening of receptor-operated channels, or release of calcium from intracellular stores (Bur- goyne, 1991). In PC12 cells, membrane depolarization by KCl (Greene and Rein, 1977a; DiVirgilio et al., 1987) resulted in an influx of Ca ++ through depolarization-induced activation of volt- age-sensitive calcium channels, thereby triggering exocytosis (DiVirgilio et al., 1987). One of the secretagogues largely investigated in PC12 cells is acetylcholine. Acetylcholine- induced catecholamine release in PC12 cells is mediated both by nicotinic and muscarinic receptors (Zerby and Ewing, 1996). Stimulation of nicotinic receptor in PC12 cells induced catecholamine release (Greene and Rein, 1977b) by mem- brane depolarization, which triggers the influx of extracellu- lar calcium through voltage-dependent calcium channels (Zerby and Ewing., 1996). Muscarinic agonists such as Received for publication October 1, 1997. 1 Affiliated with the David R. Bloom Center for Pharmacy at the Hebrew University. ABBREVIATIONS: PX, pardaxin; BPB, 4-bromophenacyl bromide; CCH, carbachol; [Ca] o , extracellular calcium; [Ca] i , cytosolic-free calcium; Dan, dantrolene; NDGA, nordihydroguaiaretic acid; AA861, 2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone; AA, arachidonic acid; 5-HETE, 5-hydroxyeicosatetranoic acid; Fura-2-AM, acetoxymethyl ester of Fura-2; DMEM, Dulbecco’s modified Eagle’s medium; PC12, pheochromocytoma cells; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetra-acetic acid; PLA 2 , phospholipase A 2 ; IP 3 , inositol 1,4,5- trisphosphate. 0022-3565/99/2882-0399$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 2 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:399 –406, 1999 399