0026-895X/90/060827-06$02.00/0 Copyright © by The American Society for Pharmacology and Expérimental Therapeutics AU rights of reproduction in any form reser\ed. MOLECULAR PHARMACOLOGY, 37:827832 Amiloride Analogs Induce the Phosphorylation of Elongation Factor-2 in Vascular Endothelial Cells D. DEMOLLE, M. LECOMTE, G. BOUTHERIN-FALSON, E. J. CRAGOE, JR.,' A. C. NAIRN,^ and J. M. BOEYNAEMS Institute of Interdisciplinary Research, School of t^edicine, Université Libre de Bruxelles, Bruxelles, Belglum Received January 12, 1989; Accepted February 23, 1990 SUMMARY 5-(A/-Ethyl-A/-isopropyl)amiloride (EIPA), a potent inhibitor of Na*/ H* antiport, reduced [^^S]methionine incorporation in proteins and induced the phosphorylation of a M, 95,000 protein In bovine aortic endothelial cells. This protein was previousiy shown to become phosphorylated in response to ATP, bradykinin, and A23187 (1) and was identified as elongation factor-2 (2). The action of EIPA was independent of changes in cytosolic pH, because it was neither mimicked by sodium acétate nor inhibited by ammonium chloride, and it was reproduced by 2',4'-dime- thylbenzamil, an analog of amiloride that is inactive on the Na*/ H* antiport. Furthermore, EIPA enhanced the Ca^*-dependent phosphorylation of a similar M, 95,000 protein in a cell-free System, rabbit reticulocyte lysate, where an inhibitory effect of amiloride on protein synthesis has aiready been described (3). Because phosphorylation decreases the activity of elongation factor-2, our observation might explain why amiloride analogs inhibit protein synthesis. Adenine nucleotides, ATP and ADP, released during platelet aggregation are able to modulate the activity of aortic endothe lial cells; they induce an immédiate stimulation of prostacyclin (prostaglandin I2) release (4) and a delayed mitogenic effect.^ The responses of endothelial cells to ATP are mediated by P2y purinergic receptors, which are coupled to the hydrolysis of phosphatidylinositol bisphosphate, generating inositol tris phosphate and a subséquent increase of cytosolic calcium (5). This is accompanied by the phosphorylation of multiple pro teins (1), probably via the activation of CaM kinases II and III. A biphasic effect of ATP on pHi has been observed recently in BAECs (6). ATP induces a transient acidification, followed by a sustained alkalinization resulting from the activation of the Na*/H* antiport (6). An alkalinization, via the activation of the Na*/H* antiport, has been described in many Systems in response to various mediators and growth factors (for review see Réf. 7) and might play a permissive rôle for initiation of DNA synthesis (810). One step in the process of cell replica This work was performed under contract of the Ministère de la Politique Scientifique {Sciences de la Vie BlO/04) and was supported by a grant from the Fonds de la Recherche Scientifique Médicale. G. B.F. is supported by a grant (Action de Stimulation) of the European Communities. M.L. was supported by the A. and D. Van Buuren Foundation. ' 2211 Oak Terrace Drive, Lansdale, PA 19446. ^ Laboratory of Molecular and Cellular Neuroscience, The Rockefeller Univer sity, New York, New York 100216399. ^ A. Van Coevorden, P. P. Roger, J. M. Boeynaems. Submitted for publication. tion, which is controlled by pH;, seems to be the phosphoryla tion of a few key proteins; in particular, the phosphorylation of ribosomal protein S6 in response to mitogens is decreased by inhibitors of the NaVH* antiport (1113). The initial purpose of our study was to analyze the relation between pHi and protein phosphorylation in BAECs, a System that we have previously characterized in détail (1). The amilor ide analog EIPA, a potent inhibitor of the Na*/H* exchanger, was the main tool used in our study (14, 15). Surprisingly, we found that EIPA per se induced the phosphorylation of a M, 95,000 protein, which was identified as EP2. This phosphor ylation seems to be independent of changes in pHi, because it was also observed in an acellular System, rabbit reticulocyte lysates. Materials and Methods Culture of endothelial cells. BAECs were obtained by coUagenase digestion of aorta excised from a freshly slaughtered cow, as previously described (4). The cells were seeded in 100mm Pétri dishes and incubated at 37° under an atmosphère of 5% C02/95% air, in the following médium: 80% (v/v) MEM with Dvaline, 20% (v/v) fetal calf sérum, 2 mM glutamine, 100 units/ml penicillin, 100 /ig/ml strepto mycin, 2.5 Mg/ml amphotericin B. The médium was changed the follow ing day and then every 3 days. After 4 or 5 days, the primary cultures formed confluent monolayers and could be subcultured. The cells were detached by a 5min incubation in a Ca^* and Mg^*free Hanks' buffer ABBREVIATIONS: CaM, calmodulin; pH,, intracellular pH; SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(/3-aminoethyl ether)-W,A/,A/',A/'- tetraacetic acid; EF-2, elongation factor-2, BAEC, bovine aortic endothelial cell; EIPA, 5-(W-ethyl-A/-isopropyl)amiloride; MEM, minimum essential médium; DMEM, Dulbecco's modified Eagle's médium; CHARS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MIBA, 5-(A/-methyl- A/-isobutyl)amiloride; HMA, 5-(A/,A/-hexamethylene)amiloride; DMB, 2',4'-dimethylbenzamil; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 827