British Journal of Pharmacology (2010), 161, 308–320 © 2010 The Authors British Journal of Pharmacology © 2010 The British Pharmacological Society All rights reserved 0007-1188/10 www.brjpharmacol.org RESEARCH PAPER GPR55-dependent and -independent ion signalling in response to lysophosphatidylinositol in endothelial cellsbph_74 308.320 Alexander Bondarenko, Markus Waldeck-Weiermair, Shamim Naghdi, Michael Poteser, Roland Malli and Wolfgang F Graier Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Graz, Austria Background and purpose: The glycerol-based lysophospholipid lysophosphatidylinositol (LPI) is an endogenous agonist of the G-protein-coupled receptor 55 (GPR55) exhibiting cannabinoid receptor-like properties in endothelial cells. To estimate the contribution of GPR55 to the physiological effects of LPI, the GPR55-dependent and -independent electrical responses in this cell type were investigated. Experimental approach: Applying small interference RNA-mediated knock-down and transient overexpression, GPR55- dependent and -independent effects of LPI on cytosolic free Ca2+ concentration, membrane potential and transmembrane ion currents were studied in EA.hy296 cells. Key results: In a GPR55-dependent, GDPbS and U73122-sensitive manner, LPI induced rapid and transient intracellular Ca2+ release that was associated with activation of charybdotoxin–sensitive, large conductance, Ca2+-activated, K+ channels (BKCa) and temporary membrane hyperpolarization. Following these initial electrical reactions, LPI elicited GPR55-independent long-lasting Na+ loading and a non-selective inward current causing sustained membrane depolarization that depended on extracellular Ca2+ and Na+ and was partially inhibited by Ni2+ and La3+. This inward current was due to the activation of a voltage-independent non-selective cation current. The Ni2+ and La3+-insensitive depolarization with LPI was prevented by inhibition of the Na/K-ATPase by ouabain. Conclusions and implications: LPI elicited a biphasic response in endothelial cells of which the immediate Ca2+ signalling depends on GPR55 while the subsequent depolarization is due to Na+ loading via non-selective cation channels and an inhibition of the Na/K-ATPase. Thus, LPI is a potent signalling molecule that affects endothelial functions by modulating several cellular electrical responses that are only partially linked to GPR55. British Journal of Pharmacology (2010) 161, 308–320; doi:10.1111/j.1476-5381.2010.00744.x Keywords: endothelial cells; GPR55; lysophospholipid signalling; membrane potential; non-selective cation channel; patch- clamp; rimonabant Abbreviations: BKCa, large conductance, Ca2+ activated, K+ channels; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid disodium salt hydrate; DMEM, Dulbecco’s modiﬁed Eagle’s medium; EDHF, endothelium-derived hyperpo- larizing factor; GDPbS, guanosine 5′-O-(2-thiodiphosphate); GPCR, G-protein-coupled receptor; IP3, inositol 1,4,5-trisphosphate; KAsp, potassium aspartate; LP, lysophospholipid; LPI, lysophosphatidylinositol; SOCE, store-operated Ca2+ entry; TRPV1, transient receptor potential cation channel V1 Introduction Lysophospholipids (LPs) have been recognized as potent signalling molecules that modulate many cell functions in a Correspondence: Wolfgang F Graier, Molecular and Cellular Physiology Research Unit, Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21/III, 8010 Graz, Austria. E-mail: wolfgang.graier@medunigraz.at Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/authorresources/ onlineopen.html Received 4 January 2010; revised 31 January 2010; accepted 6 February 2010 variety of tissues including the cardiovascular, immune, nervous, and reproductive systems (Gardell et al., 2006). The generation of these signalling molecules is most often linked to the metabolism of membrane phospholipids by enzymes that are located at the inner side of the plasma membrane and are activated upon cell stimulation. Initially, LPs have thus been discussed to serve as second-messenger molecules that modulate intracellular signalling events (Corda et al., 2002). However, recent ﬁndings point to LPs also as intercellular signalling molecules that are involved in cell-to-cell commu- nication. This assumption is based on reports that LPs are transported to, and even produced in, the extracellular