space and that cell surface receptors that are speciﬁcally acti- vated by certain LPs have been identiﬁed. However, the diverse effects of different LPs as signalling molecules are only marginally explored on the molecular level, although they deserve particular interest in order to understand the molecular processes of lipotoxic pathways. Increased levels of LPs have been found particularly under pathological conditions in the vasculature, such as atheroscle- rosis (Ridgway et al., 1999), and hypertension (Smyth et al., 2008), indicating that LPs are also important signalling mol- ecules under pathological condition (Gardell et al., 2006). Accordingly, the development of pharmacological tools that interfere with receptor-mediated LP signalling is nowadays thought to represent a promising approach for the develop- ment of novel therapeutic strategies against vascular diseases (Gardell et al., 2006). One of the less examined LPs is lysophosphatidylinositol (LPI), a glycerol-based LP that has recently been reported to speciﬁcally interact with the G-protein-coupled receptor 55 (GPR55) (Oka et al., 2007; Waldeck-Weiermair et al., 2008) that mediates a transient Ca2+ increase upon stimulation of endothelial cells with LPI (Henstridge et al., 2008; Lauckner et al., 2008; Waldeck-Weiermair et al., 2008). This observation indicates that vascular endothelial cells are targets of LPI and might further point to LPI as an important modulator of endothelial functions under physiological and pathological conditions. Notably, GPR55 activation resulted in the sup- pression of a reconstituted neuronal M-type K+ current in GPR55 transfected HEK 293 cells (Lauckner et al., 2008), sug- gesting that GPR55 stimulation might have multiple conse- quences for the regulation of the membrane potential. It has been reported that several LPs such as lysophosphatidylcholin (LPC) exert signiﬁcant effects on plasma membrane currents of endothelial as well as smooth muscle cells (Terasawa et al., 2002; Kuhlmannm et al., 2004) suggesting that LPs are capable of modulating vascular reactivity, predominantly by affecting ionic homeostasis in vascular cells. In vascular endothelial cells, the membrane potential is crucially impor- tant for various cell functions, including Ca2+ entry (Nilius and Droogmans, 2001), nitric oxide formation and the gen- eration of endothelium-derived hyperpolarizing factor (EDHF; Chen and Suzuki, 1990; Graier et al., 1996), and free- radical production (McCarty, 1999). Moreover, changes in endothelial membrane potential in situ via myo-endothelial gap junctions inﬂuence the membrane potential of underly- ing smooth muscle cells (Beny and Pacicca, 1994) and, hence, have profound inﬂuence on vascular tone. Because little is known about the effects of LPI as a possible vascular signalling mediator on endothelial membrane potential, this study was designed to investigate the effects of LPI on intracellular Ca2+ concentration, membrane potential, and to explore the underlying ion conductance in endothe- lial cells. Methods Cell culture The human umbilical vein derived endothelial cell line, EA.hy926 (Edgell et al., 1983) at passage >45 was grown in GPR55-(in)dependent ion signalling of LPI A Bondarenko et al 309 DMEM containing 10% FCS and 1% HAT (5 mM hypoxan- thine, 20 mM aminopterin, 0.8 mM thymidine) and cells were maintained in an incubator at 37°C in 5% CO2 atmo- sphere. Cells were plated on 30 mm glass cover slips at least 2 days before use in experiments (Paltauf-Doburzynska et al., 2000). Cell transfection and small interference RNA Adherent cells of approximately 70–80% conﬂuence were tran- siently transfected with 2–3 mg cDNA of the respective vectors using TransFast™ Transfection Reagent (Promega, Mannheim, Germany) according to the instruction manual and as previ- ously described (Trenker et al., 2007). Cells were used approxi- mately 36 h after transfection. For the knock-down of GPR55 small interference RNA (siRNA) (Sense sequence of GPR55 siRNA: 5′-GGUUCUUGGCCAUCCGUUAtt-3′, Qiagen, Cam- bridge, MA) was used. Cells were transfected with either the siRNAs or the AllStars® negative control siRNA (Cat. No. 1027281, Qiagen, Cambridge, MA) as described previously (Trenker et al., 2007; Waldeck-Weiermair et al., 2008). Efﬁ- ciency of the siRNA used was positively tested in our previous work (Waldeck-Weiermair et al., 2008). For GPR55, overexpres- sion cells were transiently transfected with a vector encoding GPR55 as previously described (Waldeck-Weiermair et al., 2008). Ca2+ measurements Cytosolic free-Ca2+ was measured using Fura-2/AM as previ- ously described (Graier et al., 1996; Paltauf-Doburzynska et al., 2000). Brieﬂy, cells were loaded with 2 mM fura-2/AM for 45 min at room temperature. Prior to experiments, cells were washed and equilibrated for a further 20 min. Subse- quently, cells were illuminated on an inverted microscope (Eclipse 300 TE, Nikon, Vienna) alternatively at 340 and 380 nm (ﬁlters: 340HTI15 and 380HTI15; Omega Optical Brattleboro, VT, USA) and emitted light was collected at 510 nm (510WB40 emission; Omega Optical) using a cooled charge-coupled device camera (-30°C; Quantix KAF 1400G2, Roper Scientiﬁc, Acton, MA, USA). All Ca2+ measurements were performed with a 40¥ 1.3 N.A. oil-immersion objective (Plan Fluor, Nikon, Vienna) (Paltauf-Doburzynska et al., 1998; Frieden and Graier, 2000). Electrophysiological recordings Whole cell membrane currents and membrane potential were recorded using the perforated patch-clamp technique (Frieden and Graier, 2000; Bondarenko, 2004; Bondarenko and Sagach, 2006). The conventional whole cell technique was used only in those experiments in which cells were dialysed with Cs+ -based solution. For membrane perforation, amphothericin B (300 mM) was included into the pipette solution. Single channel activity of large conductance, Ca2+- activated, K+ channels (BKCa channels; nomenclature follows Alexander et al., 2009) was recorded in the cell-attached mode (Malli et al., 2003). Membrane currents and potential were recorded using a List EPC7 ampliﬁer (List, Germany). Borosilicate glass pipettes were pulled with a Narishige puller British Journal of Pharmacology (2010) 161 308–320