GPR55-(in)dependent ion signalling of LPI A Bondarenko et al 315 Figure 5 The initial transient response to LPI is due to a stimulation of outwardly rectifying, Ca2+-dependent, charybdotoxin-sensitive, K+ channels. Representative biphasic changes in membrane currents evoked by 5 mM LPI at a holding portential of -40 mV (n = 14) (A). Voltage ramps of the outward rectifying membrane currents under resting conditions (control, n = 5) and upon cell stimulation with 5 mM (LPI (n = 8) (B). Representative time course of the effect of LPI on single BKCa channels in the cell-attached mode at a holding potential (Vhold) of 20 mV (n = 7) (C). Representative membrane potential recording showing a lack of hyperpolarization in response to 5 mM LPI in the presence of 100 nM charybdotoxin (n = 5) (D). Statistical evaluation of LPI (5 mM)-induced membrane hyperpolarization in endothelial cells under control conditions (LPI, n = 4) and in the presence of 100 nM charybdotoxin (100 nM, n = 5) (E). *P < 0.005 versus control. Discussion The present study demonstrated that the endogenous agonist of GPR55, LPI induced a variety of electrical responses in endothelial cells. The immediate and rather transient Ca2+ elevation, and its associated K+ current and membrane hyperpolarization to LPI were mediated via GPR55. However, the slowly developing and sustained inward current that resulted in intracellular Na+ loading and membrane depolar- ization was independent of GPR55 and was due to inhibition of Na/K-ATPase and direct activation of non-selective cation channels. Thus, LPI is a potent signalling molecule affecting endothelial cells by modulating multiple electrical responses. Because, these effects are only partially linked to GPR55, the physiological meaning of LPI as an intercellular messenger within the vascular wall exceeds that of just an activator of GPR55. Accordingly, in the ongoing evaluation of the physi- ological role of GPR55 and its endogenous agonists, these biphasic responses need to be considered. LPI recently received much attention following several concordant reports that identiﬁed LPI but not other lysophospholipids, such like lysophosphatidylcholine, lyso- phosphatidylethanolamine, lysophosphatidylserine or lyso- phosphatidic acid, as endogenous activators of GPR55 (Oka et al., 2007; 2008; Henstridge et al., 2008; Waldeck-Weiermair et al., 2008). Because the physiological concentrations of LPI were found to be 1–15 mM (Xiao et al., 2001), the LPI concen- trations we used here are close to those found in vivo. There is an ongoing discussion whether or not GPR55 serves as recep- tor for endocannabinoids, such as anandamide and 2-arachidonyl glycerol, though it obviously shares some similar pharmacology with classical cannabinoid receptors (Ross, 2009), The two putative endogenous GPR55 agonists, LPI and anandamide, share certain biological activities such as stimulation of intracellular Ca2+ signalling (Howlett and Mukhopadhyay, 2000; Oka et al., 2007; Henstridge et al., 2008; Lauckner et al., 2008; Waldeck-Weiermair et al., 2008) and the initiation of mitogenic signals (Falasca and Corda, 1994; Corda et al., 2002; Waldeck-Weiermair et al., 2008). Regardless of their proliferative potential, anandamide and LPI predominantly exhibit inhibition of endothelial cell migration, tumour growth and angiogenesis (Murugesan and Fox, 1996; Flygare and Sander, 2008). Endothelial cells express functional GPR55 (Waldeck- Weiermair et al., 2008) and produce its endogenous agonist, LPI (Hong et al., 1985; Kaya et al., 1989), making it more likely that LPI could be an intra-vascular messenger. To understand the physiological role of LPI, an in-depth understanding of the signalling cascades initiated by LPI in endothelial cells is essential. In this study, LPI was found to exhibit several autonomous effects in endothelial cells of which the imme- diate, rather transient signalling depended exclusively on British Journal of Pharmacology (2010) 161 308–320