GPR55-(in)dependent ion signalling of LPI A Bondarenko et al 311 Figure 1 Effect of LPI on free intracellular Ca2+ and membrane potential of endothelial cells. Representative effect of 5 mM LPI on free intracellular Ca2+ in the presence of 2 mM extracellular Ca2+ (n = 32) (A) and in nominally Ca2+-free solution (n = 27) (B). Concentration- response correlation of LPI on cytosolic Ca2+ concentration measured at the initial transient peak (Peak Phase) and the subsequent plateau phase (Plateau Phase) (1 mM, n = 9; 3 mM, n = 9; 5 mM, n = 15; 10 mM, n = 14) (C). Representative biphasic effect of LPI (5 mM) on membrane potential in the presence of extracellular Ca2+ (n = 9) (D). Concentration-response correlation of LPI in terms of initial membrane hyperpo- larization and subsequent depolarization (1 mM, n = 17; 3 mM, n = 7; 10 mM, n = 7) (E). Representative changes in endothelial membrane potential evoked by repetitive stimulations with 5 mM LPI (n = 5) (F). Representative membrane currents evoked by repetitive stimulations by LPI (5 mM) at -40 mV holding potential (n = 3) (G). GPR55 blocker rimonabant (1 mM) (Lauckner et al., 2008; Waldeck-Weiermair et al., 2008) diminished the LPI-initiated immediate Ca2+ transient (Figure 2A). Moreover, rimonabant (1 mM) prevented the transient membrane hyperpolarization (Figure 2B,C) and outward current in response to 5 mM LPI (Figure 2D). In contrast, the sustained membrane depolariza- tion (Figure 2B,C) and inward current (Figure 2B) in response to LPI were not affected by the GPR55 blocker. However, there is some controversy over to the pharmaco- logical potential of putative inhibitors/activators of GPR55 (see Ryberg et al., 2007; Henstridge et al., 2008; Waldeck- Weiermair et al., 2008; Kapur et al., 2009; Ross, 2009; Yin et al., 2009). So, the involvement of GPR55 was further inves- tigated by altering expression of GPR55. In line with these ﬁndings and our previous work using higher LPI concentra- tions (Waldeck-Weiermair et al., 2008), intracellular Ca2+ release triggered by LPI (5 mM) was strongly enhanced in cells that over-expressed GPR55 (Figure 3A) while this response was strongly attenuated in cells treated with siRNA against GPR55 (Figure 3B). Moreover, the initial membrane hyperpo- larization in response to LPI was reduced in siRNA treated cells and augmented when GPR55 was over-expressed (Figure 3C). In contrast, the subsequent membrane depolar- ization remained unaffected [Control: 10.50 2.10 mV (n = 4); GPR55 over-expressing cells: 12.16 2.20 mv (n = 6)]. To further assess the mechanistic differences between LPI-triggered initial hyperpolarization and the long-lasting membrane depolarization, the effect of the phospholipase C inhibitor U73122 (Figure 4A) and of the G protein inhibitor GDPbS (Figure 4B) was tested in conventional patch clamp experiments. U73122 as well as GDPbS prevented LPI- (3 mM) induced membrane hyperpolarization while the depolarizing effect of LPI remained unaffected in the presence of GDPbS. Moreover, in inside-out experiments LPI in the bath activated a non-selective ion current (Figure 4C), thus, indicating that LPI, besides its stimulatory activity at GPR55, might act inde- pendently from a receptor directly on plasma membrane ion channels. British Journal of Pharmacology (2010) 161 308–320