GPR55-(in)dependent ion signalling of LPI A Bondarenko et al 317 Figure 7 Dependency of the LPI-evoked electrical responses on extracellular Ca2+. Original tracing of membrane potential ﬂuctuations in response to 5 mM LPI in the absence of extracellular Ca2+ (n = 16) (A). Representative effect of re-adding Ca2+ in the continued presence of 5 mM LPI on membrane potential (n = 3) (B). Original tracing of the effect of 5 mM LPI on endothelial membrane potential in the presence of 2 mM Ni2+ (n = 7) (C). Effect of 2 mM Ni2+ on the LPI-induced inward current measured at -75mV (n = 10) (D). Effect of 100 mM La3+ on LPI-evoked depolarization (n = 4) (E). Statistical evaluation of the effects of different ionic conditions on the depolarization evoked by 5 mM LPI in endothelial cells: Control (n = 14), in the absence of extracellular Ca2+ (0 Ca2+, n = 5), in the presence of 2 mM Ni2+ (Ni2+, n = 10) or 100 mM La3+ (La3+, n = 4), and on Ca2+ re-addition (Ca2+, n = 3) (F). *P < 0.05 versus control. indicating that LPI, in a GPR55-independent manner, induces a Ca2+/Na+-permeable non-selective cation channel in endot- helial cells that favours Na+ to enter the cell and, thus, to initiate depolarization in the absence of an increase in cyto- solic free Ca2+ concentration. Removal of extracellular Ca2+ or the application of inhibi- tors of (non-selective) Ca2+-permeable cation channels (Ni2+, La3+) only prevented about 50% of the depolarization to LPI. These data point to an additional GPR55-independent effect of LPI besides the activation of a non-selective cation channel. Because Ni2+ is also an inhibitor of the plasma mem- brane Na+/Ca2+ exchanger (Iwamoto et al., 1999), this carrier can be excluded. Notably, LPI failed to trigger membrane depolarization in the absence of Na+, indicating that the remaining membrane depolarization upon LPI relies on Na+. Because other LPs including LPC and sphingosine exhibit inhibitory properties on the Na/K-ATPase (Oishi et al., 1990), we also evaluated the Na/K-ATPase as a putative target of LPI. Implementation of K+ re-addition protocols (Bondarenko and Sagach, 2006) demonstrated that, in the presence of LPI, a K+ -induced hyperpolarization was strongly reduced, indicating that LPI inhibited the Na/K-ATPase. In line with these ﬁnd- ings, LPI-induced Ni2+/La3+-insensitive depolarization was pre- vented by ouabain, an inhibitor of the Na/K-ATPase. Moreover, only the combination of Ni2+/La3+ and ouabain completely prevented the sustained deplolarization induced by LPI. Although the function of GPR55 as a common binding site for LPI and anandamide was only demonstrated in regard to inositol 1,4,5-trisphosphate (IP3)- dependent intracellular Ca2+ signalling (Oka et al., 2007; Lauckner et al., 2008; Waldeck- Weiermair et al., 2008), it is tempting to speculate that several other physiological effects of both compounds are due to their common action on GPR55. However, these compounds considerably differ in terms of their GPR55-independent potential to affect distinct ion channels and carriers and their role in the control of blood vessel tone. In particular, LPI was described as a phospholipase A2-derived messenger for the activation of SOCE (Smani et al., 2003; 2007; Singaravelu et al., 2006), and to activate non-selective cation channels and to prevent Na/K-ATPase activity (this study). Anandamide was described to activate arachidonic acid-regulated Ca2+-selective channels (Shuttleworth et al., 2004) and the transient receptor potential cation channel, TRPV1 (Zygmunt et al., 1999), and to trigger formation of 5,6- epoxyeicosatrienoic acid as a messenger for Ca2+-inﬂux (Graier et al., 1995; Hoebel et al., 1997). Thus, the differences in the physiological effects of these two putative endogenous GPR55 agonists are likely to be due to their different repertoire of GPR55-independent effects. Our present data on LPI-induced depolarization might explain the discrepancy between anan- damide and LPI in their potential to control endothelium- dependent blood vessel tone: anandamide may act as an EDHF per se or be involved in its formation/release (see Randall and Kendall, 1998), while LPI inhibits acetylcholine- induced, endothelium-dependent, hyperpolarization of rat mesenteric arteries (Fukao et al., 1995). In view of the reported more than 10-fold elevation of LPI in cancer-derived ascites ﬂuid (Xiao et al., 2001), our data presented here deserve further attention. In particular, our ﬁndings that the GPR55-dependent effects of LPI tend to be more sensitive, compared with the GPR55-independent LPI effects, suggest a considerable shift in the biological activity of LPI, under certain conditions. However, this important aspect requires more extensive evaluation and exceeds the scope of the present work. In conclusion, our results indicate that LPI exhibits GPR55- dependent and GPR55-independent effects in endothelial British Journal of Pharmacology (2010) 161 308–320