314 GPR55-(in)dependent ion signalling of LPI A Bondarenko et al Figure 4 LPI-induced hyperpolarization but not depolarization is sensitive to inhibition of phospholipase C and to GDPbS. In conventional whole cell conﬁguration, the effect of the inhibitor of phospholipase C U73122 (10 mM) (Control: n = 5; U73122: n = 6) (A) or 1 mM GDPbS in the pipette (Control: n = 7; GDPbS: n = 12) (B) on LPI- (3 mM) evoked initial hyperpolarization and subsequent depolarization was evaluated. *P < 0.005 versus control. The effect of 10 mM LPI in the bath in the inside-out conﬁguration (PS#4) in the presence of 100 nM free Ca2+ (EB#4) on plasma membrane currents at +70 and -70 mV membrane potentials (left) and the respective voltage-current relationship before (1) and after (2) LPI application (right) (n = 6) were recorded (C). independent from GPR55 as indicated by the lack of an effect of GPR55 knock-down on LPI-triggered cytosolic Na+ eleva- tion (Figure 8C). The concentration dependency that indi- cated a great difference in the effects initiated by 5 versus 10 mM LPI (Figure 8D, left) was similar to that obtained for Ca2+ entry/plateau phase (Figure 1C) and membrane depolar- ization (Figure 1E). In the absence of extracellular Na+, LPI failed to increase cytosolic Na+ levels (Figure 8D, right). LPI-evoked sustained membrane depolarization is partially due to inhibition of Na/K-ATPase Because in our experiments above, LPI-induced sustained membrane depolarization dependent on Ca2+/Na+ inward cur- British Journal of Pharmacology (2010) 161 308–320 rents (Figures 6–8), further experiments were performed in order to assess the possible involvement of the Na/K-ATPase in LPI-induced depolarization. First, 5 mM LPI prevented, in a reversible manner, membrane hyperpolarization upon re-adddition of extracellular K+ (Figure 9A), a standard proto- col that reveals Na/K-ATPase-dependent repolarization upon the re-addition of K+ (Bondarenko and Sagach, 2006). Further- more, preincubation of endothelial cells with 250 mM ouabain, which yielded strong membrane depolarization, reduced LPI (5 mM)-induced sustained depolarization from 20.1 2.5–8.5 1.5 mV (n = 9) (Figure 9B,C). The subsequent inhibition of the non-selective cation channels by 100 mM La3+ in the presence of ouabain completely prevented LPI- induced, sustained membrane depolarization (Figure 9B,C).