the absence of extracellular Ca2+, LPI produced a more pro- nounced initial hyperpolarization with the mean amplitude of -15.4 2.7 mV (n = 5) (Figure 7A). However, the subse- quent depolarization in Ca2+-free solution was reduced by about 42 4% (n = 5) compared with the depolarizing effect GPR55-(in)dependent ion signalling of LPI A Bondarenko et al 313 Figure 3 The effects of LPI on initial Ca2+ signalling and membrane hyperpolarization essentially depend on GPR55 expression. Intracel- lular Ca2+ signalling in response to 5 mM LPI was recorded in cells transiently transfected with the respective controls (same vector encoding nuclear GFP) or a GPR55 encoding vector (Control: n = 17; GPR55ox: n = 24) (A). In addition, cells were transfected with siRNA control (AllStar®) or siRNA against GPR55 (Control: n = 12; siRNA GPR55: n = 19) (B). In conventional whole cell conﬁguration, the LPI- (5 mM) induced hyperpolarization was measured in cells transfected either siRNA control (n = 4), siRNA against GPR55 (n = 5) or a vector encoding GPR55 (n = 6) (C). *P < 0.005 versus control. of LPI in Ca2+ containing buffer (Figure 7A,F). Ca2+ re-addition in the continued presence of LPI produced a hyperpolariza- tion followed by a depolarization above the level attained in the presence of LPI in Ca2+-free solution (Figure 7B,F). Similar results were obtained if Ca2+ entry was prevented by 2 mM Ni2+ (Figure 7C,F). Addition of Ni2+ caused a gradual depolar- ization pointing to a basal Ca2+ entry as a determinant of the resting membrane potential in this cell type. Subsequent addi- tion of 5 mM LPI evoked a slightly increased amplitude of the transient hyperpolarization (18.7 2.4 mV, n = 7) followed by a sustained depolarization that was signiﬁcantly reduced by 51 4% compared with the corresponding experiments that were performed in the absence of Ni2+ (Figure 7C,F). In line with these ﬁndings, the LPI-induced inward current (repeti- tive stimulation) was reduced upon addition of 2 mM Ni2+ into the bath by 50 6% (Figure 7D). Next, we examined the sensitivity of the depolarization phase to La3+, a known blocker of store-operated and non-selective cation channels (Nilius and Droogmans, 2001). In these experiments, an addi- tion of 100 mM La3+ during the sustained depolarization upon a repetitive LPI-administration (5 mM) reduced membrane depolarization by 53 7% (Figure 7E,F). These results indicate that the lack of Ca2+ entry slightly facilitates LPI-induced hyperpolarization and reduces the sub- sequent sustained depolarization phase, indicating that Ca2+ entry partially (approximately 50%) accounts for the depolar- izing effect of LPI. Together with the current-potential rela- tionship of the current that is associated with the sustained depolarization phase upon LPI stimulation (Figure 6B), these results point to the involvement of Ca2+ permeable, non- selective channels, activated by LPI action and contributing to LPI-induced endothelial cell depolarization. In order to study whether or not Na+ entry via such non- selective cation channels also contributes to the LPI-induced depolarization, experiments in the absence of extracellular Na+ (Na+ substituted for choline) and nominal Ca2+-free buffer were performed. Under such conditions, LPI-induced sus- tained depolarization was reduced by 94 3% compared with the respective control (Figure 8A). In voltage-clamp experiments (PS#2, holding potential -40 mV, voltage ramps from -80 to +80 mV), LPI-induced inward current was abol- ished upon substitution of extracellular Na+ with equimolar choline (Figure 8B). These data suggest that Ca2+- and Na+- permeable non-selective cation channels account for the slowly developing but sustained depolarization upon stimu- lation of LPI. In line with the ﬁndings above, LPI resulted in a slow accumulation of intracellular Na+ (Figure 8C), This effect was British Journal of Pharmacology (2010) 161 308–320