310 GPR55-(in)dependent ion signalling of LPI A Bondarenko et al (Narishige Co. Ltd, Tokyo, Japan), ﬁre-polished and had a resistance of 4–6 MW. The signals obtained were low pass ﬁltered at 1 kHz, and digitized with a sample rate of 10 kHz using a Digidata 1200A A/D converter (Axon Instruments, Foster City, CA, USA). Data collection and analysis were performed using Clampex and Clampﬁt software of pClamp (version 8.2, Axon Instruments). The holding potential in whole cell experiments was -40 mV. Voltage ramps of 1 s duration from -80 mV to +80 mV were deliv- ered every 5 s. Solutions The standard external solution contained (in mM): 145 NaCl, 5 KCl, 1.2 MgCl2, 10 HEPES, 10 glucose, 2.4 CaCl2 (extracellular buffer #1, EB#1). In Ca2+-free solutions, MgCl2 was increased to 2.2 mM and 1 mM EGTA was added. Patch pipettes were ﬁlled with a solution containing (in mM): 100 potassium aspartate (KAsp), 40 KCl, 10 HEPES, 2 MgCl2, 0,2 EGTA (pipette solution #1, PS#1). For studying the Na+- dependency of LPI-induced responses, bath solution con- tained: 150 NaCl, 1,2 MgCl2, 10 glucose, 10 HEPES, 1 EGTA (extracellular buffer #2, EB#2) and after current development in response to LPI, Na+ was omitted by substituting with choline. In these experiments a Cs+-based pipette solution contained the following components (in mM): 100 CsMeSO3, 40 CsCl, 10 HEPES, 2 MgCl2, 10 EGTA (pipette solution #2, PS#2). For single channel recordings of BKCa channels in the cell-attached mode, the bath solution con- tained (in mM): 150 KCl, 10 HEPES, 10 glucose, 5 EGTA and 4931 mM CaCl2 to set free Ca2+ concentration at 10 mM (cal- culated by the programme CaBuf, G. Droogmans, Leuven, Belgium; ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf. zip) (extracellular buffer #3, EB#3) Pipette solution contained (mM): 100 KAsp, 40 KCl, 10 HEPES, 2 MgCl2, 5 EGTA and 1.924 CaCl2 to set free Ca2+ concentration to 100 nM (pipette solution #3, PS#3). Pipette solution in inside-out experi- ments for the activation of non-selective cation channels contained (in mM): 140 NaCl, 5 KCl, 2,4 CaCl2, 1,2 MgCl2, 10 HEPES and 10 D-glucose (pipette solution #4, PS#4). The respective bath solution contained (in mM): 140 CsCl, 1 MgCl2, 10 HEPES, 5 EGTA and free Ca2+ concentration was set to 100 nM by adding 1.924 CaCl2 (extracellular buffer #4, EB#4). The pH of the pipette solutions was adjusted to 7.25 with either KOH or CsOH. The pH of the external solutions was adjusted to 7.40 with either NaOH or CsOH (for Na+-free solution). Intracellular Na+ measurements Cytosolic free Na+ was measured using CoroNa™ Green (Meier et al., 2006) following the standard procedure (Ohta et al., 2008). Brieﬂy, cells were loaded with 5 mM CoroNa™ Green/AM for 30 min at room temperature followed by an equilibration period of 20 min. After washing the cells three times, cells were illuminated on an inverted micro- scope at 433 nm (433DF15, Omega Optical) and emission was collected at 535 nm (535AF26, Omega Optical, Brattle- boro, USA). Changes in cytosolic Na+ were expressed as DF430/F0. British Journal of Pharmacology (2010) 161 308–320 Statistics Analysis of variance was performed and statistical signiﬁcance was veriﬁed using Scheffe’s post hoc F-test. Level of signiﬁ- cance was deﬁned as P < 0.05. Materials Fura-2/AM and CoroNa™ Green/AM, gramicidin and cell culture chemicals were obtained from Invitrogen (Vienna, Austria). Fetal bovine serum was from PPA Laboratories (Linz, Austria). LPI, Dulbecco’s modiﬁed Eagle’s medium (DMEM) and all other chemicals were purchased from Sigma (Vienna, Austria). Results LPI elicits biphasic Ca2+ elevation, accompanied by diverse changes in membrane potential In the presence of extracellular Ca2+, cell stimulation with 5 mM LPI induced a transient rise in cytosolic free [Ca2+], which returned to the basal level within 2–4 min even in the presence of 2 mM extracellular Ca2+ (Figure 1A). The comparison of LPI-induced Ca2+ signalling in the presence of extracellular Ca2+ with its effect in nominal Ca2+-free solution (Figure 1B) indi- cated that LPI mainly mobilized Ca2+ from internal Ca2+ stores, whereas Ca2+ entry contributed only marginally to the cytsolic Ca2+ elevation in this early phase while the sustained Ca2+ rise reﬂected Ca2+ entry. The concentration-response analysis in respect of cytosolic Ca2+ elevation in response to LPI revealed the initial intracellular Ca2+ mobilization to be more sensitive than the sustained Ca2+ entry (Figure 1C). The initial cytosolic Ca2+ elevation upon LPI in the presence of extracellular Ca2+ was accompanied by a transient hyper- polarization that reached maximal amplitude of 11.4 1.7 mV (n = 9) within 100 s. Following the initial hyperpolar- ization, a slowly developing sustained depolarization of 20.1 2.5 mV (n = 9) above the resting membrane potential occurred within 250–300 s (Figure 1D). The concentration- response analyses revealed similar sensitivities of the initial hyperpolarization and subsequent depolarization (Figure 1E) compared with the respective Ca2+ signals (Figure 1C). Upon repetitive applications, the LPI-induced initial hyper- polarization was markedly reduced or absent while the sus- tained depolarization remained unchanged (Figure 1F). In agreement with these ﬁndings, LPI failed to initiate repeti- tively the respective outward current that accompanied mem- brane hyperpolarization upon the ﬁrst stimulation while a sustained inward current always occurred upon any LPI stimulation (Figure 1G). GPR55 is involved in the initial hyperpolarization but not the sustained depolarization in response to LPI Because in the cell model used, GPR55 was found to be con- stitutively expressed and to account for a fast, transient Ca2+ elevation upon stimulation with LPI (Waldeck-Weiermair et al., 2008), the contribution of this receptor to LPI-induced Ca2+ signalling and electrical responses in endothelial cells was further explored. In agreement with these ﬁndings, the