Purinergic Signalling (2006) 2:545–557 pipette solution) contained 70 mM K-gluconate, 50 mM KCl, 10 mM NaCl, 0.5 mM CaCl2, 3 mM MgCl2, 10 mM HEPES, 10 mM EGTA, and 2 mM ATP. The pH was adjusted to 7.2 with KOH. For measuring current–voltage (I–V) relationships, a Cs-based inter- nal solution was employed, which contained 98 mM CsOH, 40 mM CsCl, 98 mM aspartate, 2 mM MgCl2, 5 mM HEPES, 5 mM EGTA, and 2 mM ATP. pH was adjusted to 7.2 with CsOH. The external solution was the HEPES-buffered saline, pH7.4, described above. When N-methyl-D-glucamine (NMDG+) or sucrose was substituted for external Na+ or NaCl, respectively, the osmolarity of the solution was kept constant as measured using an osmometer (Vogel OM801, Ger- many). To obtain the Ca2+/Na+ permeability ratio (PCa/PNa), we measured the reversal potential of the ATP-activated current (EATP) in the presence of 1 or 110 mM of external Ca2+ by stepping the holding potential (Vh) between 10 and 30 mV, using incre- ments of 10 mV. The 1 and 110 mM Ca2+ solutions contained 155 or 0 mM NaCl, respectively, in addition to 10 mM HEPES with pH adjusted to 7.4 with NaOH or Ca(OH)2, respectively. The values of PCa/PCs and PNa/PCs were calculated using the constant ﬁeld equation as described by Lewis ([25]), taking the activity coefﬁcients of Na+, Cs+, and Ca2+ as 0.75, 0.75, and 0.3, respectively. Measurement of [Ca]in Dissociated AP neurons on a glass coverslip were incubated with 1–2 mM fura-2 acetoxymethyl ester (fura-2/AM), 0.1% dimethyl sulfoxide, and 1% bovine serum albumin for 45 min at 37 -C. The coverslips were then mounted in a superfusion chamber and placed on the stage of an inverted microscope (Dia- phot-TMD, Nikon, Japan). Cells were continuously superfused at a rate of 1 ml/min with HEPES-buffered saline at 24–26 -C via a polyethylene tube placed 1–2 mm away from the cells. Cells were viewed with a 40 Fluor objective lens (Nikon), and a single cell (10–12 mm diameter) was ﬁxed in a window positioned between the photo- multiplier and the microscope. The changes in ﬂuores- cence ratios at 340 and 380 nm excitation wavelengths were measured using a CAM-200 spectrometer (Jasco, Japan). The absolute value of [Ca]in was calculated using the formulae as given by Grynkiewicz et al. [26]: [Ca]in = b Kd(R j Rmin)/(Rmax j R). Calibra- tion constants were determined in separate experi- ments with the same experimental set-up, as described previously (Sorimachi et al. [27]). 547 Student’s paired t test was used to evaluate differ- ences between mean values obtained from the same cells, and Student’s unpaired t test was used for data obtained from different groups of cells. Reagents The following reagents were used: Fura-2/AM [Dojindo, Kumamoto, Japan], ACh [Horai Chem. Co., Japan], PPADS (pyridoxal-phosphate-6-azo- phenyl-20, 40-disulphonic acid) [RBI, USA.], pronase [Calbiochem., USA], thermolysin, ATP, 2-methylthio- ATP, ATPgS, a,b-methylene-ATP, b,g-methylene- ATP, ADP, nitrendipine, nicardipine, suramin [all from Sigma, Aldrich, Tokyo, Japan], w-conotoxin- MVIIA, w-conotoxin-MVIIC and w-agatoxin-IVA [all from Peptide Institute, Osaka, Japan]. Results ATP-induced current Rapid application of ATP (100 mM) to isolated AP neurons voltage clamped at a Vh of j70 mV induced an inward current, which desensitized slowly (Fig. 1A). The ATP-induced current (IATP) was recorded at various Vhs between j70 and +50 mV (Fig. 1: N = 6), and the relative current amplitude was plotted against Vh exhibiting a strong inward rectiﬁcation. The EATP, estimated from the intersection of the current response and the zero voltage-axis (Fig. 1B), was 22.7 T 0.9 mV (N = 18). To investigate the ATP concentration-response relationship, care was taken to adjust the pH of the solutions containing higher concentrations of ATP to 7.3, since addition of ATP reduced pH and a small decrease in pH to 7.1 increased the IATP to 189 T 8% (N = 6) of control (pH7.3), as previously reported for recombinant P2X2 receptors (King et al. [28]). The responses to different ATP concentrations were ﬂanked by responses to the control concentration of 100 mM, and normalized responses were expressed relative to the average of these control responses (Fig. 1C; Ca2mM). We also examined the effects of 0.1 and 10 mM Ca2+ on IATP. As summarized in Fig. 1C, an increase in the external concentration of Ca2+ shifted the concentration-response curve for ATP to the right, with the half-maximum effective concen- tration (EC50) values at 0.1, 2 and 10 mM Ca2+ being 30, 70, and 190 mM, respectively.