Introduction 1 2 3 4 5 6 7 8 9 in vivo 10 2+ in 11 12 in 13 14 15 16 17 18 19 20 21 22 23 24 23 Experimental procedures Preparation of AP neurons 2 2 2 2 2 2 3 2 4 l in Electrophysiological recordings 2 2 I–V 2 N d + + 2+ + P Ca P Na E ATP 2+ V h 2+ 2 P Ca P Cs P Na P Cs 25 + + 2+ in Dissociated AP neurons on a glass coverslip were incubated with 1–2 µM 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 (Diaphot-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. in 26 \documentclass[12pt]{minimal}
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$$\lbrack Ca \rbrack_{in} = b \times Kd(R - R_{min})/(R_{max} - R)$$
\end{document} 27 Student’s paired t test was used to evaluate differences 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-azophenyl-2′, 4′-disulphonic acid) [RBI, USA.], pronase [Calbiochem., USA], thermolysin, ATP, 2-methylthio-ATP, ATPγS, α,β-methylene-ATP, β,γ-methylene-ATP, ADP, nitrendipine, nicardipine, suramin [all from Sigma, Aldrich, Tokyo, Japan], ω-conotoxin-MVIIA, ω-conotoxin-MVIIC and ω-agatoxin-IVA [all from Peptide Institute, Osaka, Japan]. Results ATP-induced current V h 1A I ATP V hs 1 N V h E ATP 1B N Fig. 1 A V h B I ATP V h C 2+ 2+  I ATP N 2 28 1C 2+ I ATP 1C 2+ 50 2+ N N I ATP I ATP N N N I ATP in in 2A 2+ in 2B in 2+ N in in 1 Fig. 2 in A in 2+ B  Table 1 2+ in + Stimulus Blockers Percent of control ATP100 M Suramin 10 M N  20 M N  50 M N  PPADS 10 M N  20 M N  50 M N  100 M N 110 mMKCl Nitrendipine 2 M N  −CT.M C 2 M N  −CT.M A 2 M N  −CT.M A 2 M + −CT.M C 2 M N ATP100 M Nitrendipine 2 M N  Nicardipine 2 M N  2+ N  −CT.M A 2 M + −CT.M C 2 M N in 2+ μ ω ω 2+ in 2+ 2+ 29 2+ in 2+ in 1 2+ in N + + + 2+ 2+ I ATP I ATP 2+ + N 3A N + 30 2+ N 2+ + I ATP 2+ + + N N 3B I ATP 2+ N N 3C I ATP Fig. 3 + 2+ A I ATP + + + 2+ V h B I ATP + + 2+ C I ATP 2+  E ATP I ATP 2+ 2+ E ATP 2+ N P Ca P Cs E ATP 2+ N P Na P Cs P Ca P Na 2+ Negative interaction between P2XR and nAChR channels Requirement of actual current flow through receptor channels for cross-inhibition 18 30 19 20 21 22 23 24 I ACh I ATP 4A I ACh I ACh I ACh N N N N P I ACh I ATP N I ACh N I ATP I ACh 4B I ATP 4C I ACh N I ATP N I ACh N N N N I ACh I ACh I ATP 4D I ATP I ACh I ACh I ATP I ATP I ACh Fig. 4 I ACh I ATP A B. B ordinate I ATP abscissae V h C I ATP D. D ordinate I ACh abscissae V h  I ATP I ACh 31 I ACh N I ACh 5A I ACh I ATP N 5A I ATP N P 5A I ACh+ATP+dTc I ACh+ATP+dTc N I ATP I ACh+ dTc 5B I ACh+ATP+dTc 5A N I ATP 5C I ATP I ACh I ACh I ACh 32 33 34 I ACh N I ACh N 5D I ATP N N P Fig. 5 I ATP A I ATP I ACh V h I ACh+ATP I ACh+ATP+dTc I ATP I ACh+dTc C I ATP I ACh+ATP+dTc I ATP I ACh+ATP+dTc I ATP I ACh I ACh+5HT I ACh V h  I ACh+ATP V h 6A I ACh+ATP N P I ACh I ATP I ACh I ATP I ACh+ATP I ACh I ACh I ATP I ACh+ATP N N N I ACh+ATP I ACh I ATP I ACh+ATP I ACh I ATP I ACh+ATP Fig. 6 I ACh I ATP A V h I ACh I ATP B V h I ACh I ATP C E ACh E ATP V h I ACh+ATP  2+ I ACh+ATP N P I ACh I ATP 2+ 2+ 35 36 in V h I ACh+ATP V h N P N P I ACh I ATP V h N 6B + E ACh+ATP I ACh I ATP E ACh E ATP N N I ACh+ATP 6C + + I ACh I ATP I ACh +ATP + + 2+ 7 + 16 I ACh 2+ I ATP + 2+ 3 I ACh+ATP I ACh I ATP N P 7 Fig. 7 I ACh+ATP + 2+ I ACh I ATP I ACh+ATP + + 2+ + V h  I ATP I ACh+ATP + 2+ + 16 3A 8B I ATP+ACh N I ATP I ACh E ACh N E ATP I ATP I ATP+ACh N I ATP I ACh 8D 30 I ATP + 2+ 30 I ATP I ATP V h Fig. 8 I ACh A I ACh V h B + + 2+ I ACh I ACh+ATP C I ACh V h D I ACh I ATP I ACh+ATP V h  Discussion in in 2 4 6 37 2 38 39 I ATP E ATP 1B I ATP 2+ 1C 2+ 40 18 41 2 42 43 I ATP + + 2+ 3A + I ATP + 18 30 2+ I ATP 3B C 2+ 2+ P Ca P Na 2+ 18 41 42 43 2+ in 2+ in + in 2+ 2+ 1 2+ 12 vs 16 18 30 20 21 19 22 23 24 I ATP I ACh 4A B 4C D I ACh+ATP + 2+ 7 + V h E ACh E ATP 6C V h E ACh E ATP 2+ 2+ I ACh+ATP V h I ACh+ATP I ACh I ATP 6B 20 21 I ACh+ATP I ACh I ATP 19 22 2 6A 4 I ACh+ATP I ATP I ACh I ACh+ATP V h E ACh E ATP I ACh I ATP 6C I ACh+ATP I ATP I ACh 8B E ACh 8D I ATP 5B 5C 30 20 21 22 24 23