548 Purinergic Signalling (2006) 2:545–557 Fig. 1 Current–voltage relationship for ATP-induced currents. A) Representative currents in response to 200 mM ATP recorded at Vhs between j70 and +50 mV. B) Current–voltage relationship for the IATP. All responses are normalized to the peak current amplitude obtained at a Vh of j70 mV, and each point is the mean T S.E.M. of ﬁve neurons. C) Averaged concentration–current relationships for ATP in the presence of 0.1, 2, and 10 mM external Ca2+. All responses were normalized to the mean of two control responses induced by 100 mM ATP in saline containing 2 mM external Ca2+ before and after the test response. Each point is the average T S.E.M of responses from four to six neurons. The purinergic agonists, 2-methylthio-ATP (50 mM) and ATPgS (50 mM) induced currents that were 41 T 3% (N = 4), and 41 T 4% (N = 5), respectively, of the IATP in response to 100 mM ATP. ADP (0.5 mM) did not produce any response. The IATP in response to 50 mM ATP was inhibited by the P2 antagonists; suramin (10 mM and 20 mM) and PPADS (50 mM), with the response being 35 T 2% (N = 5), 25 T 2% (N = 4), and 56 T 5% (N = 5), of the control IATP, respectively. ATP-induced [Ca]in increase ATP also increased [Ca]in in a dose-dependent manner (Fig. 2A). These responses were all recorded in the presence of 2 mM external Ca2+, and the [Ca]in increases by various concentrations of ATP were normalized to that induced by 100 mM ATP. The normalized and averaged responses to ATP, 2-meth- ylthio-ATP and ATPgS are shown in Fig. 2B. There was no ATP-induced [Ca]in increase in the absence of external Ca2+ (N = 6; data not shown). Furthermore, neither ADP, a,b-methylene-ATP nor b,g-methylene- ATP increased [Ca]in when tested at concentrations of 200 mM(data not shown). The ATP-induced [Ca]in increases were inhibited by suramin and PPADS (Table 1). Effects of Ca2+ channel antagonists on ATP-induced [Ca]in increase Previous results, using cultured rabbit AP neurons, demonstrated the presence of a w-conotoxin-M C- sensitive Ca2+ response, but that did not involve L- or N-type Ca2+ channels (Hay et al. [29]). We also inves- tigated the effects of various Ca2+ channel antagonists on [Ca]in increases induced by high KCl (110 mM) and ATP (100 mM). Antagonist for the L-type (ni- trendipine, nicardipine), N-type (w-conotoxin-M A), or P/Q-type (w-conotoxin-M C) Ca2+ channels each substantially inhibited these [Ca]in increases (Table 1). In contrast, the selective P-type Ca2+ channel antago- nist, w-agatoxin IVA (1 mM), did not have any inhibi- tory effect on the [Ca]in increases (98 T 5% of control, N = 4). ATP-induced current in the absence of external Na+ When external Na+ was completely replaced by NMDG+, and in the absence of external Ca2+ (0 Ca2+ plus 0.5 mM EGTA), the IATP was markedly reduced to 8 T 1% of the control IATP recorded in the pres- ence of external Ca2+ and 150 mM Na+ (N = 9, Fig. 3A). This current was further reduced to 4 T 1% (N = 3) in the presence of 20 mM suramin. The small