Purinergic Signalling (2006) 2:545–557 results are in good agreement with previous histo- chemical ﬁndings, which demonstrated the presence of P2X2, P2X4, and P2X6 receptor mRNAs (Collo et al. [37]), and of P2X2 receptor immunoreactivity in AP neurons (Kanjhan et al. [38]; Atkinson et al. [39]). The IATP showed strong inward rectiﬁcation and the EATP was 22.7 T 0.9 mV (Fig. 1B). The amplitude of IATP varied inversely with the extracellular Ca2+ concentrations (Fig. 1C). The inhibitory effect of increasing extracellular Ca2+ has also been reported in rat sensory neurons (Krishtal et al. [40]), PC-12 cells (Nakazawa et al. [18]), ventromedial hypothalamic neurons (Sorimachi et al. [41]), and in cells expressing recombinant P2X2 receptors (Evans et al. [42]; Vir- ginio et al. [43]), where an allosteric alteration of the ATP binding sites has been suggested to be the underlying mechanism. The IATP was markedly reduced, but still persisted when NMDG+ was substituted for external Na+ even in the absence of Ca2+ (Fig. 3A). Since ATP did not induce a current when sucrose was substituted for external NaCl, our result suggests that NMDG+ could permeate through P2XR. A substantial IATP has been similarly reported in PC-12 cells and sympathetic neurons when glucosamine was substituted for Na+ (Nakazawa et al. [18]; Nakazawa [30]). The addition of Ca2+ to NMDG-Cl- and sucrose-substituted saline increased the IATP (Fig. 3B and C, respectively), indicating that Ca2+ also permeates through P2XR channel. We quantiﬁed the relatively high Ca2+ per- meability, obtaining a PCa/PNa ratio of 3.0. A relatively high permeability of P2XR to Ca2+ has been reported in previous studies (Nakazawa et al. [18]; Sorimachi et al. [41]; Evans et al. [42]; Virginio et al. [43]). Although direct inﬂux of extracellular Ca2+ through P2XR channel may thus contribute to the ATP- induced [Ca]in increase, membrane depolarization and the secondary activation of voltage-dependent Ca2+ channels could also make a signiﬁcant contribu- + tion to the [Ca]in increase. In fact, the high K - and ATP-induced [Ca]in increases were substantially inhibited by a range of Ca2+ channel antagonists, including those which block L- and N-type Ca2+ channels (Table 1). This is in contrast to a previous study, which reported an absence of the L- and N-type Ca2+ channels in rabbit AP neurons (Hay et al. [12]). The discrepancies between their results and ours could be, at least in part, accounted for by the use of different AP preparations (cultured rabbit neurons vs. acutely dissociated rat neurons). The present results, in combination with our previous demonstration of nAChR on AP neurons (Sorimachi and Wakamori [16]), suggest that both 555 ATP and ACh may act as excitatory neurotransmitters in AP neurons, although their release from presyn- aptic nerve terminals has not yet been reported. We also report a negative functional interaction between P2XRs and nAChRs in AP neurons, as has been ob- served in a variety of peripheral neurons and in re- combinant expression systems (Nakazawa et al. [18]; Nakazawa [30]; Zhou and Galligan [20]; Barajas- Lopez et al. [21]; Searl et al. [19]; Khakh et al. [22, 23]; Boue-Grabot et al. [24]). This is the ﬁrst report, that we are aware of, of such interactions occurring in central neurons. When ACh was applied in the pres- ence of ATP, there was a positive correlation between the peak amplitude of inward IATP and the amount of occlusion of inward IACh (Fig. 4A and B). The converse was also true when ATP was applied in the presence of ACh (Fig. 4C and D). Non-additivity of the IACh + ATP was observed even when the inward current was carried by NMDG+ and/or Ca2+ (Fig. 7). Co-application of two agonists did not seem to change the driving force for Na+, because at a Vh between EACh and EATP an outward current due to the acti- vation of nAChR, followed by an inward current due to the activation of P2XR, was observed (Fig. 6C). Occlusion was also observed at a Vh of j10 mV, which is closer to EACh or EATP. Furthermore, the removal of external Ca2+ did not alter the occlusion, and thus a Ca2+-mediated mechanism does not contribute to the current occlusion. The IACh + ATP occlusion was observed at all nega- tive holding potentials when the current was inward but was not observed for outward currents at a Vh of +40 mV. Here the outward IACh + ATP was not different from the predicted sum of IACh and IATP (Fig. 6B). The same dependence on current direction has been reported in guinea-pig enteric and submucosal neurons (Zhou and Galligan [20]; Barajas-Lopez et al. [21]), suggesting that the current occlusion was triggered by the inward movement of cations through two channels. Some investigators have shown that the amplitude of IACh + ATP is equal to or even smaller than that of either IACh or IATP. For instance, the concentration of one agonist causing little or no inward current pro- duced dramatic occlusion of the inward current gen- erated by the other agonist (Searl et al. [19]). Khakh et al. [22] using Xenopus oocytes co-expressing P2X2 and a3b4 nAChR channels, provided several lines of evidence which indicated that occlusion was mostly mediated by the inhibition of the nAChR channel by activation of the P2XR. However, we found that concomitant application of two agonists always caused a larger current than either agonist (Fig. 6A), and that the amount of occlusion of one