ATP acts via various subtypes of ionotropic P2X receptors and/or metabotropic P2Y receptors. The translocation of nNOSNT-YFP by ATP was completely blocked by the non-selective P2 receptor antagonist suramin and significantly reduced by the P2X antagonist PPADS or the P2Y antagonist RB-2 (Figure
2). Conversely, 2-MeSATP, a P2X receptor agonist or UTP, a P2Y
2 and P2Y
4 receptor agonist, stimulated the translocation of nNOSNT-YFP to the plasma membrane (Figure
3A). However, the increase in the translocation of nNOSNT-YFP by 2-MeSATP or UTP was not as great as that obtained with ATP itself. Co-stimulation with 2-MeSATP and UTP increased the translocation of nNOSNT-YFP in a concentration-dependent manner, with the increase being comparable to that obtained with ATP and with similar EC
50 values (Figure
3B). ATP, 2-MeSATP, and UTP induced the increase in [Ca
2+]i with EC
50 values of 3.5, 10.8, and 2.84 μM, respectively, in PC12N cells (Figure
4A); and ATP could increase the [Ca
2+]i in the absence of extracellular Ca
2+(Figure
4B), demonstrating that P2Y receptors as well as P2X receptors were involved in the increase in the [Ca
2+]i of PC12N cells. Furthermore, it is intriguing that, whereas the non-selective P2 receptor antagonist suramin fully suppressed the ATP-induced increase in [Ca
2+]i, PPADS and RB-2 reduced it only partially (Figure
4C). Consistent with the results of a previous study [
26], P2X
1, P2X
3, and P2X
4 mRNAs were detected by RT-PCR in both undifferentiated and NGF-differentiated PC12 cells; and P2X
6 mRNA was detected in NGF-differentiated PC12 cells (Figure
7). Since P2X
2 receptors were cloned from NGF-differentiated PC12 cells [
27], P2X receptors except for P2X
7 are present in NGF-differentiated PC12 cells. As for metabotropic P2Y receptors, besides P2Y
12 receptors that couple to Gi/o, the other P2Y receptors P2Y
1, P2Y
2, P2Y
4, P2Y
6, and P2Y
11 are coupled to the Gq
/11 proteins, leading to stimulation of phosphoinositide metabolism, release of intracellular Ca
2+ stores and activation of PKC. P2Y
2, P2Y
4, and P2Y
6 mRNAs were found in both undifferentiated and NGF-differentiated PC12 cells. Since the P2Y
6 receptor was not activated by UTP, ATP and UTP may act on P2Y
2 and P2Y
4 receptors in NGF-differentiated PC12N cells. Arslan
et al. extensively characterized Ca
2+ responses in PC12 cells and suggested that P2X
2/P2X
2b, P2X
4, and P2Y
2 receptors contribute to the ATP-induced [Ca
2+]i increase in NGF-differentiated PC12 cells [
26]. Thus the presence of multiple P2X and P2Y receptors in PC12 cells suggests that several P2 receptor subtypes are involved in the increase in [Ca
2+]i and nNOS translocation and that the effects of ATP on them seem to be additive.
Like PC12 cells, many P2X and P2Y receptors mRNAs were detected by RT-PCR in the spinal cord (Figure
7). To clarify the involvement of P2 receptors in nNOS translocation in the spinal cord, we examined the effect of ATP and UTP on [Ca
2+]i in primary cultured spinal neurons. While ATP and UTP increased the magnitude of [Ca
2+]i levels in a concentration-dependent manner, ATP was more potent than UTP (Figure
9A), suggesting that both P2X and P2Y receptors are involved in [Ca
2+]i changes by ATP in the spinal cord. This notion was supported by the effect of antagonists on the ATP-evoked [Ca
2+]i responses (Figure
9B). While the P2Y receptor antagonist RB-2 inhibited 41%, the non-selective P2 receptor antagonist suramin reduced the increase in 78% of the cells responsive to 1 μM ATP. Interestingly, A-317491, a selective P2X
3 antagonist, reduced only 6.8% of the ATP-responsive cells. These results are consistent with the previous studies that P2X
3 receptors are expressed in DRG neurons and are transported to the central process in the spinal cord [
28,
29].