Extracellular purine nucleotides elicit a diverse range of biological responses through binding to specific cell surface receptors. The ionotrophic P2X subclass of purinoreceptors respond to ATP by stimulation of calcium ion permeability; however, it is unknown how P2X purinoreceptor activation is linked to intracellular signaling pathways. We report that stimulation of PC12 cells with ATP results in the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 and was wholly dependent upon extracellular calcium ions. Treatment of the cells with adenosine, AMP, ADP, UTP, or ␣,␤-methylene ATP was without effect; however, MAP kinase activation was abolished by pretreatment with suramin and reactive blue 2. The calcium-activated tyrosine kinase, Pyk2, acts as an upstream regulator of the MAP kinases and became tyrosine phosphorylated following treatment of the cells with ATP. We have ruled out the involvement of depolarization-mediated calcium influx because specific blockers of voltage-gated calcium channels did not affect MAP kinase activation. These data provide direct evidence that calcium influx through P2X2 receptors results in the activation of the MAP kinase cascade. Finally, we demonstrate that a different line of PC12 cells respond to ATP through P2Y2 purinoreceptors, providing an explanation for the conflicting findings of purine nucleotide responsiveness in PC12 cells.Adenine nucleotides act through specific cell surface receptors to provoke a variety of biological responses (1, 2). In the nervous system, extracellular ATP has been postulated to serve as a classical neurotransmitter and to also act as a co-transmitter through its coordinate packaging and release with norepinephrine and acetylcholine (3). The analysis of these roles of ATP has been complicated by its catabolism into other biologically active species and the unexpected diversity of cell surface purinoreceptors. Purinoreceptors have been classified into two primary classes, the P1 receptors are responsive to adenosine, whereas the P2 class receptors respond to a variety of purine nucleotides, including ATP. Presently, twelve P2 subtypes have been identified and assigned to two mechanistically distinct subclasses of purinoreceptors (1). The metabotrophic purinoreceptors of the P2Y subclass (formerly P2u, P2t, and P2y) initiate their biological actions by G-protein-dependent activation of phospholipase C and subsequent elevation of intracellular calcium levels through liberation of calcium from internal stores (4). The P2X purinoreceptors comprise a distinct subclass of receptors that are ligand-gated calcium channels functionally related to glutamate and nicotinic acetylcholine receptors. The ionotrophic P2X receptors regulate intracellular calcium levels through the ligand-stimulated increase in calcium permeability; thus their actions are dependent upon extracellular calcium ions. Although the ability of the P2X receptors to evoke a rise in intracellular calcium levels is well documented, it is entirely unclear how these rec...

No abstract