Purinergic Signalling (2012) 8 (Suppl 1):S61–S79 activation of P2X1 by ATP can synergize with low levels of known platelet activators, such as thrombin and collagen, to increase platelet aggregation. Indeed, the importance of P2X1 activation in platelets seems particularly important under conditions of high shear stress, suggesting that P2X1 is an attractive antithrombotic target [47]. P2X1 receptors have a key role as neurotransmitter receptors at sympathetically innervated smooth muscle cells in urinary bladder, vascular smooth muscle cells, and skeletal muscle, among others. Indeed, the localization of P2X1 in rat pulmonary artery smooth muscle cells and human skeletal muscle cells was demonstrated using anti- P2X1 receptor antibody [51, 52]. The localization of P2X1 receptor in afferent arteriole vascular smooth muscle cells is of particular importance as it demonstrates a critical role for this receptor in renal autoregulation, a process by which the kidney is able to maintain constant renal blood flow and glomerular filtration rate over changes in arterial pressure. Zhao et al. [53] nicely showed P2X1 immunoreactivity on the vascular smooth muscle cells of microdissected renal interlobular artery and afferent arteriole using anti-P2X1 receptor antibody. P2X2 The P2X2 channel is mostly expressed in the central and peripheral nervous system with predominant expression in dorsal ganglia neurons, mesenteric ganglia neurons, taste buds, and astrocytes and plays a key role in synaptic transmission, pain, and taste perception. Indeed, using anti-P2X2 receptor antibody (#APR-003), Ohta et al. [54] showed the localization of P2X2 in rat cultured myenteric neurons, implying that purinergic signaling is involved in the regulation of GI functions. Similarly, Florenzano et al. [55] demonstrated the co- localization of P2X2 with orexin in hypothalamic neurons, suggesting that P2X2 may be involved in arousal and wakefulness (Fig. 7a–c). The involvement of P2X2 in taste transduction was demonstrated by showing the localization of P2X2 in taste buds using Alomone Labs’ respective antibody [56]. P2X2 forms functional heteromers with P2X3 channels, and the P2X2/P2X3 unit has been described as a major player in pain transmission (see also below). Using anti-P2X2 receptor antibody, the presence of the receptor was demonstrated in rat dissociated DRG neurons from normal and inflamed animals [57]. P2X3 P2X3 and P2X2/P2X3 receptors are selectively localized on sensory neurons in trigeminal, nodose, and DRG. P2X3 receptors are now recognized as major players in mediating S71 the primary sensory effects of ATP and, as such, are of major importance in nociception and mechanosensory transduction. Hence, P2X3 and P2X2/P2X3 represent unique targets for novel analgesic agents that function as receptor antagonists [58]. Current theories predict that chronic pain is brought about by a gain in the expression and function of sensory nociceptors that enhance their signal flow to the brain to ensure the long-lasting nature of this process. In this regard, both P2X3 and P2X2/P2X3 receptors were found to be modulated by several other receptors and ion channels known to be involved in pain perception. For instance, a selective, slow upregulation of nociceptive P2X3 receptors on trigeminal neurons by the neuropeptide calcitonin gene- related peptide, a known endogenous pain mediator, was demonstrated using anti-P2X3 receptor antibody (#APR- 016) [59]. Similarly, using anti-P2X3 receptor and anti- P2X2 receptor antibodies, it was shown that the neutraliza- tion of endogenous nerve growth factor, another key pain transmission molecule, decreased P2X3 receptor-mediated currents and enhanced the expression of P2X2 subunits by P2X3-immunoreactive neurons [60]. In addition, using Alomone Labs antibodies in immu- nohistochemical studies, Banerjee et al. [61] showed that P2X3 exhibited a significant upregulation both in DRGs and in nodose ganglia of rats having chronic esophagitis, suggesting that the upregulation of P2X3 receptors in sensory neurons following inflammation may contribute to altered sensation from the esophagus (Fig. 7a). P2X4, P2X5, and P2X6 P2X4 is widely expressed in many neuronal and non- neuronal tissues. Localization studies at both protein and mRNA levels have demonstrated a wide expression pattern for the P2X4 channel: rat brain, kidney, lung, heart, spinal cord, and more were shown to express the receptor [62]. Although its role in most of these tissues remains elusive, due to the lack of highly specific antagonist, recent studies suggest an important role for P2X4 in chronic inflammation and neuropathic pain by spinal cord microglia. Following spinal cord injury or peripheral nerve injury, the upregula- tion of P2X4 receptor in activated microglia was detected. Partial nerve injury (PNI) induced a restricted de novo expression of P2X4 by activated microglia. Immunohisto- chemical staining, comparing P2X4 expression in the spinal cord of sham-operated mice versus PNI mice using the specific anti-P2X4 receptor antibody (#APR-002), indicated that sham-operated mice expressed low, barely detectable levels of P2X4, while PNI mice, 10 days following the injury, expressed significant levels of P2X4 (Fig. 7b, c) [63]. It seems that the activation of the microglia following nerve injury is not mediated by P2X4 since the activation of