Chapter Four - Purinergic Mechanisms and Pain
Introduction
The purinergic signaling hypothesis was presented in 1972, namely that adenosine 5′-triphosphate (ATP) is an extracellular signaling molecule (Burnstock, 1972). ATP was shown to be a transmitter in nonadrenergic, noncholinergic nerves supplying the gut (Burnstock, Campbell, Satchell, & Smythe, 1970), and bladder (Burnstock, Dumsday, & Smythe, 1972). Later, ATP was recognized as a cotransmitter in nerves in both peripheral and central nervous systems (Burnstock, 2007a). Receptors for purines and pyrimidines were shown to be widely expressed on non-neuronal as well as nerve cells (see Burnstock & Knight, 2004).
Until recently, apart from vesicular release from nerves (e.g., Pankratov, Lalo, Verkhratsky, & North, 2006), it was assumed that the source of extracellular ATP acting on purinoceptors was damaged or dying cells. However, it is now recognized that ATP release from healthy cells is a physiological mechanism (see Bodin and Burnstock, 2001, Dubyak, 2006). ATP is released from most cell types during mechanical deformation in response to shear stress, stretch, or osmotic swelling, as well as to hypoxia and various agents. Exocytotic vesicular release of ATP from nerves and from endothelial cells (Burnstock, 1999), urothelial cells (Knight, Bodin, De Groat, & Burnstock, 2002), osteoblasts (Romanello et al., 2005), fibroblasts (Boudreault & Grygorczyk, 2004), and astrocytes (Montana, Malarkey, Verderio, Matteoli, & Parpura, 2006) is one of the ATP transport mechanisms. In addition, there is evidence for ATP transport via connexin or pannexin hemichannels, ATP-binding cassette transporters, plasmalemmal voltage-dependent anion channels, and P2X7 receptors (Dubyak, 2006, Lazarowski, 2012, Scemes et al., 2007). ATP release from endothelial cells is increased during acute inflammation (Bodin & Burnstock, 1998). While adenosine is largely produced by ectoenzymatic breakdown of ATP, there may be subpopulations of neurons and/or glial cells that release adenosine directly (Wall & Dale, 2007). Probes for real-time measurement of ATP and adenosine release in biological tissues have been described (Llaudet et al., 2005, Nakamura et al., 2006).
After release, nucleotides undergo rapid enzymatic degradation (Yegutkin, 2014, Zimmermann, 2001). Ecto-nucleotidase families include the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, CD39), ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPP), alkaline phosphatases, and ecto-5′-nucleotidase (CD73). E-NTPDases and E-NPPs hydrolyze ATP and adenosine 5′-diphosphate (ADP) to adenosine monophosphate that is further hydrolyzed by CD73 to adenosine.
Two families of purinoceptors called P1 and P2 (for adenosine and ATP/ADP, respectively) were recognized in 1978 (Burnstock, 1978). In 1985 a proposal suggesting a pharmacological basis for distinguishing two types of P2 receptor (P2X and P2Y) was made (Burnstock & Kennedy, 1985). On the basis of studies of transduction mechanisms and the cloning of nucleotide receptors, it was proposed that purinoceptors should belong to two major families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G protein-coupled receptors (Abbracchio & Burnstock, 1994). Currently, seven P2X subunits (P2X1–7) and eight P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) are recognized, including receptors that are sensitive to pyrimidines as well as purines (Burnstock, 2007b, Ralevic and Burnstock, 1998). Four subtypes of P1 receptors were cloned, namely, A1, A2A, A2B, and A3 (see Fredholm, IJzerman, Jacobson, Klotz, & Linden, 2001). Selective agonists and antagonists are available for the P1 receptor subtypes (Jacobson & Gao, 2006). P2X1–7 receptor subunits have been cloned and characterized, with intracellular NH2- and COOH-termini and two transmembrane (TM)-spanning regions, one involved with channel gating and the other lining the ion pore (Chen et al., 1995, Egan et al., 2006, Lewis et al., 1995, North, 2002). P2X receptors involve three subunits that form a stretched trimer (Nicke et al., 1998). P2X receptor subunits can combine to form either homomultimers or heteromultimers (Burnstock, 2007b, North, 2002, Volonté et al., 2006). Heteromultimers have been established for P2X2/3, P2X4/6, P2X1/5, and P2X2/6 receptors. P2X7 receptors possess small cation channels, but on prolonged exposure to high concentrations of agonist, large channels (pores) are activated that allow the passage of larger molecular weight molecules (Garcia-Marcos, Pochet, Marino, & Dehaye, 2006). P2X7 receptors are localized on immune cells and glia. They mediate proinflammatory cytokine release, cell proliferation, and apoptosis. The metabotropic P2Y receptors have a subunit topology of an extracellular NH2-terminus and intracellular COOH-terminus and seven TM-spanning regions (Abbracchio et al., 2006). P2Y1, P2Y12, and P2Y13 receptors are activated principally by nucleoside diphosphates, while P2Y2, P2Y4, and P2Y6 receptors are activated by both purine and pyrimidine nucleotides. P2Y receptor subtypes can form heterodimeric complexes (Ecke et al., 2008). Adenosine A1 receptors can form a heteromeric complex with P2Y1 receptors (Fischer & Krügel, 2007), while dopamine D1 and adenosine A1 receptors have been shown to form functionally interactive heteromeric complexes. Selective agonists and antagonists for most of the P2Y receptor subtypes are already available (Abbracchio et al., 2006).
There were early reports that pain was produced by injection of ATP into human skin blisters (Bleehen and Keele, 1977, Collier et al., 1966). Claims were made for ATP involvement in migraine (Burnstock, 1981) and for involvement in pain pathways in the spinal cord (Jahr and Jessell, 1983, Salter and Henry, 1985). P2X3 ionotropic ion channel purinergic receptors were cloned in 1995 (Chen et al., 1995) and shown to be localized predominantly on small nociceptive sensory neurons in dorsal root ganglia (DRG) together with P2X2/3 heteromultimer receptors (Bradbury et al., 1998, Burnstock, 2007a, Burnstock, 2009a). Burnstock (1996) put forward a unifying purinergic hypothesis for the initiation of pain, proposing that ATP released as a cotransmitter with noradrenaline, and neuropeptide Y from sympathetic nerve terminal varicosities might be involved in sympathetic pain (causalgia and reflex sympathetic dystrophy). Further, it was suggested that ATP released from vascular endothelial cells of microvessels during reactive hyperemia is associated with pain in migraine, angina, and ischemia; and that high levels of ATP are released from tumor cells, damaged during abrasive activity to reach P2X3 receptors on nociceptive sensory nerves. The involvement of purinergic signaling in pain has been reinforced by an increasing number of papers. Nociceptive fibers expressing P2X3 receptors were shown to arise largely from the population of small neurons in the DRG that labeled with the isolectin B4 (IB4) (Bradbury et al., 1998, Vulchanova et al., 2001). Decreased sensitivity to noxious stimuli associated with the loss of IB4-binding neurons expressing P2X3 receptors indicated that these sensory neurons are essential for the signaling of acute pain (Vulchanova et al., 1996). The central projections of these primary afferent neurons are in inner lamina II of the dorsal horn where there are peripheral projections to skin, tooth pulp, tongue, and subepithelial regions of visceral organs (see Fig. 1; Burnstock & Wood, 1996). P2X3 and P2X2/3 receptors on sensory neurons are the predominant P2 receptor subtypes involved in the initiation of nociception. However, P2Y receptors are also present (Nakayama et al., 2004, Ruan and Burnstock, 2003), which are involved in modulation of pain transmission (Gerevich et al., 2004, Malin and Molliver, 2010). P2Y receptors potentiate pain induced by chemical or physical stimuli via capsaicin-sensitive, transient receptor potential vanilloid 1 (TRPV1) channels (Lakshmi & Joshi, 2005). It was proposed that the functional interaction between P2Y2 receptors and TRPV1 channels of nociceptors could underlie ATP-induced inflammatory pain. In mice-lacking TRPV1 receptors, ATP-induced hyperalgesia was abolished. The involvement of adenosine has also been proposed (Burnstock and Sawynok, 2010, Sawynok and Liu, 2003). Reviews concerned with different aspects of purinergic signaling and pain are available (Bele and Fabbretti, 2015, Burnstock, 2001b, Burnstock, 2006, Burnstock, 2009c, Burnstock, 2012, Burnstock and Sawynok, 2010, Donnelly-Roberts et al., 2008, Hanani, 2012, Jarvis, 2010, Krames, 2014, Magni and Ceruti, 2014, Tsuda, Beggs, et al., 2013, Tsuda et al., 2010).
Section snippets
Purinergic Mechanosensory Transduction and Pain
It was proposed that purinergic mechanosensory transduction occurred in visceral tubes and sacs, including ureter, bladder, and gut (Burnstock, 1999). ATP released from epithelial cells during distension was suggested to act on P2X3 homomeric and P2X2/3 heteromeric receptors on subepithelial sensory nerves initiating impulses in sensory pathways to pain centers in the central nervous system (Fig. 2A). There is evidence supporting this hypothesis in various organs.
Neuropathic and Inflammatory Pain
In a seminal study published in Nature in 2003, expression of P2X4 receptors on spinal cord microglia was shown to be increased in neuropathic pain, which was reduced after use of P2X4 antisense oligonucleotides (Tsuda et al., 2003). An explosion of work focused on purinergic signaling in neuropathic pain has followed (Burnstock, 2006, Inoue, 2007, McGaraughty and Jarvis, 2006, Nakatsuka and Gu, 2006). P2X7 and P2Y12 receptors on microglia have also been shown to be involved in neuropathic pain
Cancer Pain
Purinergic mechanisms are of interest in relation to cancer pain (Burnstock, 1996, Gilchrist et al., 2005, Mantyh et al., 2002). The unusually high levels of ATP contained in tumor cells (Maehara, Kusumoto, Anai, Kusumoto, & Sugimachi, 1987) may be released by mechanical stress to activate P2X3 receptors on nearby nociceptive sensory nerve fibers (Burnstock, 1996). In a bone cancer pain model, there is increased expression of P2X3 receptors on calcitonin gene-related peptide (CGRP)
Migraine
The involvement of ATP in migraine was first considered in relation to the vascular theory of this disorder, where it was suggested that ATP released from endothelial cells during reactive hyperemia activated P2X3 receptors on nociceptive sensory fibers in the adventitia following cerebral vascular vasospasm (not associated with pain) (Burnstock, 1989). P2X3 receptor involvement in neuronal dysfunction in brain areas that mediate nociception such as the trigeminal nucleus and thalamus has also
Therapeutic Developments
There is much interest in developing selective P2X3, P2X2/3, P2X4, P2X7, and P2Y12 receptor antagonists that are orally bioavailable, can cross the blood–brain barrier, and do not degrade in vivo for the treatment of acute and neuropathic pain (see Carter et al., 2009, Ford, 2012, Gever et al., 2006, Gever et al., 2010, Jahangir et al., 2009). Reviews about the involvement and therapeutic potential of purinergic drugs for gastrointestinal diseases and pain are available (Moynes et al., 2014,
Conclusion
The subtypes of purinoceptors involved in acute and chronic neuropathic and inflammatory pain are summarized in Table 1. Therapeutic strategies involving purinergic drugs are being explored, involving P2X3, P2X2/3, P2X4, P2X7, and P2Y12 receptor antagonists and A1 receptor agonists. While there is clearly purinergic multi-receptor involvement in pain pathology, the precise roles and interactions with other antinociceptive agents are still to be resolved.
Conflict of Interest
The author confirms that these article contents have no conflict of interest.
Acknowledgment
The author thanks Dr. Gillian E. Knight for her excellent editorial assistance.
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