Kevin J. Lynch

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP‐ATP and enhancement by the P2X3 allosteric modulator, cibacron blue

Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X3 mRNA in sensory neurones has led to the hypothesis that activation of P2X3 and/or P2X2/3 receptors contributes to nociception. The local administration of the P2X receptor agonist, BzATP (100 – 1000 nmol paw−1, s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. The co‐administration of the potent P2X receptor antagonist, TNP‐ATP (30 – 300 nmol paw−1), but not an inactive analogue, TNP‐AMP, with BzATP into the rat hindpaw attenuated BzATP‐induced nociception. Similarly, co‐administration of TNP‐ATP, but not TNP‐AMP, with 5% formalin reduced both acute and persistent nociception in this test. Co‐administration of cibacron blue (30 and 100 nmol paw−1), a selective allosteric enhancer of P2X3 and P2X2/3 receptor activation, with BzATP (30 and 100 nmol paw−1) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co‐administration of cibacron blue (30 and 100 nmol paw−1) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. The ability of TNP‐ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X3 containing channels contributes specifically to both acute and persistent nociception in the rat.

2', 3'-O-(2,4,6,trinitrophenyl)-ATP and A-317491 are competitive antagonists at a slowly desensitizing chimeric human P2X3 receptor.

Rapid desensitization of ligand‐gated ion channel receptors can alter the apparent activity of receptor modulators, as well as make detection of fast‐channel activation difficult. Investigation of the antagonist pharmacology of ATP‐sensitive homomeric P2X3 receptors is limited by agonist‐evoked fast‐desensitization kinetics. In the present studies, chimeric receptors were created using the coding sequence for the N‐terminus and the first transmembrane domain of either the nondesensitizing human P2X2a or fast‐desensitizing P2X3 receptor joined to the sequence encoding the extracellular loop, second transmembrane domain, and C‐terminus of the other receptor (designated P2X2–3 and P2X3–2, respectively). These clones were stably transfected into 1321N1 astrocytoma cells for biophysical and pharmacological experiments using both electrophysiological and calcium‐imaging methods. Chimeric P2X2–3 and P2X3–2 receptors were inwardly rectifying and agonist responses showed desensitization properties similar to the wild‐type human P2X2a and P2X3 receptors, respectively. The P2X2–3 chimera displayed an agonist pharmacological profile similar to the P2X3 wild‐type receptor being activated by low concentrations of both ATP and α,β‐meATP. In contrast, the P2X3–2 chimera had markedly reduced sensitivity to both agonists. The P2X3 receptor antagonists TNP‐ATP and A‐317491 were shown to be potent, competitive antagonists of the P2X2–3 chimera (Ki=2.2 and 52.1 nM, respectively), supporting the hypothesis that rapid receptor desensitization can mask the competitive antagonism of wild‐type homomeric P2X3 receptors.

Potent desensitization of human P2X3 receptors by diadenosine polyphosphates

In this study, the receptor desensitizing effects of diadenosine polyphosphates at recombinant human P2X3 (hP2X3) receptors were examined. Administration of Ap3A, Ap4A, Ap5A or Ap6A inhibited the hP2X3 receptor-mediated response to a subsequent application of 3 muM alphabeta-methyleneATP (alphabeta-meATP), in a concentration-dependent manner, with IC50 values 2707, 42, 59 and 46 nM, respectively. These agonists did not desensitize alphabeta-meATP responses mediated by the slowly desensitizing heteromeric human P2X2/3 receptor. hP2X3 receptor desensitization was reversible and was not observed following the increase in intracellular Ca2+ levels produced by carbachol. A similar pattern of desensitization evoked by Ap5A was also observed using electrophysiological recordings of Xenopus oocytes expressing hP2X3 receptors. These data demonstrate that diadenosine polyphosphates, found endogenously in the central nervous system, can readily desensitize hP2X3 receptors at nanomolar concentrations that are 10-fold lower than are required to produce agonist-induced receptor activation. Thus, P2X3 receptor desensitization by diadenosine polyphosphates may provide an important modulatory mechanism of P2X3 receptor activation in vivo.

Chapter 3. Recent advances in development of novel analgesics

Publisher Summary This chapter presents an overview of the advances in development of novel analgesics. Nonsteroidal anti-inflammatory drugs (NSAIDs), opioids, and a variety of adjuvant agents have been mainstays of pain therapy for decades. All these agents suffer from drawbacks in clinical use. A number of advances have been made in understanding of the neurobiology of pain in recent years. Distinct mechanisms contribute to physiological pain, to pain arising from tissue damage (inflammatory or nociceptive pain) and to pain arising from injury to the nervous system (neuropathic pain). The introduction of selective cyclooxygenase-2 (COX-2) inhibitors into clinical practice represents the first step towards novel, safer, and more effective pain therapies. COX-2 inhibitors are described in the chapter, along with efforts to identify safer opioids with a focus largely on novel, potential near-term analgesics. Some newly emerging molecular targets that may represent future advances in the search for novel pain therapies are highlighted.

Lack of specificity of [35S]-ATPγS and [35S]-ADPβS as radioligands for ionotropic and metabotropic P2 receptor binding

Adenosine‐5′‐O‐3‐thio[35S]triphosphate ([35S]‐ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X1, P2X2, P2X3, and P2X4. Similarly, adenosine‐5′‐O‐2‐thio[35S]diphosphate ([35S]‐ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [35S]‐ATPγS and [35S]‐ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X4 transfected 1321N1 astrocytoma cells. [35S]‐ATPγS (0.2 nM) and [35S]‐ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [35S]‐ATPγS and [35S]‐ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y1 and P2Y2 receptor subtypes, to 1321N1 cells stably transfected with the human P2X4 receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X4 receptor, as measured by cytosolic calcium flux. These data indicate that [35S]‐ATPγS and [35S]‐ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone. Drug Dev. Res. 48:84–93, 1999.