Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X 2 and P2X 3 subunits. P2X 2 and P2X 3 subunits can form homomultimeric P2X 2 , homomultimeric P2X 3 , or heteromultimeric P2X 2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood. Here we describe null mutant mice lacking the P2X 2 receptor subunit (P2X 2 −/− ) and double mutant mice lacking both P2X 2 and P2X 3 subunits (P2X 2 /P2X 3 Dbl−/− ), and compare these with previously characterized P2X 3 −/− mice. In patch-clamp studies, nodose, coeliac and superior cervical ganglia (SCG) neurones from wild-type mice responded to ATP with sustained inward currents, while dorsal root ganglia (DRG) neurones gave predominantly transient currents. Sensory neurones from P2X 2 −/− mice responded to ATP with only transient inward currents, while sympathetic neurones had barely detectable responses. Neurones from P2X 2 /P2X 3 Dbl−/− mice had minimal to no response to ATP. These data indicate that P2X receptors on sensory and sympathetic ganglion neurones involve almost exclusively P2X 2 and P2X 3 subunits. P2X 2 −/− and P2X 2 /P2X 3 Dbl−/− mice had reduced pain-related behaviours in response to intraplantar injection of formalin. Significantly, P2X 3 −/− , P2X 2 −/− , and P2X 2 /P2X 3 Dbl−/− mice had reduced urinary bladder reflexes and decreased pelvic afferent nerve activity in response to bladder distension. No deficits in a wide variety of CNS behavioural tests were observed in P2X 2 −/− mice. Taken together, these data extend our findings for P2X 3 −/− mice, and reveal an important contribution of heteromeric P2X 2/3 receptors to nociceptive responses and mechanosensory transduction within the urinary bladder.

1 We have studied the behavioural eects of intraplantar injections of adenosine 5'-triphosphate (ATP) and related compounds in freely moving rats and investigated whether these nociceptive eects are augmented in the presence of in¯ammatory mediators. 2 We ®nd that in normal animals ATP and analogues produce dose-dependent nocifensive behaviour (seen as bursts of elevation of the treated hindpaw), and localized thermal hyperalgesia. The rank order of potency was: a,b-methyleneadenosine 5'-triphosphate (a,b-methylene ATP) 42-methylthioadenosine triphosphate (2-methylthio ATP)4ATP. After neonatal treatment with capsaicin, to destroy small calibre primary sensory neurones, nocifensive behaviour was largely absent. 3 The eects of ATP analogues were assessed in three models of peripheral sensitization: 2 h after dilute intraplantar carrageenan (0.25% w v 71 ); 24 h after irradiation of the hindpaw with ultraviolet (U.V.) B; immediately following prostaglandin E 2 (PGE 2 ) treatment. In all models the eect of a,bmethylene ATP was greatly augmented. After carrageenan, signi®cant hindpaw-lifting behaviour activity was induced by injection of only 0.05 nmol of a,b-methylene ATP, some 100 times less than necessary in normal skin. 4 Our data suggest that it is much more likely that endogenous levels of ATP will reach levels capable of exciting nociceptors in in¯amed versus normal skin. Our data also suggest the involvement of P2X 3 receptor subunits in ATP-induced nociception.

Despite the considerable interest in the possibility that ATP may function as a peripheral pain mediator, there has been little quantitative study of the pain-producing effects of ATP in humans. Here we have used iontophoresis to deliver ATP to the forearm skin of volunteers who rated the magnitude of the evoked pain on a visual analogue scale. ATP consistently produced a modest burning pain, which began within 20 s of starting iontophoresis and was maintained for several minutes. Persistent iontophoresis of ATP led to desensitization within 12 min but recovery from this was almost complete 1 h later. Different doses of ATP were delivered using different iontophoretic driving currents. Iontophoresis of ATP produced a higher pain rating than saline, indicating that the pain was specifically caused by ATP. The average pain rating for ATP, but not saline, increased with increasing current. Using an 0.8 mA current, subjects reported pain averaging 27.7 +/- 2.8 (maximum possible = 100). Iontophoresis of ATP caused an increase in blood flow, as assessed using a laser Doppler flow meter. The increase in blood flow was significantly greater using ATP than saline in both the iontophoresed skin (P < 0.01) and in the surrounding skin, 3 mm outside the iontophoresed area (P < 0.05). The pain produced by ATP was dependent on capsaicin-sensitive sensory neurons, since in skin treated repeatedly with topical capsaicin pain was reduced to less than 25% of that elicited on normal skin (2.1 +/- 0.4 compared with 9.3 +/- 1.5 on normal skin). Conversely, the pain-producing effects of ATP were greatly potentiated in several models of hyperalgesia. Thus, with acute capsaicin treatment when subjects exhibited touch-evoked hyperalgesia but no ongoing pain, there was a threefold increase in the average pain rating during ATP iontophoresis (22.7 +/- 3.1) compared with pre-capsaicin treatment (7.8 +/- 2.6). Moreover, ATP iontophoresed into skin 24 h after solar simulated radiation (2 x minimal erythymic dose) resulted in double the pain rating of normal skin, increasing from 15.3 +/- 4.1 to 32.7 +/- 4.1. The pain response to saline was not significantly altered after UV irradiation at any time-point studied. We conclude that ATP produces pain by activating capsaicin-sensitive nociceptive afferents when applied to skin. The possibility that ATP activates nociceptors indirectly via its degradation products cannot be ruled out. The effects of ATP are dose-dependent and responses desensitize only slowly. In inflammatory conditions, ATP may be a potent activator of nociceptors and an endogenous mediator of pain.