Peripheral adenosine 5′-triphosphate enhances nociception in the formalin test via activation of a purinergic p_2X receptor

Abstract

The pronociceptive effects of adenosine 5′-triphosphate (ATP) were examined in the low concentration formalin model (0.5%) by coadministration of ATP, ATP analogs (α,β-methylene-ATP and 2-methylthio-ATP) and antagonists (suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid) with formalin and determining effects on the expression of flinching behaviours. Coadministration of ATP (5–500 nmol) with formalin enhanced phase 2 (12–60 min after injection) but not phase 1 (0–10 min after injection) responses. α,β-methylene-ATP (0.5–50 nmol) but not 2-methylthio-ATP (50–500 nmol) produced a similar enhancement of activity, generating an order of potency of α,β-methylene-ATP, ATP≫2-methylthio-ATP. This enhancement was primarily expressed in the latter part of phase 2, 30–60 min after injection. Coadministration of suramin 50–500 nmol, a non-selective P_2X and P_2Y purinoceptor antagonist and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid 5–500 nmol, a selective P_2X purinoceptor antagonist, dose-dependently inhibited the augmentation of the formalin response by ATP 50 nmol, but did not reduce the response to formalin itself. Pretreatment for 30 min with higher doses of suramin inhibited the response to formalin (0.5%, 1.5%) and this appeared to be by a systemically mediated action as it was seen following administration into the contralateral paw. The results of this study provide evidence in support of a P_2X purinoceptor mediated augmentation of the pain signal by ATP. The delayed time-course of the effect suggests that it may occur in concert with other mediators that are recruited by the inflammatory process, rather than reflecting a direct depolarization of sensory nerves. Other behavioural paradigms may be required to examine the fast onset, direct effect. Suramin appears to exert both local and systemic effects on the expression of pain behaviours in response to formalin.

Introduction

There is an increasing interest in the role of locally released adenosine 5′-triphosphate (ATP) with a subsequent activation of cell surface P₂ receptors in the regulation of the inflammatory response (Dubyak and El-Moatassim, 1993) and in pain initiation at sensory nerve terminals (Burnstock and Wood, 1996). ATP released under inflammatory conditions could originate from a cytosolic source in a number of cells following lysis or hypoxic-stress, or from more discrete stores in cells such as platelets, mast cells, or sensory or sympathetic nerves. ATP has long been known to stimulate sensory nerve endings, producing an algogenic or pain initiating response (Keele and Armstrong, 1964; Bleehen and Keele, 1977). ATP depolarizes sensory neuron cell bodies by activation of cation channels (Jahr and Jessell, 1983; Krishtal et al., 1988; Bean, 1990); a similar action is presumed to occur at the peripheral sensory nerve terminal accounting for the effect on sensory transmission. Recently, P_2X purinoceptor subtypes have been cloned, directly implicated in the fast depolarization of sensory neurons by ATP and proposed to play a selective role in nociceptive activation (Chen et al., 1995; Lewis et al., 1995; Burnstock and Wood, 1996).

While a direct activation of sensory neurons may mediate some aspects of pronociceptive effects of ATP, additional indirect mechanisms via interactions with inflammatory mediators or inflammatory cells may occur (Green et al., 1991; Dubyak and El-Moatassim, 1993). The formalin test is a pain model with two distinct components, an initial phase which reflects a direct sensory nerve activation and a later phase which may reflect an inflammatory component (reviewed in Tjølsen et al., 1992). A variation of this test which uses lower concentrations of formalin (0.5–1.0%) has recently been used to evaluate pronociceptive effects of adenosine (Karlsten et al., 1992; Doak and Sawynok, 1995). In the present study, we have determined whether the low concentration formalin model can reveal pronociceptive effects of ATP and whether it can be used to ascertain the role of particular P₂ receptor subtypes in pronociception by evaluating the effect of selective agonists (α,β-methylene-ATP, 2-methylthio-ATP) and antagonists (suramin; pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid or PPADS) on the response to formalin (see Section 4for a consideration of receptor selectivity). Such a model could be useful for examining potential interactions of ATP with other inflammatory mediators in modulating the sensory afferent pain signal.