Distinct neurochemical mechanisms are activated following administration of different P2X receptor agonists into the hindpaw of a rat

Abstract

Nocifensive behaviors induced by the intradermal injection of three different P2X receptor agonists, ATP, BzATP or α,β-meATP, into a hindpaw were measured in rats that were injected intrathecally with either an NMDA (MK-801) or an NK-1 (L-703,606) receptor antagonist or were pretreated systemically with the VR1 agonist resiniferatoxin (RTX). The same procedures were performed in animals injected intradermally with either capsaicin or formalin. Spinal infusion of MK-801 (10–50 nmol/10 μl) similarly reduced the number of nociceptive events triggered by each of the P2X agonists and was also effective against capsaicin and formalin induced behaviors. Intrathecal administration of L-703,606 (50–100 nmol/10 μl) had its greatest antinociceptive effect against capsaicin-induced behaviors followed by ATP and BzATP. L-703,606 was completely ineffective against behaviors induced by formalin or the other P2X agonist, α,β-meATP. Pretreatment with RTX 2 days prior to testing significantly decreased the number of nociceptive events caused by each of the P2X agonists as well as capsaicin and formalin (capsaicin>BzATP>ATP>formalin>α,β-meATP). The remaining nociceptive events in RTX animals injected with α,β-meATP were significantly higher than in animals injected with either ATP or BzATP. Intradermal administration of different P2X receptor agonists induced similar levels of nocifensive behaviors and activity at spinal NMDA receptors. Capsaicin-sensitive fibers were likely activated following injection of BzATP and ATP, but not α,β-meATP, and appeared to trigger the spinal release of substance P. The differences in mechanisms employed by the different P2X agonists may be a function of respective selectivity for P2X receptor subtypes.

Introduction

Spontaneous, short-lasting, nocifensive behaviors can be elicited by local administration of agents such as adenosine triphosphate (ATP), capsaicin or formalin into the rat hindpaw. The mechanisms induced by capsaicin and formalin have been well described, and despite similar overt behavioral responses, injections of these two agents evoke different pharmacological and physiological events in the central nervous system. Capsaicin-evoked nociceptive behaviors, such as licking, biting and lifting of the injured paw, can be greatly attenuated by intrathecal administration of excitatory amino acid and substance P antagonists as well as by pretreatment with doses of capsaicin that desensitize VR1 neurons [11], [25], [26], [33]. In contrast, similar behaviors observed during the first phase of the formalin test are only weakly affected by intrathecal administration of substance P or excitatory amino acid antagonists [7], [14] or by systemic capsaicin [23]. Therefore, different mechanisms may underlie overtly similar nocifensive behaviors.

Intraplantar administration of ATP also results in short-lasting licking, biting and lifting of the injured paw [2], [13], [18]. Unlike formalin and capsaicin, ATP is an endogenous agent that can act as a fast neurotransmitter and is thought to be a key mediator of nociception following tissue injury [3], [4], [9]. ATP is a non-selective agonist with varied potencies for each of the seven ionotropic P2X and seven metabotropic P2Y receptor subtypes currently identified [1], [17], [24]. It is possible that several subtypes of P2 receptors play significant roles in ATP-mediated nociception. P2X_1–6 mRNA and protein are expressed in the dorsal root ganglion [8], [36], and recent evidence has demonstrated the presence of presynaptic P2X₇ mRNA in the dorsal horn [10]. Most attention has focused on the P2X₃ receptor due to its high level of expression and specific localization on nociceptive neurons [4], [21]. P2Y receptors may also be involved in DRG nociceptive transmission [38] since mRNA for P2Y₁, P2Y₂ and P2Y₆ are found in DRG neurons [20]. Furthermore, P2Y₂ receptors have been located on small diameter capsaicin-sensitive DRG neurons [20].

However, the relative contribution of specific P2 receptor subtypes to ATP-induced nocifensive behaviors is not entirely clear. While an important nociceptive role for the P2X₃ receptor has emerged [6], [16], [18], [27], [29], [30], studies conducted with P2X₃ deficient animals have shown that administration of ATP into a paw still resulted in spontaneous pain behaviors [6] and that ATP continued to evoke inward currents in a subset of sensory DRG neurons [39]. Thus, activation of P2X₃ receptors alone cannot account for all of the nociceptive activity evoked by ATP.

Local administration of other P2 receptor agonists besides ATP, such as 4-benzoylbenzoyl ATP (BzATP) and α,β-methylene ATP (α,β-meATP), also evoke nocifensive behaviors. These agonists are considered preferential P2X agonists [17], [24] with different relative selectivities for P2X receptor subtypes. BzATP has agonist activity at all P2X receptor subtypes, while α,β-meATP has agonist activity at all P2X receptor subtypes except P2X₂ and P2X₇[1], [17]. Given the differences in agonist potencies for particular P2X receptor subtypes, the possibility that different mechanisms can be recruited following injection of different P2X agonists into a paw was explored. Therefore, nocifensive behaviors induced by ATP, BzATP and α,β-meATP were measured in animals that were intrathecally injected with either an NK-1 or an NMDA receptor antagonist or were pretreated with systemic resiniferatoxin (RTX). For comparison, similar experiments were conducted in animals injected with either capsaicin or formalin into a paw.