Functional expression of P2X₇ receptors in non-neuronal cells of rat dorsal root ganglia

Abstract

The P2X₇ receptor is an ATP-sensitive ligand-gated cation channel, expressed predominantly in cells with immune origin. Recent studies have demonstrated that P2X₇ may play an important role in pain signaling. In the present study, the expression of P2X₇ receptors in non-neuronal cells and neurons isolated from dorsal root ganglia was characterized using patch clamp, pharmacological and confocal microscopy approaches. In small diameter DRG neurons, 100 μM 2′, 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) evoked an inward current, which was inhibited completely by 1 μM A-317491, a potent and selective P2X₃ receptor antagonist. In contrast, BzATP evoked concentration-dependent increases in inward currents in non-neuronal DRG cells with an EC₅₀ value of 26 ± 0.14 μM, which were resistant to the blockade by A-317491. The activity to evoke cationic currents by P2X receptor agonists in non-neuronal cells showed a rank order of BzATP > ATP > α,β-meATP. Pyridoxal-phosphate-6-azophenyl-,2′,4′-disulphonic acid (PPADS) and Mg²⁺ produced concentration-dependent inhibition of BzATP-evoked currents in non-neuronal cells. Confocal microscopy revealed positive immunoreactivity of anti-P2X₇ receptor antibodies on non-neuronal cells. No anti-P2X₇ immunoreactivity was observed on DRG neurons. Further electrophysiological studies showed that prolonged agonist activation of P2X₇ receptors in non-neuronal cells did not lead to cytolytic pore formation. Taken together, the present study demonstrated functional expression of P2X₇ receptors in non-neuronal but not in small diameter neurons from rat DRG. Modulation of P2X₇ receptors in non-neuronal cells might have impact on peripheral sensory transduction under normal and pathological states.

Introduction

P2X₇ receptors belong to a superfamily of ATP-sensitive ionotropic P2X receptors that are composed of seven cloned receptor subtypes (P2X₁–P2X₇). Expression of homomeric or heteromeric P2X receptors such as P2X₂ and P2X₃ (P2X_2/3) form ion channels which are permeable to non-selective cations (Na⁺, K⁺, Ca²⁺) in response to the binding of extracellular adenosine triphosphate (ATP). Unlike other P2X receptors, homomeric P2X₇ receptors are the only subtype for which 2′, 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) is a more potent agonist than ATP [26]. Although P2X₁, P2X₂, P2X₃ and P2X_2/3 receptors are sensitive to inhibition by non-selective P2X antagonists such as suramin, pyridoxal-phosphate-6-azophenyl-,2′,4′-disulphonic acid (PPADS) and trinitrophenyl–adenosine triphosphate (TNP–ATP), both P2X₄ and P2X₇ are at least 50- to 100-fold less sensitive to these antagonists [21], [26]. A unique feature that distinguishes P2X₇ receptors from other P2X receptors is the ability to rapidly form large pores that allows permeation of large molecules following exposure to agonists. The mechanisms underlying pore formation and its functional role remain to be elucidated; however, the pore formation appears to depend on signal transduction in host cells [11], [28], [30], [32].

The P2X₁ to P2X₆ receptors are expressed throughout the peripheral and central nervous systems and play important roles in cellular function [26]. Fast-desensitizing P2X₃ and slow-desensitizing P2X_2/3 receptors are preferentially expressed on peripheral C-afferents and play an important role in nociception [2], [19], [23]. Inhibition of P2X₃ currents by a selective antagonist, A-317491, effectively reduced hyperalgesia and mechanical allodynia in inflammatory and neuropathic pain models and also alleviated persistent pain in the formalin- and acetic acid-induced abdominal constriction tests [20], [23]. Furthermore, expression of P2X₃ in bladder afferents has been shown to play a major role in mechanosensory transduction of bladder function [44].

In contrast, P2X₇ receptors are found predominantly in astrocytes, macrophages and most immune cells where they can trigger a series of cellular responses such as membrane permeabilization, cytokine release, cell proliferation or apoptosis [4], [6], [27], [40]. In central neuronal tissues, the expression of P2X₇ receptors has been demonstrated in brain microglia and astrocytes [6], [36]. However, it has also been reported that P2X₇ receptors are expressed in the presynaptic terminals and play important roles in controlling neurotransmitter release [10], [25], [31], [35], [39].

Recent studies have demonstrated that the inflammatory and neuropathic hypersensitivity to both mechanical and thermal stimuli is completely absent in P2X₇ knockout mice while normal nociceptive processing is preserved [5]. Intraplantar administration of oxidized ATP, an irreversible P2_Z/P2X₇ inhibitor, concentration-dependently relieved inflammatory pain induced by injection of complete Freund's adjuvant in rat hind paw [8], [9]. Taken together, these studies demonstrated the important roles of P2X₇ in pain signaling. However, investigation of functional expression of P2X₇ receptors in ancillary supporting cells or neurons, particularly the small diameter neurons that might be involved in a variety of pain sensation in dorsal root ganglia, has been difficult because of lack of selective P2X₇ modulators.

Using selective and potent P2X₃ antagonist, A-317491, to eliminate predominant P2X₃ currents in peripheral sensory neurons as a pharmacological tool, together with patch clamp recording and immunocytochemistry approaches, the present studies examined the functional expression of P2X₇ receptors in sensory neurons and non-neuronal cells isolated from L4/L5 spinal dorsal root ganglia.