Nucleotide homeostasis and purinergic nociceptive signaling in rat meninges in migraine-like conditions

Extracellular ATP is suspected to contribute to migraine pain but regulatory mechanisms controlling pro-nociceptive purinergic mechanisms in the meninges remain unknown. We studied the peculiarities of metabolic and signaling pathways of ATP and its downstream metabolites in rat meninges and in cultured trigeminal cells exposed to the migraine mediator calcitonin gene-related peptide (CGRP). Under resting conditions, meningeal ATP and ADP remained at low nanomolar levels, whereas extracellular AMP and adenosine concentrations were one-two orders higher. CGRP increased ATP and ADP levels in meninges and trigeminal cultures and reduced adenosine concentration in trigeminal cells. Degradation rates for exogenous nucleotides remained similar in control and CGRP-treated meninges, indicating that CGRP triggers nucleotide release without affecting nucleotide-inactivating pathways. Lead nitrate-based enzyme histochemistry of whole mount meninges revealed the presence of high ATPase, ADPase, and AMPase activities, primarily localized in the medial meningeal artery. ATP and ADP induced large intracellular Ca²⁺ transients both in neurons and in glial cells whereas AMP and adenosine were ineffective. In trigeminal glia, ATP partially operated via P2X7 receptors. ATP, but not other nucleotides, activated nociceptive spikes in meningeal trigeminal nerve fibers providing a rationale for high degradation rate of pro-nociceptive ATP. Pro-nociceptive effect of ATP in meningeal nerves was reproduced by α,β-meATP operating via P2X3 receptors. Collectively, extracellular ATP, which level is controlled by CGRP, can persistently activate trigeminal nerves in meninges which considered as the origin site of migraine headache. These data are consistent with the purinergic hypothesis of migraine pain and suggest new targets against trigeminal pain.     

P2X<Subscript>3</Subscript> Receptor Involvement in Pain States

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X₃ and P2X_2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X₃ receptor in different states of pain.     

The Mechanism of Functional Up-Regulation of P2X3 Receptors of Trigeminal Sensory Neurons in a Genetic Mouse Model of Familial Hemiplegic Migraine Type 1 (FHM-1)

A knock-in (KI) mouse model of FHM-1 expressing the R192Q missense mutation of the Cacna1a gene coding for the α1 subunit of Ca_V2.1 channels shows, at the level of the trigeminal ganglion, selective functional up-regulation of ATP -gated P2X3 receptors of sensory neurons that convey nociceptive signals to the brainstem. Why P2X3 receptors are constitutively more responsive, however, remains unclear as their membrane expression and TRPV1 nociceptor activity are the same as in wildtype (WT) neurons. Using primary cultures of WT or KI trigeminal ganglia, we investigated whether soluble compounds that may contribute to initiating (or maintaining) migraine attacks, such as TNFα, CGRP, and BDNF, might be responsible for increasing P2X3 receptor responses. Exogenous application of TNFα potentiated P2X3 receptor-mediated currents of WT but not of KI neurons, most of which expressed both the P2X3 receptor and the TNFα receptor TNFR2. However, sustained TNFα neutralization failed to change WT or KI P2X3 receptor currents. This suggests that endogenous TNFα does not regulate P2X3 receptor responses. Nonetheless, on cultures made from both genotypes, exogenous TNFα enhanced TRPV1 receptor-mediated currents expressed by a few neurons, suggesting transient amplification of TRPV1 nociceptor responses. CGRP increased P2X3 receptor currents only in WT cultures, although prolonged CGRP receptor antagonism or BDNF neutralization reduced KI currents to WT levels. Our data suggest that, in KI trigeminal ganglion cultures, constitutive up-regulation of P2X3 receptors probably is already maximal and is apparently contributed by basal CGRP and BDNF levels, thereby rendering these neurons more responsive to extracellular ATP.     

Effects of LPS on P2X3 receptors of trigeminal sensory neurons and macrophages from mice expressing the R192Q Cacna1a gene mutation of familial hemiplegic migraine-1

A knockin (KI) mouse model with the R192Q missense mutation in the Cacna1a gene commonly detected in familial hemiplegic migraine was used to study whether trigeminal ganglia showed a basal inflammatory profile that could be further enhanced by the lipopolysaccharide (LPS) toxin. Adenosine-5′-triphosphate (ATP)-gated purinergic ionotropic receptor 3 (P2X3) currents expressed by the large majority of trigeminal sensory neurons were taken as functional readout. Cultured R192Q KI trigeminal ganglia showed higher number of active macrophages, basal release of tumor necrosis factor alpha (TNFα), and larger P2X3 receptor currents with respect to wild type (WT) cells. After 5 h application of LPS in vitro, both WT and R192Q KI cultures demonstrated significant increase in macrophage activation, very large rise in TNFα mRNA content, and ambient protein levels together with fall in TNFα precursor, suggesting potent release of this inflammatory mediator. Notwithstanding the unchanged expression of P2X3 receptor protein in WT or R192Q KI cultures, LPS evoked a large rise in WT neuronal currents that recovered faster from desensitization. Basal R192Q KI currents were larger than WT ones and could not be further augmented by LPS. These data suggest that KI cultures had a basal neuroinflammatory profile that might facilitate the release of endogenous mediators (including ATP) to activate constitutively hyperfunctional P2X3 receptors and amplify nociceptive signaling by trigeminal sensory neurons.     

In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro

Chronic pain is supported by sterile inflammation that induces sensitisation of sensory neurons to ambient stimuli including extracellular ATP acting on purinergic P2X receptors. The development of in vitro methods for drug screening would be useful to investigate cell crosstalk and plasticity mechanisms occurring during neuronal sensitisation and sterile neuroinflammation. Thus, we studied, at single-cell level, membrane pore dilation based on the uptake of a fluorescent probe following sustained ATP-gated P2X receptor function in neurons and non-neuronal cells of trigeminal ganglion cultures from wild-type (WT) and R192Q Ca_V2.1 knock-in (KI) mice, a model of familial hemiplegic migraine type 1 characterised by neuronal sensitisation and higher release of soluble mediators. In WT cultures, pore responses were mainly evoked by ATP rather than benzoyl-ATP (BzATP) and partly inhibited by the P2X antagonist TNP-ATP. P2X7 receptors were expressed in trigeminal ganglia mainly by non-neuronal cells. In contrast, KI cultures showed higher expression of P2X7 receptors, stronger responses to BzATP, an effect largely prevented by prior administration of Ca_V2.1 blocker ω-agatoxin IVA, small interfering RNA (siRNA)-based silencing of P2X7 receptors or the P2X7 antagonist A-804598. No cell toxicity was detected with the protocols. Calcitonin gene-related peptide (CGRP), a well-known migraine mediator, potentiated BzATP-evoked membrane permeability in WT as well as R192Q KI cultures, demonstrating its modulatory role on trigeminal sensory ganglia. Our results show an advantageous experimental approach to dissect pharmacological properties potentially relevant to chronic pain and suggest that CGRP is a soluble mediator influencing purinergic P2X pore dilation and regulating inflammatory responses.     

The role of TRPV1 in different subtypes of dorsal root ganglion neurons in rat chronic inflammatory nociception induced by complete Freund's adjuvant

Background

The aberrant release of the neurotransmitters, glutamate and calcitonin-gene related peptide (CGRP), from trigeminal neurons has been implicated in migraine. The voltage-gated P/Q-type calcium channel has a critical role in controlling neurotransmitter release and has been linked to Familial Hemiplegic Migraine. Therefore, we examined the importance of voltage-dependent calcium channels in controlling release of glutamate and CGRP from trigeminal ganglion neurons isolated from male and female rats and grown in culture. Serotonergic pathways are likely involved in migraine, as triptans, a class of 5-HT₁ receptor agonists, are effective in the treatment of migraine and their effectiveness may be due to inhibiting neurotransmitter release from trigeminal neurons. We also studied the effect of serotonin receptor activation on release of glutamate and CGRP from trigeminal neurons grown in culture.

Results

P/Q-, N- and L-type channels each mediate a significant fraction of potassium-stimulated release of glutamate and CGRP. We determined that 5-HT significantly inhibits potassium-stimulated release of both glutamate and CGRP. Serotonergic inhibition of both CGRP and glutamate release can be blocked by pertussis toxin and NAS-181, a 5-HT_1B/1D antagonist. Stimulated release of CGRP is unaffected by Y-25130, a 5-HT₃ antagonist and SB 200646, a 5-HT_2B/2C antagonist.

Conclusion

These data suggest that release of both glutamate and CGRP from trigeminal neurons is controlled by calcium channels and modulated by 5-HT signaling in a pertussis-toxin dependent manner and probably via 5-HT₁ receptor signaling. This is the first characterization of glutamate release from trigeminal neurons grown in culture.     

Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review

Previously, it was believed that the lumbar intervertebral disc was innervated segmentally by dorsal root ganglion (DRG) neurons via the sinuvertebral nerves. Recently, it was demonstrated using retrograde tracing methods that the lower disc (L5-L6) is innervated predominantly by upper (L1 and L2) DRG neurons via the sympathetic trunks. Furthermore, we investigated the expression of various pain-related molecules such as calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), P2X(3) receptor and vanniloid receptor 1 (VR1) in DRG neurons innervating the disc using a combination of immunostaining with the retrograde tracing method. This review outlines the distribution and immunocytochemical characterization of DRG neurons innervating the disc. Small nociceptive DRG neurons are classified into nerve growth factor (NGF)-dependent neurons and glial cell line-derived neurotrophic factor (GDNF)-dependent neurons and they can be distinguished by their reactivity for CGRP and IB4, respectively. We found that about half of the neurons innervating the disc were CGRP-immunoreactive (-ir), whilst, only 0.6% of the DRG neurons were IB4-positive, thereby indicating that NGF-dependent neurons are the main subpopulation which transmits and modulates nociceptive information from the disc. In addition, we also demonstrated P2X(3)- and VR1-immunoreactivity in DRG neurons innervating the disc and noted that they were mainly localized in NGF-dependent neurons. It is well known that NGF has sensitizing effects on DRG neurons, with a recent study demonstratng the presence of NGF in the painful intervertebral disc. Therefore, it is suggested that NGF is involved in the generation of discogenic low back pain.     

The role of purinergic signaling in the etiology of migraine and novel antimigraine treatment

Etiopathogenesis of migraine involves different structures of the central nervous system: the trigeminal nerve with nuclei located in the brain stem, vascular system, and the cerebral cortex as well as diverse mechanisms and pathological processes. The multidirectional action of purines in different cell types (blood vessels, neurons, and satellite glial cells) and through different types of purinergic receptors contributes to the etiopathogenesis of migraine pain. Adenosine triphosphate (ATP) and its derivatives are involved in initiation and propagation of migrenogenic signals in several ways: they participate in vasomotor mechanism, cortical spreading depression, and in fast transmission or cross-excitation based on the satellite glial cells in trigeminal ganglion. Contribution of purinergic signaling in the conduction of pain is realized through the activation of P1 and P2 receptors expressed widely in the central nervous system: on the neurons and glial cells as well as on the smooth muscles and endothelium in the vascular system. Therefore, the purinergic receptors can be an excellent target for pharmacologists constructing new antimigraine therapeutics. Moreover, the mechanisms facilitating ATP and adenosine degradation may prevent vasodilatation and thus avoid a secondary central sensitization during a migraine attack. Thus, agonists and antagonists of P receptors as well as ecto-enzymes metabolizing nucleotides/nucleosides could gain the growing attention as therapeutic agents.     

Role of sodium ferulate in the nociceptive sensory facilitation of neuropathic pain injury mediated by P2X(3) receptor

Neuropathic pain usually is persistent and no effective treatment. ATP plays an important role in the initiation of pain. P2X(3) receptors are localized in the dorsal root ganglion (DRG) neurons and activated by extracellular ATP. Sodium ferulate (SF) is an active principle from Chinese herbal medicine and has anti-inflammatory activities. This study observed the effects of SF on the nociceptive facilitation of the primary sensory afferent after chronic constriction injury (CCI) mediated by P2X(3) receptor. In this study, the content of ATP in DRG neurons was measured by high-performance liquid chromatography (HPLC). P2X(3) agonist-activated currents in DRG neurons was recorded by the whole-cell patch-clamp skill. The expression of P2X(3) mRNA in DRG neurons was analyzed by in situ hybridization. The ATP content of DRG was increased after CCI. In CCI rats treated with SF, the content of ATP in DRG neurons was reduced. SF decreased the increment of P2X(3) agonist-activated currents and P2X(3) mRNA expression in DRG neurons during CCI. SF may inhibit the initiation of pain and primary afferent sensitization mediated by P2X(3) receptor during CCI.     

Purinoceptor-mediated calcium signaling in primary neuron-glia trigeminal cultures

Receptors for extracellular nucleotides (the P2X-calcium channels and the phospholipase C-coupled P2Y receptors) play key roles in pain signaling, but little is known on their function in trigeminal ganglia, whose hyperactivation leads to the development of migraine pain. Here we characterize calcium signaling via P2X(3) and P2Y receptors in primary mouse neuron-glia trigeminal cultures. Comparison with intact ganglion showed that, in dissociated cultures, sensory neurons retain, at least in part, their physical relationships with satellite glia. RT-PCR indicated expression of P2X(2)/P2X(3) (confirmed by immunocytochemistry) and of all cloned P2Y receptors. Single-cell calcium imaging with subtype-selective P2-agonists/antagonists revealed presence of functional neuronal P2X(3), as well as of ADP-sensitive P2Y(1,12,13) and UTP-activated P2Y(2)/P2Y(4) receptors on both neurons and glia. Calcium responses were much higher in glia, that also responded to UDP, suggesting functional P2Y(6) receptors. To study whether trigeminal ganglia P2 receptors are modulated upon treatment with pro-inflammatory agents, cultures were acutely (up to 3 min) or chronically (24 h) exposed to bradykinin. This resulted in potentiation of algogenic P2X(3) receptor-mediated calcium responses followed by their down-regulation at 24 h. At this exposure time, P2Y receptors responses in satellite glia were instead upregulated, suggesting a complex modulation of P2 receptors in pain signaling.     

大鼠初级感觉神经元P2X3受体的表达及其与SP的关系

目的研究在大鼠初级感觉神经元细胞上P2X3受体的表达情况及其与P物质的关系。方法取SD大鼠背根神经节(DRG)和三叉神经节(TG)固定后切片;用抗P2X3受体抗体和抗SP抗体进行免疫组织化学反应,并通过两种不同的显色方法同时进行P2X3受体和SP的双标。结果P2X3免疫反应阳性细胞主要集中在小细胞和中等细胞(其中在TG,P2X3-ir阳性神经元约占整个细胞的24.8%;在DRG约31.7%的神经元是P2X3-ir阳性),并且在DRG和TG细胞上均存在有P2X3受体和SP共存(TG上的双标细胞占P2X3-ir阳性细胞总数的36.26%,DRG上占46.81%)。结论由于ATP门控阳离子通道受体P2X3本身就与伤害性感受的初级传入有关,而它与SP的共存可提示当组织中的ATP释放时可以通过P2X3受体作用于含SP的伤害性感觉神经末梢上,促使SP释放引起痛觉过敏。     

2′,3′-<Emphasis Type="Italic">O</Emphasis>-Substituted ATP derivatives as potent antagonists of purinergic P2X3 receptors and potential analgesic agents

Blocking membrane currents evoked by the activation of purinergic P2X3 receptors localized on nociceptive neurons represents a promising strategy for the development of agents useful for the treatment of chronic pain conditions. Among compounds endowed with such antagonistic action, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP analogue, whose inhibitory activity on P2X receptors has been previously reported. Based on the results of molecular modelling studies performed with homology models of the P2X3 receptor, novel adenosine nucleotide analogues bearing cycloalkyl or arylalkyl substituents replacing the trinitrophenyl moiety of TNP-ATP were designed and synthesized. These new compounds were functionally evaluated on native P2X3 receptors from mouse trigeminal ganglion (TG) sensory neurons using patch clamp recordings under voltage clamp configuration. Our data show that some of these molecules are potent (nanomolar range) and reversible inhibitors of P2X3 receptors, without any apparent effect on trigeminal GABA_A and 5-HT₃ receptors, whose membrane currents were unaffected by the tested compounds.     

B-Type Natriuretic Peptide-Induced Delayed Modulation of TRPV1 and P2X3 Receptors of Mouse Trigeminal Sensory Neurons

Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,β-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Epoxyeicosatrienoic acids are endogenous regulators of vasoactive neuropeptide release from trigeminal ganglion neurons

Epoxyeicosatrienoic acids (EETs) are bioactive eicosanoids produced from arachidonic acid by cytochrome P450 epoxygenases. We previously described the expression of cytochrome P450-2J epoxygenase in rat trigeminal ganglion neurons and that EETs signaling is involved in cerebrovascular dilation resulting from perivascular nerve stimulation. In this study, we evaluate the presence of the EETs signaling pathway in trigeminal ganglion neurons and their role in modulating the release of calcitonin gene-related peptide (CGRP) by trigeminal ganglion neurons. Liquid chromatography tandem mass spectrometry identified the presence of each of the four EETs regio-isomers within primary trigeminal ganglion neurons. Stimulation for 1 h with the transient receptor potential vanilloid-1 channel agonist capsaicin (100 nmol/L) or depolarizing K(+) (60 mmol/L) increased CGRP release as measured by ELISA. Stimulation-evoked CGRP release was attenuated by 30 min pre-treatment with the EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol/L). K(+) stimulation elevated CGRP release 2.9 ± 0.3-fold above control levels, whereas in the presence of 14,15-EEZE K(+)-evoked CGRP release was significantly reduced to 1.1 ± 0.2-fold above control release (p < 0.01 anova, n = 6). 14,15-EEZE likewise attenuated capsaicin-evoked CGRP release from trigeminal ganglion neurons (p < 0.05 anova, n = 6). Similarly, pre-treatment with the cytochrome P450 epoxygenase inhibitor attenuated stimulation-evoked CGRP release. These data demonstrate that EETs are endogenous constituents of rat trigeminal ganglion neurons and suggest that they may act as intracellular regulators of neuropeptide release, which may have important clinical implications for treatment of migraine, stroke and vasospasm after subarachnoid hemorrhage.     

Calcitonin gene-related peptide enhances release of native brain-derived neurotrophic factor from trigeminal ganglion neurons

Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is coexpressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1-1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose-dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8-37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity.     

Amino acid variations resulting in functional and nonfunctional zebrafish P2X<Subscript>1</Subscript> and P2X<Subscript>5.1</Subscript> receptors

Several zebrafish P2X receptors (zP2X(1), zP2X(2), and zP2X(5.1)) have been reported to produce little or no current although their mammalian orthologs produce functional homomeric receptors. We isolated new cDNA clones for these P2X receptors that revealed sequence variations in each. The new variants of zP2X(1) and zP2X(5.1) produced substantial currents when expressed by Xenopus oocytes, however the new variant of zP2X(2) was still nonfunctional. zP2X(2) lacks two lysine residues essential for ATP responsiveness in other P2X receptors; however introduction of these two lysines was insufficient to allow this receptor to function as a homotrimer. We also tested whether P2X signaling is required for myogenesis or synaptic communication at the zebrafish neuromuscular junction. We found that embryonic skeletal muscle expressed only one P2X receptor, P2X(5.1). Antisense knockdown of P2X(5.1) eliminated skeletal muscle responsiveness to ATP but did not prevent myogenesis or behaviors that require functional transmission at the neuromuscular junction.     

The C-terminal Src Inhibitory Kinase (Csk)-mediated Tyrosine Phosphorylation Is a Novel Molecular Mechanism to Limit P2X3 Receptor Function in Mouse Sensory Neurons

On sensory neurons, sensitization of P2X₃ receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X₃ receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X₃ receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X₃ receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X₃ receptor function and outlines a potential new target for trigeminal pain suppression.ATP-activated P2X₃ receptors are expressed almost exclusively by mammalian sensory neurons to play an important role in the transduction of painful stimuli to the central nervous system (1). Activation of P2X₃ receptors by ATP released during acute and chronic pain is thought to send nociceptive signals to central pain-related networks (2). In view of the multitude of environmental stimuli normally reaching sensory terminals, the question then arises how inappropriate activation of P2X₃ receptors is normally prevented. This process may contribute to suppression of continuous pain sensation in conjunction with central synaptic inhibition.The molecular pathways triggered by algogenic substances and responsible for modulating P2X₃ receptor structure and function remain incompletely understood. This topic is of particular interest because it can provide original clues for novel approaches related to treat pain. The nerve growth factor, NGF,² is one of the most powerful endogenous substances which elicit pain and inflammation via the tyrosine kinase receptor TrkA (3). This neurotrophin stimulates an intracellular cascade that elicits PKC-dependent P2X₃ receptor phosphorylation with ensuing facilitation of receptor currents. Conversely, suppression of NGF signaling powerfully down-regulates P2X₃ receptor function (4). These observations are consistent with the raised NGF levels in acute or inflammatory pain conditions (3). The molecular mechanisms underlying these effects remain, however, unclear.A dynamic balance between tyrosine phosphorylation and dephosphorylation is a major factor controlling the activity of many neurotransmitter receptors (5). TrkA stimulation activates intracellular signaling including Src tyrosine kinases (6) that, in neurons, are important modulators of ligand-gated receptors like nicotinic (7), NMDA receptors (8), and TRPV1 receptors (9). All these receptors are involved in mediating various types of pain in the spinal cord and sensory ganglia. There is, however, no available data on the role of tyrosine phosphorylation on P2X₃ receptor function.The fundamental regulator of Src signaling is the C-terminal Src kinase (Csk) that blocks it via tyrosine phosphorylation (Tyr-527, Refs. 10, 11). We explored whether tyrosine phosphorylation might regulate P2X₃ receptors of sensory neurons by focusing on the P2X₃ C-terminal domain Tyr-393 residue, which is included in a region with significant similarity with the Csk-phosphorylating region of Src. Our data demonstrate that Csk activation induced an increased tyrosine (Tyr-393 residue) P2X₃ receptor phosphorylation with decreased receptor function, observed both in mouse trigeminal sensory neurons as well as a cell expression system. We, thus, propose that Csk-mediated P2X₃ receptor inhibition is a novel mechanism to limit overactivation of P2X₃ receptors.