Antidepressants Inhibit P2X4 Receptor Function: a Possible Involvement in Neuropathic Pain Relief

BACKGROUND: Neuropathic pain is characterized by pain hypersensitivity to innocuous stimuli (tactile allodynia) that is nearly always resistant to known treatments such as non-steroidal anti-inflammatory drugs or even opioids. It has been reported that some antidepressants are effective for treating neuropathic pain. However, the underlying molecular mechanisms are not well understood. We have recently demonstrated that blocking P2X(4 )receptors in the spinal cord reverses tactile allodynia after peripheral nerve injury in rats, implying that P2X(4 )receptors are a key molecule in neuropathic pain. We investigated a possible role of antidepressants as inhibitors of P2X(4 )receptors and analysed their analgesic mechanism using an animal model of neuropathic pain. RESULTS: Antidepressants strongly inhibited ATP-mediated Ca(2+ )responses in P2X(4 )receptor-expressing 1321N1 cells, which are known to have no endogenous ATP receptors. Paroxetine exhibited the most powerful inhibition of calcium influx via rat and human P2X(4 )receptors, with IC(50 )values of 2.45 μM and 1.87 μM, respectively. Intrathecal administration of paroxetine produced a striking antiallodynic effect in an animal model of neuropathic pain. Co-administration of WAY100635, ketanserin or ondansetron with paroxetine induced no significant change in the antiallodynic effect of paroxetine. Furthermore, the antiallodynic effect of paroxetine was observed even in rats that had received intrathecal pretreatment with 5,7-dihydroxytryptamine, which dramatically depletes spinal 5-hydroxytryptamine. CONCLUSION: These results suggest that paroxetine acts as a potent analgesic in the spinal cord via a mechanism independent of its inhibitory effect on serotonin transporters. Powerful inhibition on P2X(4 )receptors may underlie the analgesic effect of paroxetine, and it is possible that some antidepressants clinically used in patients with neuropathic pain show antiallodynic effects, at least in part via their inhibitory effects on P2X(4 )receptors

Behavioral phenotypes of mice lacking purinergic P2X4 receptors in acute and chronic pain assays

A growing body of evidence indicates that P2X receptors (P2XRs), a family of ligand-gated cation channels activated by extracellular ATP, play an important role in pain signaling. In contrast to the role of the P2X3R subtype that has been extensively studied, the precise roles of others among the seven P2XR subtypes (P2X1R-P2X7R) remain to be determined because of a lack of sufficiently powerful tools to specifically block P2XR signaling in vivo. In the present study, we investigated the behavioral phenotypes of a line of mice in which the p2rx4 gene was disrupted in a series of acute and chronic pain assays. While p2rx4-/- mice showed no major defects in pain responses evoked by acute noxious stimuli and local tissue damage or in motor function as compared with wild-type mice, these mice displayed reduced pain responses in two models of chronic pain (inflammatory and neuropathic pain). In a model of chronic inflammatory pain developed by intraplantar injection of complete Freund's adjuvant (CFA), p2rx4-/- mice exhibited attenuations of pain hypersensitivity to innocuous mechanical stimuli (tactile allodynia) and also of the CFA-induced swelling of the hindpaw. A most striking phenotype was observed in a test of neuropathic pain: tactile allodynia caused by an injury to spinal nerve was markedly blunted in p2rx4-/- mice. By contrast, pain hypersensitivity to a cold stimulus (cold allodynia) after the injury was comparable in wild-type and p2rx4-/- mice. Together, these findings reveal a predominant contribution of P2X4R to nerve injury-induced tactile allodynia and, to the lesser extent, peripheral inflammation. Loss of P2X4R produced no defects in acute physiological pain or tissue damaged-induced pain, highlighting the possibility of a therapeutic benefit of blocking P2X4R in the treatment of chronic pain, especially tactile allodynia after nerve injury

Reduced spinal microglial activation and neuropathic pain after nerve injury in mice lacking all three nitric oxide synthases

<p>Abstract</p> <p>Background</p> <p>Several studies have investigated the involvement of nitric oxide (NO) in acute and chronic pain using mice lacking a single NO synthase (NOS) gene among the three isoforms: neuronal (nNOS), inducible (iNOS) and endothelial (eNOS). However, the precise role of NOS/NO in pain states remains to be determined owing to the substantial compensatory interactions among the NOS isoforms. Therefore, in this study, we used mice lacking all three NOS genes (<it>n/i/eNOS^-/-</it>mice) and investigated the behavioral phenotypes in a series of acute and chronic pain assays.</p> <p>Results</p> <p>In a model of tissue injury-induced pain, evoked by intraplantar injection of formalin, both <it>iNOS^-/-</it>and <it>n/i/eNOS^-/-</it>mice exhibited attenuations of pain behaviors in the second phase compared with that in wild-type mice. In a model of neuropathic pain, nerve injury-induced behavioral and cellular responses (tactile allodynia, spinal microglial activation and Src-family kinase phosphorylation) were reduced in <it>n/i/eNOS^-/-</it>but not <it>iNOS^-/-</it>mice. Tactile allodynia after nerve injury was improved by acute pharmacological inhibition of all NOSs and nNOS. Furthermore, in MG-5 cells (a microglial cell-line), interferon-γ enhanced NOSs and Mac-1 mRNA expression, and the Mac-1 mRNA increase was suppressed by L-NAME co-treatment. Conversely, the NO donor, sodium nitroprusside, markedly increased mRNA expression of Mac-1, interleukin-6, toll-like receptor 4 and P2X4 receptor.</p> <p>Conclusions</p> <p>Our results provide evidence that the NOS/NO pathway contributes to behavioral pain responses evoked by tissue injury and nerve injury. In particular, nNOS may be important for spinal microglial activation and tactile allodynia after nerve injury.</p

Intrathecal delivery of PDGF produces tactile allodynia through its receptors in spinal microglia

Neuropathic pain is a debilitating pain condition that occurs after nerve damage. Such pain is considered to be a reflection of the aberrant excitability of dorsal horn neurons. Emerging lines of evidence indicate that spinal microglia play a crucial role in neuronal excitability and the pathogenesis of neuropathic pain, but the mechanisms underlying neuron-microglia communications in the dorsal horn remain to be fully elucidated. A recent study has demonstrated that platelet-derived growth factor (PDGF) expressed in dorsal horn neurons contributes to neuropathic pain after nerve injury, yet how PDGF produces pain hypersensitivity remains unknown. Here we report an involvement of spinal microglia in PDGF-induced tactile allodynia. A single intrathecal delivery of PDGF B-chain homodimer (PDGF-BB) to naive rats produced a robust and long-lasting decrease in paw withdrawal threshold in a dose-dependent manner. Following PDGF administration, the immunofluorescence for phosphorylated PDGF β-receptor (p-PDGFRβ), an activated form, was markedly increased in the spinal dorsal horn. Interestingly, almost all p-PDGFRβ-positive cells were double-labeled with an antibody for the microglia marker OX-42, but not with antibodies for other markers of neurons, astrocytes and oligodendrocytes. PDGF-stimulated microglia in vivo transformed into a modest activated state in terms of their cell number and morphology. Furthermore, PDGF-BB-induced tactile allodynia was prevented by a daily intrathecal administration of minocycline, which is known to inhibit microglia activation. Moreover, in rats with an injury to the fifth lumbar spinal nerve (an animal model of neuropathic pain), the immunofluorescence for p-PDGFRβ was markedly enhanced exclusively in microglia in the ipsilateral dorsal horn. Together, our findings suggest that spinal microglia critically contribute to PDGF-induced tactile allodynia, and it is also assumed that microglial PDGF signaling may have a role in the pathogenesis of neuropathic pain

Purinergic P2Y(6) receptors heterodimerize with angiotensin AT1 receptors to promote angiotensin II-induced hypertension

The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y6 receptor (P2Y6R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR), promoted Ang II–induced hypertension in mice. In mice, deletion of P2Y6R attenuated Ang II–induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein–dependent vascular hypertrophy but reduced β-arrestin–dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y6R heterodimers by the P2Y6R antagonist MRS2578 suppressed Ang II–induced hypertension in mice. Furthermore, P2Y6R abundance increased with age in vascular smooth muscle cells. The increased abundance of P2Y6R converted AT1R-stimulated signaling in vascular smooth muscle cells from β-arrestin–dependent proliferation to G protein–dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II

Modification of neuropathic pain sensation through microglial ATP receptors

Neuropathic pain that typically develops when peripheral nerves are damaged through surgery, bone compression in cancer, diabetes, or infection is a major factor causing impaired quality of life in millions of people worldwide. Recently, there has been a rapidly growing body of evidence indicating that spinal glia play a critical role in the pathogenesis of neuropathic pain. Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors. Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia through P2 purinoceptors expressing on microglia. It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion. In this review, we describe the modification of neuropathic pain sensation through microglial P2X4 and P2X7, with the possibility of P2Y6 and P2Y12 involvement

Recombinant Mouse PAP Has pH-Dependent Ectonucleotidase Activity and Acts through A1-Adenosine Receptors to Mediate Antinociception

Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A1-adenosine receptor (A1R) activation. In this study, we purified the secretory isoform of mouse (m)PAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mPAP dephosphorylated AMP, and to a much lesser extent, ADP at neutral pH (pH 7.0). In contrast, mPAP dephosphorylated all purine nucleotides (AMP, ADP, ATP) at an acidic pH (pH 5.6). The transmembrane isoform of mPAP had similar pH-dependent ectonucleotidase activity. A single intraspinal injection of mPAP protein had long-lasting (three day) antinociceptive properties, including antihyperalgesic and antiallodynic effects in the Complete Freund's Adjuvant (CFA) inflammatory pain model. These antinociceptive effects were transiently blocked by the A1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (CPX), suggesting mPAP dephosphorylates nucleotides to adenosine to mediate antinociception just like human and bovine PAP. Our studies indicate that PAP has species-conserved antinociceptive effects and has pH-dependent ectonucleotidase activity. The ability to metabolize nucleotides in a pH-dependent manner could be relevant to conditions like inflammation where tissue acidosis and nucleotide release occur. Lastly, our studies demonstrate that recombinant PAP protein can be used to treat chronic pain in animal models

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis