Neurotropin alleviates rat osteocarcinoma pain via P2X3 receptor activation in the midbrain periaqueductal gray.

OBJECTIVES: Clinically effective analgesia treatment for patients afflicted with osteocarcinoma lessens the intensity of pain. The midbrain periaqueductal gray (PAG) plays a critical role in pain modulation, and activation of P2X3 receptors in this region mediates pain processing. Neurotropin is a small molecule drug used for analgesic treatment of a number of chronic pain conditions. The present study aims at determining whether P2X3 receptor activation in PAG is responsible for the analgesic effect of neurotropin in rats with osteocarcinoma pain. MATERIALS AND METHODS: The tibia of female Sprague-Dawley rats was inoculated with breast carcinoma cells to establish the osteocarcinoma pain model. The effects of intraperitoneal injection of 6, 12, and 18 neurotropin units (NU)/kg on pain threshold and receptor expression of P2X3 in the ventrolateral PAG (vlPAG) were assessed. The P2X3 receptor antagonist A-317491 (1.5 nmol/0.3 µl) was administered into vlPAG with a high-dose neurotropin (18 NU/kg) to determine the role of this receptor in the analgesic effect. RESULTS: The pain thresholds of the rats with osteocarcinoma pain continuously decreased, whereas P2X3 receptor expression in vlPAG only slightly increased after osteocarcinoma cell inoculation. Neurotropin substantially elevated the pain threshold and P2X3 receptor expression in vlPAG in a dose-dependent manner. A-317491 microinjection into vlPAG significantly reduced the analgesic effects of neurotropin in the rats with osteocarcinoma pain. CONCLUSION: Through these findings, it is shown that vlPAG P2X3 receptor activation participates in neurotropin-mediated analgesia mechanism in osteocarcinoma pain.

Neferine alleviates P2X3 receptor in rat dorsal root ganglia mediated neuropathic pain.

Chronic neuropathic pain is caused by tissue damage or nervous system inflammation and is characterized by sensitivity to painful stimuli. P2X3 receptors play an important role in facilitating pain transmission. Neferine is a bisbenzylisoquinline alkaloid isolated from seed embryos of lotus, which has anti-inflammatory and anti-oxidation pharmacological functions. The present research investigated whether neferine relieves neuropathic pain related to the P2X3 receptor in rat dorsal root ganglia (DRGs). Chronic contraction injury (CCI) in rats was used as a model for neuropathic pain. The results indicated that the expression of P2X3 receptor was significantly increased in the DRGs of CCI rats and that mechanical allodynia and thermal hyperalgesia were also enhanced in CCI rats. Neferine markedly lowered the upregulated P2X3 receptor and interleukin-1beta, inhibited the phosphorylation and activation of ERK1/2 in the DRGs of CCI rats, and relieved neuropathic pain. Therefore, neferine alleviates neuropathic pain by downregulating the expression of P2X3 receptor.

The therapeutic potential of purinergic signalling.

This review is focused on the pathophysiology and therapeutic potential of purinergic signalling. A wide range of diseases are considered, including those of the central nervous system, skin, kidney, musculoskeletal, liver gut, lower urinary tract, cardiovascular, airways and reproductive systems, the special senses, infection, diabetes and obesity. Several purinergic drugs are already on the market, including P2Y12 receptor antagonists for stroke and thrombosis, P2Y2 receptor agonists for dry eye, and A1 receptor agonists for supraventricular tachycardia. Clinical trials are underway for the use of P2X3 receptor antagonists for the treatment of chronic cough, visceral pain and hypertension, and many more compounds are being explored for the treatment of other diseases. Most experiments are 'proof of concept' studies on animal or cellular models, which hopefully will lead to further clinical trials. The review summarises the topic, mostly referring to recent review articles.

Effect of A-317491 delivered by glycolipid-like polymer micelles on endometriosis pain.

Endometriosis is a common gynecological disease with a lack of effective clinical treatment. Current therapy often results in endometriosis pain recurrence and serious side effects. P2X3 receptor, an adenosine triphosphate (ATP)-gated ion channel, might be implicated in endometriosis pain. In this study, chitosan oligosaccharide-g-stearic acid (CSOSA) polymer micelles-coated nanostructured lipid carriers (NLCs) were developed as a novel delivery system for A-317491, a selective P2X3 receptor antagonist for endometriosis pain therapy. A-317491-loaded NLC (NLC/A-317491) could be coated by CSOSA micelles to form CSOSA/NLC/A-317491 nanoparticles. Pheochromocytoma PC12 cells, which highly expressed P2X3 receptors, were used as a cell model, and the CSOSA/NLC/A-317491 partly blocked the Ca2+ influx induced by ATP stimulation. In nude mouse and rat endometriotic models, CSOSA/NLC could accumulate into endometriotic lesions after vein injection. In endometriotic rats, CSOSA/NLC/A-317491 reversed mechanical and heat hyperalgesia with long-term efficacy, which might be attributed to the massive CSOSA/NLC/A-317491 distribution in the endometriotic lesions. In conclusion, A-317491 delivered by CSOSA/NLC nanoparticles attenuated endometriosis pain in rats, and CSOSA/NLC/A-317491 could be used as an effective treatment strategy for P2X3-targeted therapy in endometriosis pain.

P2X receptors up-regulate the cell-surface expression of the neuronal glycine transporter GlyT2.

Glycinergic inhibitory neurons of the spinal dorsal horn exert critical control over the conduction of nociceptive signals to higher brain areas. The neuronal glycine transporter 2 (GlyT2) is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft and its activity modulates intra and extracellular glycine concentrations. In this report we show that the stimulation of P2X purinergic receptors with ßγ-methylene adenosine 5'-triphosphate induces the up-regulation of GlyT2 transport activity by increasing total and plasma membrane expression and reducing transporter ubiquitination. We identified the receptor subtypes involved by combining pharmacological approaches, siRNA-mediated protein knockdown, and dorsal root ganglion cell enrichment in brainstem and spinal cord primary cultures. Up-regulation of GlyT2 required the combined stimulation of homomeric P2X3 and P2X2 receptors or heteromeric P2X2/3 receptors. We measured the spontaneous glycinergic currents, glycine release and GlyT2 uptake concurrently in response to P2X receptor agonists, and showed that the impact of P2X3 receptor activation on glycinergic neurotransmission involves the modulation of GlyT2 expression or activity. The recognized pro-nociceptive action of P2X3 receptors suggests that the fine-tuning of GlyT2 activity may have consequences in nociceptive signal conduction.

A317491 relieved HIV gp120-associated neuropathic pain involved in P2X3 receptor in dorsal root ganglia.

Glycoprotein 120 (gp120) is an HIV envelope glycoprotein. Gp120 can directly stimulate the primary sensory afferent neurons and cause hyperalgesia. The P2X3 receptor in dorsal root ganglia (DRG) is involved in the transmission of pain. In this study, we aimed to explore the role of the P2X3 receptor in gp120-induced neuropathic pain. Our data showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared to those in the control group. The expression levels of the P2X3 mRNA and protein in rats treated with gp120 were higher than those in the control group. The P2X3 antagonist A317491 decreased mechanical hyperalgesia and thermal hyperalgesia and the up-regulated expression levels of P2X3 mRNA and protein in rats treated with gp120. A317491 decreased ERK1/2 phosphorylation levels in the gp120-treated rat DRG. In addition, P2X3 agonist α,ß-methylene ATP (α,ß-meATP)-activated currents in DRG neurons cultured with gp120 were higher than those in control neurons. The inhibitory effect of A317491 on α,ßme-ATP-induced currents in DRG neurons from the gp120-treated neurons was larger than that for control neurons. Molecular docking data showed that A317491 may be acted in the gp120 protein to inhibit the gp120 initiated the P2X3 activation, decrease the sensitizing DRG primary afferents and reduce the signal transmission of neuropathic pain in gp120-treated rats. Therefore, the inhibition of the P2X3 receptor in rat DRG neurons relieved gp120-induced mechanical hyperalgesia.

Effect of Electroacupuncture at Hegu (LI 4) on P2X2 and P2X3 Receptors in Trigeminal Ganglion in Pulpalgia Rats / 上海针灸杂志

Objective To explore the effect of electroacupuncture at Hegu (LI 4) on the expressions of P2X2 and P2X3 receptors in experimental pulpalgia rats.Method Forty-two male SD rats were randomized into a normal group (group N), a control group (group C), a pulpalgia model group (group M), an antagonist group (group A), an electroacupuncture group (group E), and an antagonist+electroacupuncture group (group AE), 7 rats in each group. Group N didn’t receive any interventions; group C received injection of normal saline into pulp cavity of the same dose as the injection in group M, and the cavity was then blocked by dental fillings 5-6 min later; in group M, maxillary first and second molar teeth were drilled (drill bit of 1 mm in diameter) to expose pulp and lipopolysaccharide (LPS) solution 5μg/μL was injected into the holes (1~3μL for each hole), and the holes were then covered by dental fillings 5-6 min later; group A received the same modeling method as that in group M, but A-317491 was injected together with LPS (0.5 mg/kg); group E received electroacupuncture at bilateral Hegu (LI 4) with needles retained for 30 min, once a day, totally for 3 times; group AE received the same electroacupuncture intervention after receiving the same treatments as that in group A. The rats’ behaviors and weight were observed for 30 min after intervention each day. The rats were sacrificed on the 4th day, and the mRNA expressions of P2X2 and P2X3 receptors andβ-actin in trigeminal ganglion were detected by using RT-PCR. The mRNA expressions were then compared among the groups.Result The behavioral changes in group M, E, and AE were more significant than that in group C and N (P<0.01); the behavioral changes in group A, E, and AE were less significant than that in group M (P<0.01). The weight in group C was significantly lower than that in group N (P<0.01); the weights in group M, A, E and AE were significantly lower than that in group C and N (P<0.01); the weights in group E and AE were significantly higher than that in group M and A (P<0.01,P<0.05); the weight in group A was slightly higher than that in group M (P<0.05); the weights in group AE was significantly higher than that in group E (P<0.01). The mRNA expressions of P2X2 receptor in group M, A, and AE were significantly higher than that in group N and C (P<0.01); the mRNA expressions of P2X2 receptor in group A, E, and AE were lower than that in group M (P<0.05); the mRNA expression of P2X2 receptor in group A was lower than that in group E (P<0.05); the mRNA expression of P2X2 receptor in group E was higher than that in group AE (P<0.05). The mRNA expressions of P2X3 receptor in group M, A, and E were significantly higher than that in group C (P<0.05) and group N (P<0.01); the mRNA expressions of P2X3 receptor in group A, E, and AE were significantly lower than that in group M (P<0.01); the mRNA expression of P2X3 receptor in group AE was markedly lower than that in group E (P<0.01).Conclusion The expressions of P2X2 and P2X3 receptors in trigeminal ganglion were increased in LPS-induced pulpalgia rats. Electroacupuncture at Hegu (LI 4) and injection of A-317491 both can down-regulate the mRNA expressions of P2X2 and P2X3 receptors, which is plausibly the action mechanism of electroacupuncture at Hegu (LI 4) in analgesia.

Electroacupuncture and A-317491 depress the transmission of pain on primary afferent mediated by the P2X3 receptor in rats with chronic neuropathic pain states.

P2X is a family of ligand-gated ion channels that act through adenosine ATP. The P2X3 receptor plays a key role in the transmission of neuropathic pain at peripheral and spinal sites. Electroacupuncture (EA) has been used to treat neuropathic pain effectively. To determine the role of EA in neuropathic pain mediated through the P2X3 receptor in dorsal root ganglion neurons and the spinal cord, a chronic constriction injury (CCI) model was used. Sprague-Dawley rats were divided into four groups: sham CCI, CCI, CCI plus contralateral EA, and CCI plus ipsilateral EA. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded. Furthermore, the expression of the P2X3 receptor was evaluated through Western blotting and immunofluorescence. The effects of EA and A-317491 were investigated through the whole-cell patch-clamp method and intrathecal administration. Our results show that the MWT and TWL of EA groups were higher than those in the CCI group, whereas the expression of the P2X3 receptor was lower than that in the CCI group. However, no significant difference was detected between the two EA groups. EA depressed the currents created by ATP and the upregulation of the P2X3 receptor in CCI rats. Additionally, EA was more potent in reducing mechanical allodynia and thermal hyperalgesia when combined with A-317491 through intrathecal administration. These results show that both contralateral and ipsilateral EA might inhibit the primary afferent transmission of neuropathic pain induced through the P2X3 receptor. In addition, EA and A-317491 might have an additive effect in inhibiting the transmission of pain mediated by the P2X3 receptor.