Adenosine triphosphate mediates the pain tolerance effect of manual acupuncture at Zusanli (ST36) in mice.

OBJECTIVE: To investigate the mechanisms behind the effects of acupuncture in Traditional Chinese Medicine, we delved into the adenosine triphosphate/peripheral purinergic P2X receptor 3 (ATP/P2X3) receptor signaling system as an indicator of the body's energy state, commonly referred to as "Qi". METHODS: The tail-flick test was utilized to explore the impact of acupuncture on pain tolerance threshold (PTT) in mice, while also assessing adenosine (ADO) levels and adenylate energy charge (EC) at Zusanli (ST36). The study further investigated the dose-dependent effects of acupuncture on PTT and ADO levels at Zusanli (ST36). To shed light on the underlying mechanisms of acupuncture's effects, the study examined the impact of ATP, a P2X3 receptor antagonist, and adenosine disodium on PTT following acupuncture administration. RESULTS: Acupuncture at Zusanli (ST36) led to significant improvements in PTT in mice, with the most effective interventions being twirling for 2 min and needle retention for 28 min. These interventions also resulted in significant increases in ATP levels. The effects of acupuncture were further augmented by administration of different doses of ATP at Zusanli (ST36), and pretreatment with a P2X3 receptor antagonist decreased PTT. Adenylate EC peaked at 30 min after intraperitoneal injection of ATP, and pretreatment with various doses of i.p. ATP 30 min prior to acupuncture increased PTT in a dose-dependent manner. Additionally, pretreatment with an i.p. or intramuscular injection of adenosine disodium enhanced the effects of acupuncture. CONCLUSION: This research provides compelling evidence that ATP is involved in the regulation of PTT through acupuncture, revealing new avenues for achieving enhanced clinical outcomes.

Amplified P2X3 pathway activity in muscle afferent dorsal root ganglion neurons and exercise pressor reflex regulation in hindlimb ischaemia-reperfusion.

Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,ß-methylene ATP injection. P2X3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR.

Purinergic P2X3 receptors in the carotid body as new therapeutic targets for controlling heart failure.

Heart failure is associated with multiple mechanisms, including sympatho-excitation, and is one of the leading causes of death worldwide. Enhanced carotid body chemoreflex function is strongly related to excessive sympathetic nerve activity and sleep-disordered breathing in heart failure. How to reduce the excitability of the carotid body is still scientifically challenging. Both clinical and experimental evidence have suggested that targeting purinergic receptors is of great potential to combat heart failure. In a recent study, Lataro et al. (Lataro et al. in Nat Commun 14:1725, 5) demonstrated that targeting purinergic P2X3 receptors in the carotid body attenuates the progression of heart failure. Using a series of molecular, biochemical, and functional assays, the authors observed that the carotid body generates spontaneous, episodic burst discharges coincident with the onset of disordered breathing in male rats with heart failure, which was generated by ligating the left anterior descending coronary artery. Moreover, P2X3 receptor expression was found to be upregulated in the petrosal ganglion chemoreceptive neurons of rats with heart failure. Of particular note, treatment with a P2X3 antagonist rescued pathological breathing disturbances, abolished episodic discharges, reinstated autonomic balance, attenuated cardiac dysfunction, and reduced the immune cell response and plasma cytokine levels in those rats.

Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions.

In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

Estrogen inhibits bladder overactivity in rats with cyclophosphamide-induced cystitis via downregulating the expression of P2X3 receptors in bladder epithelium cells.

AIMS: The therapeutic effect of estrogen on interstitial cystitis/bladder pain syndrome is unclear. We aim to explore the effect of estrogen on bladder overactivity in rats with cyclophosphamide-induced cystitis and its underlying mechanism. METHODS: In vivo cystometry was used to determine the effect of estrogen on bladder excitability. The effect of estrogen on the expression of P2X3 receptors in bladder epithelium was detected by real-time polymerase chain reaction and western blot. Effect of P2X3 receptors in bladder urothelium on stretch-released adenosine triphosphate was performed by a Flexcell FX5000 Compression system and an Enzyme-Linked Immunosorbent Assay Kit. RESULTS: Estrogen deprivation significantly increased the urinary frequency, while supplementation with diarylpropionitrile (DPN), an estrogen receptor ß (ERß) agonist, alleviated the urinary frequency. 17ß-Estradiol and DPN decreased the expression of P2X3 receptors in urothelium cells which was partially inhibited by ERß antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol. Meanwhile, inhibiting the expression of P2X3 receptors by ERß agonist or antagonizing the function of P2X3 receptors by selective P2X3 receptor antagonist AF-353 or A-317491 significantly reduced the stretch-released ATP from urothelium cells. CONCLUSIONS: Estrogen has a direct effect on the regulation of bladder overactivity in rats with cyclophosphamide-induced cystitis by downregulating the expression of bladder epithelial P2X3 receptors through ERß and reducing the adenosine triphosphate released from urothelium during bladder filling, thereby inhibiting the generation of the micturition reflex.

Effect of lappaconitine on neuropathic pain mediated by retrograde transport of P2X3 receptor in dorsal root ganglion neurons of rats with chronic constriction injury of the sciatic nerve / 中华创伤杂志

Objective:To investigate the effect of lappaconitine (LA) on neuropathic pain (NPP) mediated by retrograde transport of purinergic P2X3 receptor (P2X3R) in dorsal root ganglion (DRG) neurons of rats with chronic constriction injury (CCI) of the sciatic nerve.Methods:Seventy-two male healthy SD rats were selected to construct the NPP model following CCI of the sciatic nerve by ligating the right sciatic nerve. according to the random number table method. The rats were divided into CCI group, CCI+LA group and normal control group according to the random number table method, with 24 rats in each group. In normal control group, the right sciatic nerve was exposed without ligation. In CCI+LA group, the rats were given 2 g/L LA (ie, 4 mg/kg intravenously for once a day for one day only) after the same treatment as CCI group. Other two groups were injected with the identical amount of normal saline in the same way. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were evaluated before injury and at 2, 6, 12 and 24 hours after injury to evaluate the symptoms of neuralgia caused by nerve injury. The proximal and distal nerve fragments were collected in the three groups at 2, 6, 12 and 24 hours after injury. Western blotting was applied to analyze the expression of P2X3R at 2, 6, 12 and 24 hours after injury and the expression of neurotrophic factor (NGF) and tyrosine kinase receptor A (TrkA) at 24 hours after injury to evaluate the effect of LA on P2X3R, NGF and TrkA.Results:There were insignificant differences in MWT and TWL among all groups before injury (all P>0.05). Compared with normal control group, MWT and TWL were significantly decreased in CCI group and CCI+LA group at 2, 6, 12 and 24 hours after injury (all P<0.05 or 0.01). There were insignificant differences in MWT and TWL between CCI group and CCI+LA group at 2 and 6 hours after injury (all P>0.05), while MWT and TWL were significantly higher in CCI+LA group than those in CCI group at 12 and 24 hours after injury (all P<0.05 or 0.01). In the proximal sciatic nerve segment, Western blotting showed similar levels of P2X3R among all groups at 2, 6, 12 and 24 hours after injury (all P>0.05). In the distal sciatic nerve segment, Western blotting showed higher expression of P2X3R in CCI group than that in normal control group at 2, 6, 12, 24 hours after injury (all P<0.01), higher expression of P2X3R in CCI+LA group than that in normal control group at 2, 6 and 12 hours after injury (all P<0.05), similar expression of P2X3R expression between CCI+LA group and normal control group at 24 hours after injury ( P>0.05), similar expression of P2X3R between CCI group and CCI+LA group at 2 and 6 hours after injury (all P>0.05), and lower expression of P2X3R in CCI+LA group than that in CCI group at 12 and 24 hours after injury ( P<0.05 or 0.01). In the proximal and distal nerve fragments, the expression of NGF was lower in normal control group than that in CCI group and CCI+LA group ( P<0.05 or 0.01), but was similar between CCI group and CCI+LA group at 24 hours after injury ( P>0.05). In the proximal and distal nerve fragments, there were insignificant differences in the expression of TrkA among all groups at 24 hours after injury (all P>0.05). Conclusion:Early LA treatment after injury can alleviate mechanical and thermal hyperalgesia in NPP rats, which may be related to the reduction of P2X3R retrograde transport in DRG neuron axonal.

Regulatory effect of mild moxibustion on P2X3 receptors in spinal cord, anterior cingulate cortex and thalamic ventral posterolateral nucleus of rats with IBS visceral hyperalgesia / 针灸推拿医学（英文版）

Objective: To observe the therapeutic effect of mild moxibustion on irritable bowel syndrome (IBS) visceral hyperalgesiamodel rats and its regulatory effect on P2X3 receptors in the spinal cord, anterior cingutate cortex (ACC) and thalamic ventral posterolateral nucleus (VPL). Methods: Thirty 8-day-old newborn rats were randomly divided into a normal group (n=6) and a modeling group (n=24) according to the completely random number table method. Rats in the normal group were bred routinely, and those in the modeling group were subjected to preparing IBS chronic visceral hyperalgesia model using colorectal distention (CRD) in stimulation method. Rats successfully modelled were re-divided into a model group, a mild moxibustion group, a P2X3 receptor antagonist group, and a normal saline group according to the completely random number table method with 6 rats in each group. Rats in each group received corresponding interventions from the 37-day old, once a day for 7 consecutive days. Immunohistochemistry and Western blot assays were used to detect P2X3 protein expressions in the spinal cord, ACC and VPL of rats. Results: Under different intensities of CRD stimulation, the abdominal withdrawal reflex (AWR) scores of the model group were significantly increased versus the normal group (all P<0.05); the AWR scores of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group (all P<0.01). The P2X3 protein expressions in rat spinal cord, ACC and VPL tissues of the model group were significantly increased versus the normal group (all P<0.01); the P2X3 protein expressions in rat spinal cord, ACC and VPL tissues of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group (all P<0.01). Conclusion: Mild moxibustion can inhibit the P2X3 receptor expressions in the spinal cord, ACC, and VPL tissues of IBS visceral hyperalgesia model rats, which may be the mechanism of mild moxibustion in relieving the central sensitization of rats with IBS visceral hyperalgesia.

Voiding Dysfunction in Old Male Rats Associated With Enlarged Prostate and Irregular Afferent-Triggered Reflex Responses.

PURPOSE: This study was conducted to evaluate the hypothesis that an enlarged prostate in old rats may lead to complications associated with voiding dysfunction involving ionotropic P2X2/3-type purinergic receptors. METHODS: Intact animals were divided into male young (MYR; 8-10 weeks old) and male old (MOR; 20 months old) rats. The animals underwent simultaneous detrusor electromyography (EMG) and suprapubic cystometry (CMG) under urethane anesthesia. Immunofluorescence techniques were used to evaluate prostatic autonomic innervation and P2X3R expression in bladder urothelial cells. The functional role of P2X3R was characterized by intramuscular application of AF-353, a selective P2X2/3R antagonist. RESULTS: The prostate index significantly increased in MOR, suggestive of an enlarged prostate affecting micturition patterns. Significant EMG and CMG differences were found between MYR and MOR. Higher immunoreactivity for P2X2/3R in the urothelial layer and for prostatic neurofilaments was seen in MOR. Systemic inhibition of P2X2/3R had minimal effects on MYR responsiveness, but improved voiding function in MOR with a marked decrease of intravesical pressure and bladder contractile responses. CONCLUSION: The data support the hypothesis that an enlarged prostate in MOR may contribute to voiding dysfunction involving activation of P2X2/3R, which enhances a prostate-bladder reflex. This reflex may increase bladder afferent transmission and activation of increased prostate innervation, leading to voiding dysfunction.

Electrophysiological Evidence for Functional Astrocytic P2X3 Receptors in the Mouse Trigeminal Caudal Nucleus.

Recently, we reported that astrocytes in the trigeminal caudal nucleus (Vc) of the brain stem express a purinergic receptor P2X3, which is involved in the craniofacial pathologic pain. Although we observed protein expression of P2X3 receptors (P2X3 Rs) in the astrocyte of the Vc, it is still unclear that astrocyte has functional P2X3Rs in Vc. To address this issue, we recorded asrtocytic P2X3Rs by using whole cell voltage-clamp recording in the Vc of the GFAP-GFP mice, which was used as a guide to astrocytes with green fluorescence. While measuring voltage ramp-induced astrocytic membrane current, we found the amplitude of the current was increased when we applied P2-purinoreceptor agonist, α,ß-meATP. This increase was blocked by co-application of A317491, P2X3R antagonist. These results demonstrate that astrocytes in the Vc express functional P2X3Rs, which might be critical in craniofacial pathologic pain.

Electrophysiological Evidence for Functional Astrocytic P2X₃ Receptors in the Mouse Trigeminal Caudal Nucleus

Recently, we reported that astrocytes in the trigeminal caudal nucleus (Vc) of the brain stem express a purinergic receptor P2X₃, which is involved in the craniofacial pathologic pain. Although we observed protein expression of P2X₃ receptors (P2X₃ Rs) in the astrocyte of the Vc, it is still unclear that astrocyte has functional P2X₃Rs in Vc. To address this issue, we recorded asrtocytic P2X₃Rs by using whole cell voltage-clamp recording in the Vc of the GFAP-GFP mice, which was used as a guide to astrocytes with green fluorescence. While measuring voltage ramp-induced astrocytic membrane current, we found the amplitude of the current was increased when we applied P2-purinoreceptor agonist, α,β-meATP. This increase was blocked by co-application of A317491, P2X₃R antagonist. These results demonstrate that astrocytes in the Vc express functional P2X₃Rs, which might be critical in craniofacial pathologic pain.

P2X3 receptor-mediated visceral hyperalgesia and neuronal sensitization following exposure to PTSD-like stress in the dorsal root ganglia of rats.

BACKGROUND: Patients with posttraumatic stress disorder (PTSD) often share co-morbidity with chronic pain conditions. Recent studies suggest a role of P2X3 receptors and ATP signaling in pain conditions. However, the underlying mechanisms of visceral hyperalgesia following exposure to PTSD-like stress conditions remain unclarified. METHODS: The behavior and hormones relevant for PTSD were studied. Visceromotor responses (VMR) and the abdominal withdrawal reflexes (AWR) to colorectal distention (CRD) were recorded to determine P2X3-receptor-mediated alteration of hyperalgesia following single-prolonged stress (SPS) exposure. Immunofluorescence, Western blotting, and patch-clamp were used. KEY RESULTS: The escape latency, adrenocorticotropic hormone and cortisol were increased on days 7-14. Visceromotor responses and AWR was reduced at day 1 in SPS rats but increased to higher levels than in controls after exposure to day 7. Intrathecal administration of the P2X3-receptor antagonist TNP-ATP abolished the CRD response. Based on immunofluorescence and Western blotting analysis, SPS-treated rats exhibited reduced P2X3 expression in dorsal root ganglia (DRG) after day 1 compared with controls. P2X3 expression in DRG was enhanced on day 7 after SPS and the increase of the P2X3 expression was maintained on day 14 and 21 compared with controls. The P2X3-receptor agonist α,ß-me ATP (10 µM) induced a fast desensitizing inward current in DRG neurons of both control and SPS-treated rats. The average peak current densities in SPS-treated group were increased 3.6-fold. TNP-ATP (100 nM) markedly blocked all fast α,ß-me ATP-induced inward currents in the DRG neurons both in control and SPS-treated rats. CONCLUSIONS & INFERENCES: The data indicate an important role of P2X3 signaling in visceral hyperalgesia following PTSD-like stress.