Comparative Analysis of P2X1, P2X2, P2X3, and P2X4 Receptor Subunits in Rat Nodose Ganglion Neurons

Nodose ganglion (NG) neurons are visceral primary sensory neurons. The transmission and regulation of visceral sensation is mediated mainly by the P2X purinoceptor (P2X receptor). Although the characteristics of different P2X receptor subunits in the NG have been studied previously, comprehensive analyses have not been performed. In this study, we used immunohistochemistry, immunocytochemistry, and whole cell patch clamp techniques to compare the expression and function of P2X1, P2X2, P2X3, and P2X4 receptor subunits in adult rat NG neurons. Polyclonal antibodies against the four P2X subunits labeled different subpopulations of NG neurons. P2X1 and P2X3 were expressed mainly in small-to-medium sized NG neurons, whereas P2X2 and P2X4 were located mostly in medium- and larger-sized NG neurons. Over 36% of NG neurons were P2X3 positive, which was higher than the other three P2X subunits. In addition, different types of currents were recorded from neurons expressing different P2X subunits. The fast type of ATP current was recorded from neurons containing P2X1–4 subunits, the intermediate type of current was recorded from neurons containing the P2X1, P2X3, and P2X4 subunits, the slow type was recorded from neurons expressing P2X1–3, and/or P2X4 subunits, whereas the very slow type was recorded from neurons containing the P2X2 and P2X3 subunits. These comparative results provide an anatomical verification of the different subunits in NG neurons, and offer direct support for the idea that various functional NG populations have distinct responses to ATP, which might be in part due to the different expression profiles of diverse P2X subunits.     

Distribution of immunoreactivity for P2X3, P2X5, and P2X6-purinoceptors in mouse retina

Previous findings have shown that P2X-purinoceptor-mediated signaling pathways regulate the release of ACh in the retina. We previously reported the existence of immunoreactivity for P2X1-, P2X2-, P2X4-, and P2X7-purinoceptors in mouse retina and speculated that P2X2 and P2X7-purinoceptors may modulate the activity of cholinergic amacrine cells. In the present study, we used an immunohistochemical technique to examine whether P2X3-, P2X5, and P2X6-purinoceptors are also important for the modulation of cholinergic amacrine cells in mouse retina. Immunoreactivity for P2X3-, P2X5-, and P2X6-purinoceptors was observed in mouse retina. Immunoreactivity for P2X3- purinoceptors was observed in the dendrites of cholinergic amacrine cells. Immunoreactivity for P2X5-purinoceptors existed in the soma of cholinergic amacrine cells. P2X6-purinoceptor immunoreactivity was not colocalized with the cholinergic amacrine cells. We concluded that, among the three P2X-purinoceptors that were examined, P2X3-purinoceptors seem to affect the function of cholinergic amacrine cells in the mouse retina.     

Substance P Synthesis and Transport in Explants of Nodose Ganglion/Vagus Nerve: Effects of Double Ligation, 2-Deoxyglucose, Veratridine, and Ouabain

Substance P (SP), the widely distributed undecapeptide, is synthesized in cell bodies of vagal sensory ganglia and transported bidirectionally toward the CNS and thoracic and abdominal viscera. In explants of the guinea pig inferior (nodose) vagal sensory ganglion and attached 2 cm of distal vagus nerve, SP is synthesized within the ganglion and transported predominantly distally. The quantity of distal transport is similar to that observed in vivo and provides an index of ongoing synthesis within the ganglion. In this report, the model is further characterized. Double ligation of the explant distal to the ganglion demonstrates that all the transported peptide is derived from the ganglion; there is no evidence of intraaxonal processing of peptide precursor. Approximately 50% of the peptide is in a rapid transport vs. an apparent stationary compartment. Not only transport, but also synthesis, of SP was blocked by 20 mM colchicine. Ongoing SP biosynthesis is dependent on a nutrient medium [medium 199 (M-199)] and is partially inhibited with added fetal bovine serum (FBS; 10%): total explant content in M-199/FBS vs. M-199, 1,785 +/- 101 (n = 8) vs. 2,254 +/- 123 pg (n = 9); p less than 0.02. Addition of 2-deoxyglucose (2-DG) decreased both total SP synthesis and transport (total explant content for 2-DG vs. control, 986 +/- 94 vs. 1,391 +/- 111; p less than 0.05). Medium supplemented with glucose to a final concentration of 600 mg/100 ml or with glucose (300 mg/100 ml) with or without insulin (50 ng/ml) did not alter explant SP content or transport. Veratridine (5 X 10(-6) M) inhibited both SP synthesis and transport; ouabain (10(-4) M) also inhibited synthesis, but less so transport. Tetrodotoxin reversed the effects of veratridine. These studies demonstrate the usefulness of this model, which can examine factors regulating both synthesis and transport of sensory neuropeptides in vitro. The results suggest that SP synthesis/transport may be under tonic inhibition, perhaps by both neural and humoral mechanisms.     

福尔马林致痛对大鼠脊髓和背根神经节的P2X3的影响

目的:探索福尔马林致痛后大鼠脊髓和背根神经节(dorsal root ganglion,DRG)的P2X3表达变化。方法:选取健康成年正常SD大鼠25只,分正常对照组和实验组;实验组为右侧足底皮下给予0.1ml 5%福尔马林,分别观察15min、30min、1h、3h后处死,采用免疫组织化学方法及图像分析技术检测脊髓腰段及L4～6背根节P2X3的表达情况。结果:与正常对照组相比,实验15min、30min、1h组脊髓后角Ⅱ层P2X3表达未见变化,实验3h组可见P2X3表达升高,但未见明显差异;实验15min、30min组DRG神经元P2X3表达未见变化,1h组开始表达上调,3h组表达明显升高,与各组相比有显著性差异。结论:福尔马林致痛能引起脊髓和背根神经节P2X3的表达上调,可能是其产生伤害性作用的机制之一。     

Effect of of ATP on Neurons of the Rat Intact Nodose Ganglion

Under intracellular recording, we studied the effect of ATP on nerve cells of the rat intact nodose ganglion. The resting membrane potential of the examined neurons was, on average, –60.3 ± 1.4 mV (n = 84); among such units, 88% were classified as C cells. Local application of 2 mM ATP to the surface of the ganglion using a modified laminar flow system led to depolarization of neurons by 7.1 ± 0.9 mV, on average (n = 19). A blocker of P2X receptors, PPADS (100 μM), suppressed these depolarization responses, decreasing their amplitude, on average, to 16 ± 3% (n = 3) of the initial value. The obtained data indicate that an overwhelming majority of neurons of the intact nodose ganglion possess functional P2X receptors on their membranes. The absence of the corresponding responses in a considerable part of neurons of intact spinal ganglia [13-15] was, apparently, determined by the fact that P2X receptors in the course of the described experiments had enough time to desensitize before ATP reached the effective concentration.     

Distribution of P2X receptors in the rat adrenal gland

The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7-immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4-7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5-7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland.     

Heterogeneity of the Functional Expression of P2X3 and P2X2/3 Receptors in the Primary Nociceptive Neurons of Rat

The properties and functional expression of the purinergic receptors in small (nociceptive) neurons acutely isolated from the DRG of rat were studied using whole-cell patch-clamp recording. The responses of small DRG neurons to ATP exhibited diverse kinetics and could be subdivided into three types: rapid, slow and mixed kinetics responses. Their affinities to agonists allowed to identify the responsible receptors as P2X3 (fast) and heteromeric P2X2/3 (slow) subtypes. The expression of different responses dramatically varied both on the neuron-to-neuron and animal-to-animal basis. Out of 744 neurons tested 24% of cells demonstrated predominance of functional P2X2/3 receptors, 44% had mixed representation and in 32% of cells P2X3 receptors dominated. All the animals tested (110) could be subdivided into 3 groups: in 19% of animals the response of each cell to ATP was mediated by P2X2/3 receptors, both types of ATP-evoked currents were found in 58% of animals and only in 23% of the animals P2X3 receptors dominated. Our results argue with exclusive role of P2X3 receptors in purinergic signaling in primary nociceptive neurons.     

The In Vitro Synthesis of RNA within the Rat Nodose Ganglion Following Vagotomy

Abstract: This report describes the application of an in vitro labelling procedure for the evaluation of changes in the uptake and incorporation of tritiated nucleotides into RNA of the rat nodose ganglion following crush injury of the cervical vagus nerve. Significant changes in the incorporation into 28S, 18S and 4S RNA were observed at 3 and 9 days after injury which confirms and extends our previous in vivo observations where [³²P]orthophosphate was used as the precursor. An early stimulation in the uptake of nucleotides, which was maximal at 2 days after injury, was also observed. Evidence is presented which indicates that this data reflects a real increase in RNA synthesis within the injured tissue concomitant with an increase in the uptake of nucleotide precursors which may reflect an increase in the nucleotide pool size. The transient nature of the rRNA synthetic responses and their occurrence prior to the peak of the chromatolytic changes suggest that there may be a shift in the distribution of ribosome types resulting in qualitative changes in protein production rather than an overall increase in protein synthesis resulting from an increased ribosome population.     

Characterization of P2X3, P2Y1 and P2Y4 receptors in cultured HEK293-hP2X3 cells and their inhibition by ethanol and trichloroethanol

Membrane currents and changes in the intracellular Ca2+ concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3 receptor (HEK293-hP2X3). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1 and to some extent P2Y4 receptors. P2 receptor agonists induced inward currents in HEK293-hP2X3 cells with the rank order of potency alpha,beta-meATP approximately ATP > ADP-beta-S > UTP. A comparable rise in [Ca2+]i was observed after the slow superfusion of ATP, ADP-beta-S and UTP; alpha,beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to alpha,beta-meATP is due to P2X3 receptor activation, while the ATP-induced rise in [Ca2+]i is evoked by P2Y1 and P2Y4 receptor activation. TCE depressed the alpha,beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca2+]i was much less sensitive to the inhibitory effect of TCE than the current response to alpha,beta-meATP. The present study indicates that in HEK293-hP2X3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X3 receptors and, in addition, moderately interferes with G protein-coupled P2Y1 and P2Y4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.     

Blockage of HCN Channels Inhibits the Function of P2X Receptors in Rat Dorsal Root Ganglion Neurons

Neurochemical Research - Hyperpolarization-activated cyclic nucleotide-gated channels and purinergic P2X receptors play critical roles in the nerve injury-induced pain hypersensitivity. Both HCN...     

P2X2 and P2X3 receptor expression in postnatal and adult rat urinary bladder and lumbosacral spinal cord

P2X receptors mediate the effects of ATP in micturition and nociception. During postnatal maturation, a spinobulbospinal reflex and voluntary voiding replace primitive voiding reflexes. This may involve changes in neuroactive compounds and receptors in bladder reflex pathways. We examined P2X2 and P2X3 receptors in bladder and spinal cord from postnatal (P0-P36, indicating number of days) and adult Wistar rats. Western blot of whole bladders for P2X2 and P2X3 expression was performed. Immunostaining for P2X2 and P2X3 receptors in urothelium and detrusor smooth muscle whole mounts and spinal cord sections was examined. Western blot demonstrated an age-dependent decrease (R(2) = 0.96, P 相似文献   

LRRN3膜蛋白在人胚脊神经节的分布和表达

目的:探讨神经系统富亮氨酸重复超家族成员LRRN3膜蛋白在人胚脊神经节的表达和分布。方法:从人胚脊神经节分离mRNA和蛋白质,用RT-PCR、Western印记杂交法和免疫组织化学方法检测LRRN3膜蛋白表达。结果:LRRN3膜蛋白C端序列RT-PCR扩增产物cDNA在各胎龄脊神经节均有表达,长度约500bp;Western印记杂交结果显示LRRN3膜蛋白存在,分子质量约78 kD;免疫组织化学和免疫荧光组织化学结果表明LRRN3阳性表达细胞均为脊神经节感觉神经细胞,部分神经细胞弱阳性表达,部分未表达。结论:LRRN3膜蛋白在脊神经节神经元的表达,推断与脊神经节感觉神经元的发育、形态构建及损伤后修复有密切的关系。     

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2’,3’-O-(2,4,6-trinitrophenyl)-adenosine 5’-triphosphate), a nonspecific P2X1–7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4–L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.     

Imaging P2X4 receptor subcellular distribution,trafficking, and regulation using P2X4-pHluorin

P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current–voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.     

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT₃ receptors, respectively. We have investigated the changes in P2X and 5-HT₃-mediated signalling in pelvic afferent neurons in mice deficient in P2X₂ and/or P2X₃ subunits by whole-cell recording of L₆–S₂ dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X₃ ^−/− neurons, whereas sustained currents were absent in P2X₂ ^−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X₂/P2X₃ ^Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT₃ receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X ₂ ^−/−, P2X₃ ^−/− and P2X₂/P2X₃ ^Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X₂/P2X₃ ^Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X₂/P2X₃ ^Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X₂/P2X₃ ^Dbl−/− preparations. These data suggest that P2X receptors containing the P2X₂ and/or P2X₃ subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X₂ or P2X₃ receptor genes. This effect is more pronounced when both subunits are absent.     

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X_{1–5, 7}) was detected in all sampled equine tissues, whereas P2X₆ receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X_{1–3, 7} antibodies, and these were used in immunohistochemistry studies. P2X_{1–3, 7} receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X₃ subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X₂ and P2X₃ receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X_1–3 receptor subunits, whereas glial cells were positive for P2X₇ and P2X_{1 and 2} receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.     

包含P2X3亚基的受体在介导痛觉中的作用

包含P2X3亚基的受体为三磷酸腺苷 (ATP)门控的阳离子通道 ,包括P2X3亚基的同源多聚体(P2X3受体 )和异源多聚体 (P2X2 /3受体 )。大量研究表明包含P2X3亚基的受体在介导多种类型痛觉中有重要作用