Contribution of nerve growth factor to upregulation of P2X₃ expression in DRG neurons of rats with femoral artery occlusion

Femoral artery occlusion augments the sympathetic nerve and pressor responses to muscle contraction and muscle metabolites injected into the arterial blood supply of the hindlimb muscles in rats. The underlying mechanism by which these reflex responses are enhanced after muscle vascular insufficiency is unclear. Purinergic P2X(3) receptor has been reported to contribute to the metabolic component of the exercise pressor reflex. Thus the purpose of this study was to examine if chronic femoral occlusion would alter the expression of P2X(3) in dorsal root ganglion (DRG) neurons of rats. Also, P2X(3)-mediated sympathetic responsiveness was examined after femoral occlusion. In addition, the role played by nerve growth factor (NGF) in regulating the expression and response of P2X(3) was examined. Western blot analysis showed that 24 h of femoral ligation increased the levels of P2X(3) (optical density: 0.93 ± 0.07 in control and 1.37 ± 0.10 after occlusion; P < 0.05 vs. control). The fluorescence immunohistochemistry further demonstrated that the occlusion elevated P2X(3) expression in DRG neurons (percentage of P2X(3)-positive cells: 33 ± 3% in control and 51 ± 3% in occlusion; P < 0.05 vs. control). Furthermore, the results showed that responses of renal sympathetic nerve activity and blood pressure to stimulation of P2X were greater in occluded rats than responses in control rats by injection of α,β-methylene ATP into the arterial blood supply of the hindlimb muscle. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased P2X(3) (optical density: 0.98 ± 0.12 in control and 1.37 ± 0.16 with NGF; P < 0.05 vs. control). The pressor response to injection of α,β-methylene ATP was increased in the rats with NGF infusion. Likewise, blocking NGF attenuated exaggeration of the reflex response induced by α,β-methylene ATP in occluded rats. The findings of this study suggest that the levels of P2X(3) in primary afferent neurons are upregulated as the blood supply to the hindlimb is deficient under ischemic conditions, leading to augmentation of the muscle reflex. NGF is closely related to increases in P2X(3) receptor expression and response.     

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.     

P2X2 receptors differentiate placodal vs. neural crest C-fiber phenotypes innervating guinea pig lungs and esophagus

The lungs and esophagus are innervated by sensory neurons with somata in the nodose, jugular, and dorsal root ganglion. These sensory ganglia are derived from embryonic placode (nodose) and neural crest tissues (jugular and dorsal root ganglia; DRG). We addressed the hypothesis that the neuron's embryonic origin (e.g., placode vs. neural crest) plays a greater role in determining particular aspects of its phenotype than the environment in which it innervates (e.g., lungs vs. esophagus). This hypothesis was tested using a combination of extracellular and patch-clamp electrophysiology and single-cell RT-PCR from guinea pig neurons. Nodose, but not jugular C-fibers innervating the lungs and esophagus, responded to alpha,beta-methylene ATP with action potential discharge that was sensitive to the P2X3 (P2X2/3) selective receptor antagonist A-317491. The somata of lung- and esophagus-specific sensory fibers were identified using retrograde tracing with a fluorescent dye. Esophageal- and lung-traced neurons from placodal tissue (nodose neurons) responded similarly to alpha,beta-methylene ATP (30 microM) with a large sustained inward current, whereas in neurons derived from neural crest tissue (jugular and DRG neurons), the same dose of alpha,beta-methylene ATP resulted in only a transient rapidly inactivating current or no detectable current. It has been shown previously that only activation of P2X2/3 heteromeric receptors produce sustained currents, whereas homomeric P2X3 receptor activation produces a rapidly inactivating current. Consistent with this, single-cell RT-PCR analysis revealed that the nodose ganglion neurons innervating the lungs and esophagus expressed mRNA for P2X2 and P2X3 subunits, whereas the vast majority of jugular and dorsal root ganglia innervating these tissues expressed only P2X3 mRNA with little to no P2X2 mRNA expression. We conclude that the responsiveness of C-fibers innervating the lungs and esophagus to ATP and other purinergic agonists is determined more by their embryonic origin than by the environment of the tissue they ultimately innervate.     

Circulating plasma serine208‐phosphorylated troponin T levels are indicator of cardiac dysfunction

Heart failure (HF) following myocardial infarction (MI) is characterized by progressive alterations of left ventricular (LV) structure and function, named LV remodelling. Although several risk factors such as infarct size have been identified, HF remains difficult to predict in clinical practice. Recently, using phosphoproteomic technology, we found that serine²⁰⁸‐phosphorylated troponin T (P‐Ser²⁰⁸‐TnT) decreases in LV of HF rats. Our aim was to determine the performance of P‐Ser²⁰⁸‐TnT as plasma biomarker of HF compared to conventional cardiac biomarkers such as B‐type natriuretic peptide (BNP), cardiac troponin I (cTnI), C‐reactive protein (CRP) or tissue inhibitor of metalloproteinase I (TIMP‐1) measured by x‐MAP technology, as well as its capacity to reflect a pharmacological improvement of HF. We observed a significant increase of BNP, TnT and cTnI levels and a significant decrease of P‐Ser²⁰⁸‐TnT and TIMP‐1 in the plasma of 2‐month‐MI rats compared with control rats with no modulation of CRP level. Circulating levels of P‐Ser²⁰⁸‐TnT were shown to be associated with most of the echocardiographic and haemodynamic parameters of cardiac function. We verified that the decrease of P‐Ser²⁰⁸‐TnT was not because of an excess of phosphatase activity in plasma of HF rats. Two‐month‐MI rats treated with the heart rate reducing agent ivabradine had improved LV function and increased plasma levels of P‐Ser²⁰⁸‐TnT. Thus, circulating phosphorylated troponin T is a highly sensitive biological indicator of cardiac dysfunction and has the potentiality of a new biomarker of HF post‐MI, and of a surrogate marker for the efficacy of a successful treatment of HF.     

Analgesic effect of electroacupuncture on chronic neuropathic pain mediated by P2X3 receptors in rat dorsal root ganglion neurons

Adenosine 5'-triphosphate disodium (ATP) gated P2X receptors, especially the subtype P2X(3), play a key role in transmission of pain signals in neuropathic pain, ATP has been documented to play a significant role in the progression of pain signals, suggesting that control of these pathways through electroacupuncture (EA) is potentially an effective treatment for chronic neuropathic pain. EA has been accepted to effectively manage chronic pain by applying the stimulating current to acupoints through acupuncture needles. To determine the significance of EA on neuropathic pain mediated by P2X(3) receptors in the dorsal root ganglion (DRG) neurons, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were recorded, and the expression of P2X(3) receptors in the DRG neurons was assessed by immunohistochemistry (IHC) and in situ hybridization (ISH). In addition, the currents which were evoked in DRG neurons isolated from rats following chronic constriction injury (CCI) by the P2X(3) receptors agonists i.e. ATP and α,β-methylen-ATP (α,β-meATP) were examined through the experimental use of whole cell patch clamp recording. The present study demonstrates that EA treatment can increase the MWT and TWL values and decrease the expression of P2X(3) receptors in DRG neurons in CCI rats. Simultaneously, EA treatment attenuates the ATP and α,β-meATP evoked currents. EA may be expected to induce an apparent induce analgesic effect by decreasing expression and inhibiting P2X(3) receptors in DRG neurons of CCI rats. There is a similar effect on analgesic effect between rats with contralateral EA and those with ipsilateral EA.     

Echocardiographic detection of congestive heart failure in postinfarction rats

In studies of congestive heart failure (CHF) treatment, it is essential to select animals with a similar degree of cardiac dysfunction. However, this is difficult to establish without hemodynamic evaluation in rat postinfarction-induced CHF. This study aimed to diagnose CHF in long-term follow-up postinfarction rats using only echocardiographic criteria through a J-tree cluster analysis and Fisher's linear discriminant function. Two sets of sham and infarcted rats were studied. The first was used to perform cluster analysis and the second to prospectively validate the results. Six months after inducing myocardial infarction (MI), rats were subjected to transthoracic echocardiography. Infarct size was measured by histological analysis. Six echocardiographic variables were used in the cluster analysis: left ventricular (LV) systolic dimension, LV diastolic dimension-to-body weight ratio, left atrial diameter-to-body weight ratio, LV posterior wall shortening velocity, E wave, and isovolumetric relaxation time. Cluster analysis joined the rats into one sham and two MI groups. One MI cluster had more severe anatomical and echocardiographic changes and was called MI with heart failure (MI/HF+, n = 24, infarct size: 42.7 ± 5.8%). The other had less severe changes and was called MI without heart failure (MI/HF-, n = 11, infarct size: 32.3 ± 9.9%; P < 0.001 vs. MI/HF+). Three rats with small infarct size (21.6 ± 2.2%) presenting mild cardiac alterations were misallocated in the sham group. Fisher's linear discriminant function was built using these groups and used to prospectively classify additional groups of sham-operated (n = 20) and infarcted rats (n = 57) using the same echocardiographic parameters. The discriminant function therefore detected CHF with 100% specificity and 80% sensitivity considering allocation in MI/HF+ and sham group, and 100% specificity and 58.8% sensitivity considering MI/HF+ and MI/HF- groups, taking into account pathological criteria of CHF diagnosis. Echocardiographic analysis can be used to accurately predict congestive heart failure in postinfarction rats.     

慢性炎症痛引起DRG感觉神经元P2X3受体表达的改变

目的探讨脊髓背根神经节（dorsal root ganglia,DRG）P2X3受体参与大鼠足底慢性炎症痛相关的热痛觉过敏机制。方法 1）用行为学的研究方法,以大鼠右侧后脚掌注射松节油加石蜡（各占50％）0．1ml建立后脚掌慢性痛模型,用热测痛的方法测量后脚掌皮下注射松节油后的痛阈,每天1次,连续测15d。2）用免疫组织化学技术观察大鼠后脚掌慢性炎症后第2天和第7天,炎症侧脊髓背根神经节（L4—6）神经元中P2X,受体阳性细胞类型的分布变化;以及正常脊髓背根神经节（L4—6）神经元中P2X,受体阳性细胞类型的分布作为对照。结果1）炎症后大鼠后脚掌侧痛阈出现降低,在第2天痛阈达到最低,后逐渐恢复,14d后恢复正常痛阈值。2）正常大鼠P2X,主要表达于DRG的中小神经元上,炎症后DRG（L4—6）,中小型P2X,受体阳性细胞数比对照组明显增加。细胞平均面积增大。结论后脚掌慢性炎症痛可以引起大鼠对伤害性热刺激的痛觉过敏,并导致脊髓背根神经节（L4—6）神经元qbP2X3受体阳性细胞数目增加,表明P2X3在DRG的中小神经元的改变可能对松节油引起脚掌炎症痛时热痛觉过敏的形成与维持起重要作用。     

Endogenous reactive oxygen species modulates voltage-gated sodium channels in dorsal root ganglia of rats

We recently reported that reactive oxygen species (ROS) plays an excitatory role in modulation of the exercise pressor reflex (EPR) in normal rats. In this study, we further tested two independent hypotheses: 1) ROS interacts with EPR-related ionotropic receptors such as the purinergic receptors (P(2)) and transient receptor potential vanilloid 1 receptors (TRPV1) to indirectly modulate the EPR function; 2) ROS directly affects excitability of muscle afferents by modulating the voltage-gated sodium (Na(v)) channels. To test the first hypothesis, we performed animal experiments to investigate the effect of the SOD mimetic 4-hydroxy-2,2,6,6-tetramethyl piperidine 1-oxyl (Tempol) on the pressor response to hindlimb intra-arterial (IA) injection of either α,β-methylene ATP (a P(2X) agonist) or capsaicin (a TRPV1 agonist) in decerebrate rats. To test the second hypothesis, we used the patch-clamp technique to determine the effect of ROS on Na(v) channels on the soma of muscle afferents. We also performed local microinjection of a sodium channel blocker, tetrodotoxin (TTX), into ipsilateral L4/L5 dorsal root ganglia (DRGs) to investigate whether the blockade of Na(v) channels by TTX affects the EPR function. We found that Tempol did not affect the pressor response to injection of either capsaicin or α,β-methylene ATP but significantly decreased the Na(v) current in small and medium-sized 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons. A membrane-permeant superoxide dismutase, polyethylene glycol (PEG)-SOD, had an effect on the Na(v) current in these neurons similar to that of Tempol. Microinjection of TTX into L4/L5 DRGs dramatically attenuated the pressor response to static contraction induced by electrical stimulation of L4/L5 ventral roots. These data suggest that ROS modulates the EPR by affecting the activity of the Na(v) channels on muscle afferents.     

Effect of lappaconitine on neuropathic pain mediated by P2X3 receptor in rat dorsal root ganglion

ATP facilitates initiation and transmission of the neuropathic pain at the dorsal root ganglion (DRG) level via the P2X receptors, especially the subtype P2X(3). Lappaconitine (LA) is an active principle isolated from Chinese herbal medicine and possesses analgesic effect. The aim of this study was to investigate the effect of LA on chronic constriction injury (CCI)-induced neuropathic pain mediated by P2X(3) receptor in the DRG neurons. In the presence of CCI and/or LA, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and P2X(3) receptor expression in the DRG neurons was evaluated by immunohistochemistry and Western blotting. Following intrathecal administration of P2X(3) receptor oligonucleotide, the effect of LA on pain thresholds was assessed. Furthermore, the effect of LA on the P2X(3) receptor agonists ATP- and α,β-meATP-induced inward currents (I(ATP) and I(α,β-meATP)) in the acutely dissociated rat DRG neurons was investigated by whole cell patch-clamp. The results included: (1) There showed reduction of pain thresholds, enhancement of I(ATP) and I(α,β-meATP) and up-regulation of P2X(3) receptor expression in rat DRG neurons when neuropathic pain occurred. (2) In the presence of LA, the decreased pain thresholds, the up-regulated P2X(3) receptor expression and the enhanced I(ATP) and I(α,β-meATP) were reversible in the CCI rats. (3) The down-regulated P2X(3) receptor expression with pretreatment of P2X(3) receptor antisense oligonucleotide significantly attenuated the analgesic effect of LA. These results indicate that the analgesic effect of LA involves decrease of expression and sensitization of the P2X(3) receptors of the rat DRG neurons following CCI.     

Prenatal expression of purinergic receptor P2X3 in human dorsal root ganglion

The dorsal root ganglion (DRG) is consisted of neurons that relay multiple types of spinal sensory stimuli to the central nervous system. Several neuroactive molecules may be involved in sensory modulation especially pain processing at the DRG, including the purinergic receptor P2X3 and calcitonin-gene-related peptide (CGRP). P2X3 receptor has been considered a promising pharmaceutical target for the development of new pain medicine. Currently, litter is known about the expression of P2X3 in the human DRG. The present study characterized the localization of P2X3 in prenatal human DRG obtained from fetuses at 4-8 gestational months, by comparing to CGRP expression as well as binding pattern of isolectin-B4 (IB4), a marker of small DRG neurons presumably relevant to nociception. P2X3 immunoreactivity (IR) appeared in most neuron-like perikarya, with their numerical density reduced during the gestational period studied. P2X3 IR was co-labeled very commonly with IB4 binding and infrequently with CGRP IR and was not colocalized with IR for the gliocyte marker glutamine synthetase. Together, the data show an early and broad expression of P2X3 in prenatal human DRG neurons, pointing to a biological role of purinergic signaling during the development of spinal sensory system.     

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。     

ATP stimulates chemically sensitive and sensitizes mechanically sensitive afferents

We examined whether ATP stimulation of P2X purinoceptors would raise blood pressure in decerebrate cats. Femoral arterial injection of the P2X receptor agonist alpha,beta-methylene ATP into the blood supply of the triceps surae muscle induced a dose-dependent increase in arterial blood pressure. The maximal increase in mean arterial pressure (MAP) evoked by 0.1, 0.2, and 0.5 mM alpha,beta-methylene ATP (0.5 ml/min injection rate) was 6.2 +/- 2.5, 22.5 +/- 4.4, and 35.2 +/- 3.9 mmHg, respectively. The P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (2 mM ia) attenuated the increase in MAP elicited by intra-arterial alpha,beta-methylene ATP (0.5 mM), whereas the P2Y receptor antagonist reactive blue 2 (2 mM ia) did not affect the MAP response to alpha,beta-methylene ATP. In a second group of experiments, we tested the hypothesis that ATP acting through P2X receptors would sensitize muscle afferents and, thereby, augment the blood pressure response to muscle stretch. Two kilograms of muscle stretch evoked a 26.5 +/- 4.3 mmHg increase in MAP. This MAP response was enhanced when 2 mM ATP or 0.1 mM alpha,beta-methylene ATP (0.5 ml/min) was arterially infused 10 min before muscle stretch. Furthermore, this effect of ATP on the pressor response to stretch was attenuated by 2 mM pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (P < 0.05) but not by the P1 purinoceptor antagonist 8-(p-sulfophenyl)-theophylline (2 mM). These data indicate that activation of ATP-sensitive P2X receptors evokes a skeletal muscle afferent-mediated pressor response and that ATP at relatively low doses enhances the muscle pressor response to stretch via engagement of P2X receptors.     

Localization of P2X and P2Y receptors in dorsal root ganglia of the cat.

The distribution of P2X and P2Y receptor subtypes in upper lumbosacral cat dorsal root ganglia (DRG) has been investigated using immunohistochemistry. Intensity of immunoreactivity for six P2X receptors (P2X(5) receptors were immuno-negative) and the three P2Y receptors examined in cat DRG was in the order of P2Y(2) = P2Y(4)>P2X(3)>P2X(2) = P2X(7)>P2X(6)>P2X(1) = P2X(4)>P2Y(1). P2X(3), P2Y(2), and P2Y(4) receptor polyclonal antibodies stained 33.8%, 35.3%, and 47.6% of DRG neurons, respectively. Most P2Y(2), P2X(1), P2X(3), P2X(4), and P2X(6) receptor staining was detected in small- and medium-diameter neurons. However, P2Y(4), P2X(2), and P2X(7) staining was present in large- and small-diameter neurons. Double-labeling immunohistochemistry showed that 90.8%, 32.1%, and 2.4% of P2X(3) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively; whereas 67.4%, 41.3%, and 39.1% of P2Y(4) receptor-positive neurons coexpressed IB(4), CGRP, and NF200, respectively. A total of 18.8%, 16.6%, and 63.5% of P2Y(2) receptor-positive neurons also stained for IB(4), CGRP, and NF200, respectively. Only 30% of DRG neurons in cat were P2X(3)-immunoreactive compared with 90% in rat and in mouse. A further difference was the low expression of P2Y(1) receptors in cat DRG neurons compared with more than 80% of the neurons in rat. Many small-diameter neurons were NF200-positive in cat, again differing from rat and mouse.     

大鼠坐骨神经结扎后疼痛受体P2X3在背根神经节内的表达变化

目的:研究坐骨神经结扎损伤后疼痛受体P2X3在相应背根神经节(dorsal root ganglia,DRG)内的表达变化情况。方法:选取健康成年SD大鼠35只,建立右侧坐骨神经结扎损伤模型,采用免疫组织化学和图像分析技术检测相应L4-6DRG内P2X3的表达情况。结果:正常大鼠L4-6DRG内有大量P2X3免疫阳性神经元,坐骨神经结扎后3d P2X3表达即下调,3,7,14,21和28d其表达呈进行性下降趋势,各时间点与正常和假手术对照比较差异均有统计学意义(P＜0．05)。结论:坐骨神经结扎后P2X3在L4-6DRG内表达明显下调,提示其可能在神经源性疼痛中发挥一定的作用。     

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to sub-populations of sensory neuron cell bodies in the L4/L5 DRG of neonatal rats. The size distribution of ¹²⁵I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 μm²). ¹²⁵I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 μm². Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 μm², respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 22–32, 1997.     

Inhibitory effect of estrogen receptor beta on P2X3 receptors during inflammation in rats

Estrogen receptor beta (ERβ) has been shown to play a therapeutic role in inflammatory bowel disease (IBD). However, the mechanism underlying how ERβ exerts therapeutic effects and its relationship with P2X3 receptors (P2X3R) in rats with inflammation is not known. In our study, animal behavior tests, visceromotor reflex recording, and Western blotting were used to determine whether the therapeutic effect of ERβ in rats with inflammation was related with P2X3R. In complete Freund adjuvant (CFA)-induced chronic inflammation in rats, paw withdrawal threshold was significantly decreased which were then reversed by systemic injection of ERβ agonists, DPN or ERB-041. In 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats, weight loss, higher DAI scores, increased visceromotor responses, and inflammatory responses were reversed by application of DPN or ERB-041. The higher expressions of P2X3R in dorsal root ganglia (DRG) of CFA-treated rats and those in rectocolon and DRG of TNBS-treated rats were all decreased by injection of DPN or ERB-041. DPN application also inhibited P2X3R-evoked inward currents in DRG neurons from TNBS rats. Mechanical hyperalgesia and increased P2X3 expression in ovariectomized (OVX) CFA-treated rats were reversed by estrogen replacements. Furthermore, the expressions of extracellular signal-regulated kinase (ERK) in DRG and spinal cord dorsal horn (SCDH) and c-fos in SCDH were significantly decreased after estrogen replacement compared with those of OVX rats. The ERK antagonist U0126 significantly reversed mechanical hyperalgesia in the OVX rats. These results suggest that estrogen may play an important therapeutic role in inflammation through down-regulation of P2X3R in peripheral tissues and the nervous system, probably via ERβ, suggesting a novel therapeutic strategy for clinical treatment of inflammation.     

Effect of muscle interstitial pH on P2X and TRPV1 receptor-mediated pressor response.

Activation of purinergic P2X receptors and transient receptor potential vanilloid type 1 (TRPV1) on muscle afferent nerve evokes the pressor response. Because P2X and TRPV1 receptors are sensitive to changes in pH, the aim of this study was to examine the effects of muscle acidification on those receptor-mediated cardiovascular responses. In decerebrate rats, the pH in the hindlimb muscle was adjusted by infusing acidic Ringer solutions into the femoral artery. Dialysate was then collected using microdialysis probes inserted into the muscles, and pH was measured. The interstitial pH was 7.53+/-0.01, 7.22+/-0.02, 6.94+/-0.04, and 6.59+/-0.03 in response to arterial infusion of the Ringer solution at pH 7.4, 6.5, 5.5, and 4.5, respectively. Femoral arterial injection of alpha,beta-methylene-ATP (P2X receptor agonist) in the concentration of 0.25 mM (volume, 0.15-0.25 ml; injection duration, 1 min) at the infused pH of 7.4, 6.5, and 5.5 increased mean arterial pressure (MAP) by 29+/-2, 24+/-3, and 21+/-3 mmHg, respectively (P<0.05, pH 5.5 vs. pH 7.4). When pH levels in the infused solution were 7.4, 6.5, 5.5, and 4.5, capsaicin (1 microg/kg), a TRPV1 agonist, was injected into the artery. This elevated MAP by 29+/-4, 33+/-2, 35+/-3, and 40+/-3 mmHg, respectively (P<0.05, pH 4.5 vs. pH 7.4). Furthermore, blocking acid-sensing ion channel (ASIC) blunted pH effects on TRPV1 response. Our data indicate that 1) muscle acidosis attenuates P2X-mediated pressor response but enhances TRPV1 response; 2) exaggerated TRPV1 response may require lower pH in muscle, and the effect is likely to be mediated via ASIC mechanisms. This study provides evidence that muscle pH may be important in modulating P2X and TRPV1 responsiveness in exercising muscle.     

The effect of a myocardial infarction on the normalized time-varying elastance curve.

It has been suggested that the shape of the normalized time-varying elastance curve [E(n)(t(n))] is conserved in different cardiac pathologies. We hypothesize, however, that the E(n)(t(n)) differs quantitatively after myocardial infarction (MI). Sprague-Dawley rats (n = 9) were anesthetized, and the left anterior descending coronary artery was ligated to provoke the MI. A sham-operated control group (CTRL) (n = 10) was treated without the MI. Two months later, a conductance catheter was inserted into the left ventricle (LV). The LV pressure and volume were measured and the E(n)(t(n)) derived. Slopes of E(n)(t(n)) during the preejection period (alpha(PEP)), ejection period (alpha(EP)), and their ratio (beta = alpha(EP)/alpha(PEP)) were calculated, together with the characteristic decay time during isovolumic relaxation (tau) and the normalized elastance at end diastole (E(min)(n)). MI provoked significant LV chamber dilatation, thus a loss in cardiac output (-33%), ejection fraction (-40%), and stroke volume (-30%) (P < 0.05). Also, it caused significant calcium increase (17-fold), fibrosis (2-fold), and LV hypertrophy. End-systolic elastance dropped from 0.66 +/- 0.31 mmHg/microl (CTRL) to 0.34 +/- 0.11 mmHg/microl (MI) (P < 0.05). Normalized elastance was significantly reduced in the MI group during the preejection, ejection, and diastolic periods (P < 0.05). The slope of E(n)(t(n)) during the alpha(PEP) and beta were significantly altered after MI (P < 0.05). Furthermore, tau and end-diastolic E(min)(n) were both significantly augmented in the MI group. We conclude that the E(n)(t(n)) differs quantitatively in all phases of the heart cycle, between normal and hearts post-MI. This should be considered when utilizing the single-beat concept.     

Sensory–sympathetic coupling in superior cervical ganglia after myocardial ischemic injury facilitates sympathoexcitatory action via P2X7 receptor

P2X receptors participate in cardiovascular regulation and disease. After myocardial ischemic injury, sensory–sympathetic coupling between rat cervical DRG nerves and superior cervical ganglia (SCG) facilitated sympathoexcitatory action via P2X₇ receptor. The results showed that after myocardial ischemic injury, the systolic blood pressure, heart rate, serum cardiac enzymes, IL-6, and TNF-α were increased, while the levels of P2X₇ mRNA and protein in SCG were also upregulated. However, these alterations diminished after treatment of myocardial ischemic (MI) rats with the P2X₇ antagonist oxATP. After siRNA P2X₇ in MI rats, the systolic blood pressure, heart rate, serum cardiac enzymes, the expression levels of the satellite glial cell (SGC) or P2X₇ were significantly lower than those in MI group. The phosphorylation of ERK 1/2 in SCG participated in the molecular mechanism of the sympathoexcitatory action induced by the myocardial ischemic injury. Retrograde tracing test revealed the sprouting of CGRP or SP sensory nerves (the markers of sensory afferent fibers) from DRG to SCG neurons. The upregulated P2X₇ receptor promoted the activation of SGCs in SCG, resulting in the formation of sensory–sympathetic coupling which facilitated the sympathoexcitatory action. P2X₇ antagonist oxATP could inhibit the activation of SGCs and interrupt the formation of sensory–sympathetic coupling in SCG after the myocardial ischemic injury. Our findings may benefit the treatment of coronary heart disease and other cardiovascular diseases.