Pharmacological characterization of 5-hydroxytryptamine2 and 5-hydroxytryptamine3 receptors in rat dorsal root ganglion cells

5-Hydroxytryptamine (5-HT) depolarized 87% of the rat dorsal root ganglion cells recorded. 5-HT increased the input resistance (Rin) in 50%, decreased Rin in 41% and produced both responses in 9% of the responding cells. When 5-HT increased the Rin, the response was mimicked by the 5-HT2 agonists alpha-methyl-5-HT, (+/-)-1-(2,5-dimethoxy-4 iodophenyl)-2-aminopropane HCl, quipazine and MK 212 (6-chloro-1-[1-piperazinyl]-pyrazine), but not by 2-methyl-5-HT or carboxamidotryptamine. The response to 5-HT was antagonized by ketanserin, spiperone and methiothepin. The unsurmountable blockade induced by higher concentrations of ketanserin was not explained by pseudo-irreversible antagonism or multiple receptor subtypes, but could result from a two-state receptor model or multiple subtypes of the 5-HT2 receptor. This conclusion is supported by the partial agonist action of DOI. Cells responding to 5-HT with depolarization and decreased Rin responded similarly to 2-methyl-5-HT and phenylbiguanide, but not to alpha-methyl-5-HT or carboxyamidotryptamine. This response was surmountably blocked by ICS 205-930 (3-tropanyl-indole-3-carboxylate) (pA2 = 10.3) and MDL 72222 (3-tropanyl-3,5-dichlorobenzoate)(pA2 = 7.8). The arylpiperazines, quipazine and MK 212, antagonized the action of 2-methyl-5-HT with IC50 values of 8 and 4 nM, respectively. These data indicate that 5-HT2 receptors mediate the increased Rin and 5-HT3 receptors mediate the decreased Rin.     

Activation of neurokinin-1 receptors increases the excitability of Guinea pig dorsal root ganglion cells

The suppression of overactive bladder symptoms in patients and overactive bladder reflexes in animal models by neurokinin (NK)-1 receptor antagonists raises the possibility that these drugs target sensory neurons. This mechanism was evaluated by examining the interactions between a specific NK-1 agonist, [Sar(9),Met(O(2))(11)]-substance P (Sar-Met-SP), and a potent NK-1 antagonist, netupitant (NTP), on small size (20-30 μm) dissociated L6 and S1 dorsal root ganglion (DRG) neurons from female guinea pigs. Current-clamp recording revealed that Sar-Met-SP (1 μM) elicited membrane depolarization (average 8.05 ± 1.38 mV) in 27% (18 of 65) of DRG neurons. In 74% of the remaining neurons (35 of 47) Sar-Met-SP decreased the rheobase for action potential (AP) generation and increased the response to a suprathreshold stimulus (3 times rheobase) without changing the membrane potential. Sar-Met-SP also induced changes in the action potential (AP) wave form, including 1) an increase in overshoot (average 5 mV, n = 35 neurons), 2) a prolongation of AP duration (from 4.64 to 5.29 ms, n = 34), and 3) a reduction in the maximal rate of AP repolarization. NTP (200 nM) reversed the Sar-Met-SP-induced changes. Ca(2+) imaging showed that application of Sar-Met-SP (1 μM) decreased the tachyphylaxis induced by repeated application of capsaicin (0.5 μM), an effect blocked by pretreatment with NTP (200 nM). These results raise the possibility that activation of NK-1 receptors in primary sensory neurons plays a role in the generation of overactive bladder and that block of NK-1 receptors in these neurons may contribute to efficacy of NK-1 antagonists in the treatment of overactive bladder symptoms.     

Role of TNF-alpha in sensitization of nociceptive dorsal horn neurons induced by application of nucleus pulposus to L5 dorsal root ganglion in rats

Herniation of the nucleus pulposus (NP) from lumbar intervertebral discs commonly results in radiculopathic pain and paresthesia (sciatica). While traditionally considered the result of mechanical compression of the dorsal root ganglion (DRG) and/or spinal nerve root, recent studies implicate pro-inflammatory mediators released from or evoked by NP, a possibility that was presently investigated. Single-unit recordings were made from L5 wide dynamic range dorsal horn neurons in pentobarbital-anesthetized rats. Autologous NP was harvested from a coccygeal disc and placed onto the exposed L5 DRG. A control group had subcutaneous adipose tissue or saline placed similarly. To test involvement of tumor necrosis factor- (TNF-), a third group received autologous NP plus local soluble TNF- receptor type 1 (0.013 μg) which binds TNF- to prevent its action. In each group, neuronal responses to graded heat (38–50 °C) and mechanical (von Frey filaments 4–76 g) stimuli were recorded prior to and at three successive hourly intervals following each treatment. Responses to noxious heat and mechanical stimuli were significantly enhanced 1 h post-NP and remained elevated thereafter. Thermally and mechanically evoked responses were not significantly affected in control rats or those treated with NP+soluble TNF- receptor type 1. These results indicate that sensitization of nociceptive spinal neuronal responses develops quickly following exposure of the DRG to NP, and that TNF- is involved. This electrophysiological model of herniated NP may prove useful in further characterizing the role of inflammatory mediators in hyperalgesia and allodynia resulting from lumbar disc herniation.     

Competitive inhibition of the capsaicin receptor-mediated current by dehydroepiandrosterone in rat dorsal root ganglion neurons

The effects of dehydroepiandrosterone (5-androsten-3beta-ol-17-one; DHEA) and related steroids on the capsaicin receptor-mediated current were studied in acutely dissociated rat dorsal root ganglion neurons using the whole-cell voltage-clamp technique. DHEA rapidly and reversibly inhibited the capsaicin-induced current in a concentration-dependent manner, with an EC(50) of 6.7 microM and a maximal inhibition of 100%. DHEA increased the capsaicin EC(50) with little effect on the capsaicin maximal response, suggesting that the blocking action of DHEA is competitive. Neither the capsaicin response nor inhibition of the capsaicin response by extracellularly applied DHEA was significantly affected by inclusion of a saturating concentration of DHEA in the electrode buffer, arguing that DHEA acted at the extracellular surface of the membrane. Moreover, DHEA did not act through protein phosphatases to inhibit the capsaicin-induced current. Furthermore, the stereoisomer of DHEA, 5-androsten-3alpha-ol-17-one, failed to inhibit the capsaicin-induced current, producing instead a potentiating effect on the capsaicin response, demonstrating that the interaction of steroids with the capsaicin receptor is stereospecific. The inhibitory action of DHEA on the capsaicin-induced current may provide a basis for reducing capsaicin receptor-mediated nociception.     

氨基酸受体在脊神经节损伤所介导痛觉过敏中的作用

目的：观察脊髓背角N-甲基-D-天冬氨酸受体或／和非-N-甲基-D-天冬氨酸受体在背根神经节炎性损伤所介导的伤侧肢体痛觉过敏中的作用。方法：实验于2002-11／2004-02在第三军医大学基础部实验室完成。日本大耳白兔42只。①分组方法：动物模型为手术显微镜下仔细显露双侧脊神经节，左侧作为损伤侧，右侧作为非损伤侧，直视下经硬脊置入无菌医用导管至蛛网膜下腔中。将兔随机分为3组，每组14只，AP-5组注入N-甲基-D-天冬氨酸受体拮抗剂AP-5（50μg／kg＋100μL生理盐水）；CNQX组注入非N-甲基-D-天冬氨酸受体拮抗剂CNQX（20μg／kg＋100μL生理盐水）；对照组为同等剂量的生理盐水。②主要观察指标：AP-5和CNQX对兔伤侧痛觉过敏的影响；AP-5和CNQX对兔脊髓背侧兴奋性氨基酸和抑制性氨基酸含量影响：CNQX对脊髓背角c-fos表达的影响。③评估标准：机械性刺激缩腿反应强度阈值为正值时，表示为实验侧肢体痛觉减退，当为负值时，表示为实验侧肢体痛觉过敏，绝对值越大，表明其损伤后所造成的变化程度越大；热辐射刺激缩腿反应潜伏期为正值时，表示实验侧肢体温度性痛觉减退，为负值时则为温度性痛觉过敏。其绝对值越大，说明损伤所造成的变化越大。结果：42只白兔均进入结果分析。①AP-5和CNQX对兔伤侧痛觉过敏的影响：AP-5组机械刺激反应阈值逐渐增高，60～180min时已显著高于用药前[（-9．28&;#177;3．9），（-11．4&;#177;8．3），（-12．3&;#177;5．7），（26．7&;#177;15．8）g，t=2．57-3．46，P〈0．05]，潜伏期反应值也迅速增高，30-180min时已较用药前显著增高（P〈0．05），对照组各点间均无显著变化。CNQX组反应阈值增高，与用药前比较无显著差异，潜伏期值在用药30－120min时显著高于用药前水平和对照组俨〈0．05）。②AP-5和CNQX对兔脊髓背侧兴奋性氨基酸和抑制性氨基酸含量影响：脊髓背侧部分氨基酸的浓度有一定变化，如注射AP-5和CNQX后，脊髓背侧天冬氨酸含量均明显高于术前水平[（4．48&;#177;0．47）（4．98&;#177;0．41），（2．38&;#177;0．13）μmol／g，P〈0．05]。③CNQX对脊髓背角c-fos表达的影响：CNQX推注后，c-fos表达神经元数量较用药前和对照组显著降低（P〈0．05）。结论：①背根神经节炎性损伤所造成脊髓背角抑制性氨基酸（如天冬氨酸）释放增加，N-甲基-D-天冬氨酸受体和非N-甲基-D-天冬氨酸受体的激活，在伤侧肢体痛觉过敏的形成和维持中具有重要作用。②同时表明非-N-甲基-D-天冬氨酸受体主要介导温度性痛觉过敏，N-甲基-D-天冬氨酸受体的激活，介导了背根神经节炎性损伤所产生机械性和温度性痛觉过敏。③AP-5和CNQX蛛网膜下腔注射，能显著减轻背根神经节炎性损伤所造成伤侧肢体痛觉过敏。     

氨基酸受体在脊神经节损伤所介导痛觉过敏中的作用

目的:观察脊髓背角N-甲基-D-天冬氨酸受体或/和非-N-甲基-D-天冬氨酸受体在背根神经节炎性损伤所介导的伤侧肢体痛觉过敏中的作用。方法:实验于2002-11/2004-02在第三军医大学基础部实验室完成。日本大耳白兔42只。①分组方法:动物模型为手术显微镜下仔细显露双侧脊神经节,左侧作为损伤侧,右侧作为非损伤侧,直视下经硬脊置入无菌医用导管至蛛网膜下腔中。将兔随机分为3组,每组14只,AP-5组注入N-甲基-D-天冬氨酸受体拮抗剂AP-5(50μg/kg+100μL生理盐水);CNQX组注入非N-甲基-D-天冬氨酸受体拮抗剂CNQX(20μg/kg+100μL生理盐水);对照组为同等剂量的生理盐水。②主要观察指标:AP-5和CNQX对兔伤侧痛觉过敏的影响;AP-5和CNQX对兔脊髓背侧兴奋性氨基酸和抑制性氨基酸含量影响;CNQX对脊髓背角c-fos表达的影响。③评估标准:机械性刺激缩腿反应强度阈值为正值时,表示为实验侧肢体痛觉减退,当为负值时,表示为实验侧肢体痛觉过敏,绝对值越大,表明其损伤后所造成的变化程度越大;热辐射刺激缩腿反应潜伏期为正值时,表示实验侧肢体温度性痛觉减退,为负值时则为温度性痛觉过敏。其绝对值越大,说明损伤所造成的变化越大。结果:42只白兔均进入结果分析。①AP-5和CNQX对兔伤侧痛觉过敏的影响:AP-5组机械刺激反应阈值逐渐增高,60～180min时已显著高于用药前[(-9.28±3.9),(-11.4±8.3),(-12.3±5.7),(26.7±15.8)g,t=2.57～3.46,P<0.05],潜伏期反应值也迅速增高,30～180min时已较用药前显著增高(P<0.05),对照组各点间均无显著变化。CNQX组反应阈值增高,与用药前比较无显著差异,潜伏期值在用药30～120min时显著高于用药前水平和对照组(P<0.05)。②AP-5和CNQX对兔脊髓背侧兴奋性氨基酸和抑制性氨基酸含量影响:脊髓背侧部分氨基酸的浓度有一定变化,如注射AP-5和CNQX后,脊髓背侧天冬氨酸含量均明显高于术前水平[(4.48±0.47)(4.98±0.41),(2.38±0.13)μmol/g,P<0.05]。③CNQX对脊髓背角c-fos表达的影响:CNQX推注后,c-fos表达神经元数量较用药前和对照组显著降低(P<0.05)。结论:①背根神经节炎性损伤所造成脊髓背角抑制性氨基酸(如天冬氨酸)释放增加,N-甲基-D-天冬氨酸受体和非N-甲基-D-天冬氨酸受体的激活,在伤侧肢体痛觉过敏的形成和维持中具有重要作用。②同时表明非-N-甲基-D-天冬氨酸受体主要介导温度性痛觉过敏,N-甲基-D-天冬氨酸受体的激活,介导了背根神经节炎性损伤所产生机械性和温度性痛觉过敏。③AP-5和CNQX蛛网膜下腔注射,能显著减轻背根神经节炎性损伤所造成伤侧肢体痛觉过敏。     

Regulation of 5-hydroxytryptamine1A receptor function in rat hippocampus by short- and long-term administration of 5-hydroxytryptamine1A agonist and antidepressants.

Single s.c. injections of the 5-hydroxytryptamine (5-HT)1A receptor agonists buspirone at 4 mg/kg, 8-hydroxy-2-(di-n-propylamino)tetralin at 1 or 4 mg/kg or ipsapirone at 10 mg/kg did not affect 5-HT inhibition of forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes. However, a single injection of buspirone at 8 mg/kg, and daily injections of each of the agonists for 8 days, resulted in a reduction in the degree of enzyme inhibition by 5-HT. Chronic administration of the antidepressants fluoxetine, zimelidine and maprotiline by i.p. injections at 15 mg/kg for 3 weeks also resulted in a decreased degree of enzyme inhibition. Chronic iprindole at the same dose had no effect. It is concluded that the antidepressant-like properties of 5-HT1A receptor agonists may be mediated partly by a postsynaptic action at the level of serotonergic second messenger transduction in the hippocampus.     

Opiate-mediated inhibition of calcium signaling is decreased in dorsal root ganglion neurons from the diabetic BB/W rat.

The effect of diabetes mellitus on opiate-mediated inhibition of calcium current density (I(D Ca) [pA pF-1]) and cytosolic calcium response ([Ca2+]i nM) to depolarization with elevated KCl and capsaicin was assessed. Experiments were performed on isolated, acutely dissociated dorsal root ganglion (DRG) neurons from diabetic, BioBreeding/Worcester (BB/W) rats and age-matched control animals. Sciatic nerve conduction velocity was significantly decreased in diabetic animals compared to controls. Mean I(DCa) and [Ca2+]i responses to capsaicin and elevated KCl recorded in DRGs from diabetic animals were significantly larger than those recorded in DRG neurons from controls. In neurons from diabetic animals, the opiate agonist dynorphin A (Dyn A; 1, 3, and 5 microM) had significantly less inhibitory effect on I(D Ca) and KCl-induced [Ca2+]i responses compared to controls. Omega-conotoxin GVIA (omega-CgTX; 10 microM) and pertussis toxin (PTX; 250 ng ml-1) abolished Dyn A-mediated inhibition of I(DCa) and [Ca2+]i in control and diabetic neurons, suggesting that Dyn A modulated predominantly N-type calcium channels coupled to opiate receptors via PTX-sensitive (Gi/o) inhibitory G proteins. These results suggest that opiate-mediated regulation of PTX-sensitive, G protein-coupled calcium channels is diminished in diabetes and that this correlates with impaired regulation of cytosolic calcium.     

The arginine-rich hexapeptide R4W2 is a stereoselective antagonist at the vanilloid receptor 1: a Ca2+ imaging study in adult rat dorsal root ganglion neurons

Vanilloid receptors (VR) integrate various painful stimuli, e.g., noxious heat, acidic pH, capsaicin, and resiniferatoxin (RTX). Although VR antagonists may be useful analgesics, the available agents capsazepine and ruthenium red lack the necessary potency and selectivity. Recently, submicromolar concentrations of the arginine-rich hexapeptide RRRRWW-NH(2) (R(4)W(2)) blocked VR-mediated ionic currents in a Xenopus expression system in a noncompetitive and nonstereoselective manner. Here, VR-antagonistic effects of L-R(4)W(2) and D-R(4)W(2), hexapeptides consisting entirely of L- and D-amino acids, were characterized in native adult rat dorsal root ganglion neurons using [Ca(2+)](i) imaging (Fura-2/acetoxymethyl ester). Fura-2 fluorescence ratio (R) was increased by RTX and capsaicin by 0.473 +/- 0.098 unit above basal levels of 0.903 +/- 0.011 (R(max), 2.289 +/- 0.031; R(min), 0.657 +/- 0.007) in a concentration-dependent manner (log EC(50): RTX, -10.04 +/- 0.05, n = 10; capsaicin, -6.60 +/- 0.10, n = 11). Agonist concentration-response curves were shifted to the right by L- and D-R(4)W(2) (0.1, 1, and 10 microM each) and by capsazepine (3, 10, 30, and 100 microM), whereas their maximal effects and slopes remained unaffected, indicating competitive antagonism. Schild analysis for L-R(4)W(2) yielded apparent dissociation constants of 4.0 nM (RTX) and 3.7 nM (capsaicin), and slopes smaller than unity (RTX, 0.38; capsaicin, 0.42). Apparent dissociation constants and slopes for D-R(4)W(2) and capsaicin were 153 nM and 0.67 versus 4.1 microM and 1.19 for capsazepine and capsaicin. Thus, VR-mediated effects in native dorsal root ganglion neurons were antagonized by L-R(4)W(2) > D-R(4)W(2) > capsazepine (order of potency). In conclusion, the R(4)W(2) hexapeptide is a potent, stereospecific, and (probably) competitive VR antagonist, although an allosteric interaction cannot be completely ruled out.     

Genetic regulation of extracellular serotonin by 5-hydroxytryptamine(1A) and 5-hydroxytryptamine(1B) autoreceptors in different brain regions of the mouse

The regulation of extracellular levels of 5-hydroxytryptamine (serotonin) (5-HT) in the striatum and ventral hippocampus was studied using in vivo microdialysis in awake, unrestrained wild-type 5-HT(1A) and 5-HT(1B) receptor knockout mice. Systemic administration of the selective serotonin reuptake inhibitor fluoxetine evoked a significant dose-dependent increase in extracellular 5-HT in both the striatum and hippocampus at both 2.5 mg/kg (i.p.) and 20 mg/kg (i.p.) in wild-type mice. In 5-HT(1A) receptor knockout mice, the response to 2.5 mg/kg fluoxetine was significantly augmented in the striatum but not the hippocampus, whereas the response to 20 mg/kg fluoxetine was significantly greater in both brain regions. In 5-HT(1B) receptor knockout mice, the increase of extracellular 5-HT was augmented in the hippocampus but not the striatum at both doses of fluoxetine. The response pattern to fluoxetine alone in 5-HT receptor mutant mice corresponded with the effects of fluoxetine given with either the 5-HT(1A) receptor antagonist WAY 100635 (0.1 mg/kg i.p.) or the 5-HT(1B/1D) receptor antagonist GR 127935 (0.056 mg/kg) in wild-type mice. These results indicate common topographical regulation of 5-HT release in different brain regions by genetic mutation and pharmacological challenges. The 5-HT(1A) autoreceptor plays a larger role in regulating 5-HT release in the striatum and possibly other brain regions innervated by the dorsal raphe nucleus, whereas the role of the 5-HT(1B) receptor is relatively greater in the hippocampus and possibly other brain regions innervated by the median raphe nucleus.     

Reduction of 5-hydroxytryptamine (5-HT)(1A)-mediated temperature and neuroendocrine responses and 5-HT(1A) binding sites in 5-HT transporter knockout mice

The aim of the present study was to determine whether alterations in 5-hydroxytryptamine (5-HT)(1A) receptors would be found in knockout mice lacking the serotonin transporter (5-HTT). Hypothermic and neuroendocrine responses to the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) were used to examine the function of 5-HT(1A) receptors. Initial studies evaluated the dose-response and time course of 8-OH-DPAT-induced hypothermia and hormone secretion in normal CD-1 mice (the background strain of the 5-HTT knockout mice). 8-OH-DPAT dose-dependently produced hypothermic responses that peaked at 20 min postinjection. 8-OH-DPAT-induced hypothermia was blocked by the 5-HT(1A) antagonist WAY-100635. 8-OH-DPAT dose-dependently increased the concentrations of plasma oxytocin, corticotropin, and corticosterone. In the 5-HTT knockout (-/-) mice, the hypothermic response to 8-OH-DPAT (0.1 mg/kg s.c.) was completely abolished. Furthermore, 5-HTT-/- mice had significantly attenuated plasma oxytocin and corticosterone responses to 8-OH-DPAT. No significant changes in the hypothermic or hormonal responses to 8-OH-DPAT were observed in heterozygous (5-HTT+/-) mice. [(3)H]8-OH-DPAT- and [(125)I]MPPI [4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-iodobenzamido]ethyl] pip erazine]-binding sites in the hypothalamus and [(125)I]MPPI-binding sites in the dorsal raphe were significantly decreased in 5-HTT-/- mice. The results indicate that lack of the 5-HTT is associated with a functional desensitization of 5-HT(1A) receptor responses to 8-OH-DPAT, which may be a consequence, at least in part, of the decrease in density of 5-HT(1A) receptors in the hypothalamus and dorsal raphe of 5-HTT-/- mice.     

A型肉毒毒素通过调控大鼠背根神经节神经元中钠离子通道缓解神经病理性疼痛的研究

目的:观察A型肉毒毒素(BoNT/A)对大鼠L5脊神经前根切断(L5 VRT)疼痛模型大鼠的镇痛效果,研究BoNT/A对未受损的背根神经节(DRG)神经元中不同类型钠通道电流和神经元兴奋性的影响。方法:建立L5 VRT神经病理性疼痛大鼠模型。实验大鼠分成3组:假手术组(Sham)、VRT+生理盐水注射组(VRT+Saline)、VRT+BoNT/A注射组(VRT+BoNT/A)。造模后第5天给药,测量不同时间点各组大鼠机械撤足阈值和热撤足潜伏期的变化。电生理膜片钳方法观察BoNT/A对DRG神经元中不同类型钠通道电流的影响,电流钳检测神经元动作电位阈值变化情况。结果:足底皮下注射BoNT/A(7U/kg)可显著缓解L5 VRT介导的机械触痛敏和热痛敏症状(P<0.01);BoNT/A干预后可显著减小L5 VRT术后DRG神经元中河豚毒素敏感型(TTX-S)和河豚毒素抵抗型(TTX-R)钠通道电流密度(P<0.05);BoNT/A可升高VRT术后原本下降的神经元动作电位阈值(P<0.05)。结论:BoNT/A可减小未损伤DRG神经元中TTX-S和TTX-R钠电流,降低神...     

坐骨神经损伤大鼠模型鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达

背景：坐骨神经损伤模型可测试伤害性的热刺激和机械刺激所引发的痛觉过敏及冷、触觉异常。目的：观察坐骨神经损伤模型大鼠鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达。方法：72只SD大鼠随机均分为假手术组、对照组和实验组。对照组和实验组制作坐骨神经损伤模型,假手术组仅暴露坐骨神经,不结扎。分别于造模后第3,10天进行鞘内移植,实验组注入30μL的神经干细胞悬液,空白组和对照组注入30μL的细胞培养液。结果与结论：与假手术组相比,对照组和实验组移植后3d机械痛阈和热痛阈逐渐降低,至移植后7d降低至最低点（P〈0.01）,于移植后21d恢复至移植前水平;实验组移植后7,14d机械痛阈和热痛阈较对照组明显上升（P〈0.01）。与对照组相比,假手术组移植后7,14,21d各组大鼠脑源性神经营养因子的表达呈低水平（P〈0.05）;移植后14,21d,实验组脑源性神经营养因子的表达量高于对照组（P〈0.05）。提示鞘内移植神经干细胞可提高脊髓背角和背根神经节中脑源性神经营养因子的表达。从而抑制了周围神经损伤产生的神经病理性疼痛。     

The P2X3 antagonist P1, P5-di[inosine-5'] pentaphosphate binds to the desensitized state of the receptor in rat dorsal root ganglion neurons

P2X3 purinergic receptors are predominantly expressed in dorsal root ganglion (DRG) neurons and play an important role in pain sensation. P2X3-specific antagonists are currently being sought to ameliorate pain in several indications. Understanding how antagonists interact with the P2X3 receptor can aid in the discovery and development of P2X3-specific antagonists. We studied the activity of the noncompetitive antagonist P1, P5-di[inosine-5'] pentaphosphate (IP5I) at the P2X3 receptor, compared with the well studied competitive antagonist TNP-ATP, using a whole-cell voltage-clamp technique in dissociated rat DRG neurons. IP5I blocked alphabeta-methylene ATP (alphabeta-meATP)-evoked P2X3 responses in a concentration-dependent manner (IC50 = 0.6 +/- 0.1 microM). IP5I effectively inhibited P2X3 currents when pre-exposed to desensitized but not unbound receptors. Furthermore, IP5I equally blocked 1 and 10 microM alphabeta-meATP-evoked currents and had no effect on the desensitization rate constant of these currents. This supports the action of IP5I as a noncompetitive antagonist that interacts with the desensitized state of the P2X3 receptor. In contrast, TNP-ATP inhibited the current evoked by 1 microM alphabeta-meATP significantly more than the one evoked by 10 microM alphabeta-meATP. It also significantly slowed down the desensitization rate constant of the current. These results suggest that TNP-ATP acts as a competitive antagonist and competes with alphabeta-meATP at the P2X3 agonist binding site. These findings may help to explain why IP5I acts selectively at the fast-desensitizing P2X1 and P2X3 subtypes of the P2X purinoceptor, while having much less potency at slow-desensitizing P2X2 and P2X(2/3) subtypes that lack the fast desensitized conformational state.     

胶原酶对大鼠背根神经节亚急性损伤的病理研究

目的研究胶原酶对大鼠脊神经背根神经节(DRG)超微结构亚急性损伤的影响,以期探讨胶原酶应用的安全性,进一步论证经皮椎间盘胶原酶化学髓核溶解术(PCCN)这项治疗方法的安全性。方法SD健康雄性大鼠27只。按随机配伍原则进行分组:正常组9只;DRG胶原酶亚急性实验模型组9只、亚急性假手术组9只。各组大鼠经腹腔注射戊巴比妥钠(45mg/kg)麻醉后,分离并辨认DRG,实验组局部滴注胶原酶1ml(1200单位/4ml),假手术组局部滴注生理盐水1ml。于注药后7～9d行DRG超微结构的电镜检测。结果实验组背根神经节观测细胞种类、细胞数量、细胞大体形态、胞膜情况、节内神经纤维情况(有髓神经纤维有无肿胀,脱髓鞘,髓鞘松解等改变)、血管等情况与正常组、假手术组比较均无明显变化和差别。实验组背根神经节细胞超微结构与正常组、相应假手术组比较均有明显变化和差别:①核仁部分偏向一侧;②线粒体大量肿胀,部分嵴断裂、空泡形成。各组均未见节细胞成群细胞坏死、细胞膜早期破裂、凋亡小体形成等节细胞凋亡相关表现和节细胞坏死相关表现。结论临床应用于PCCN治疗浓度的胶原酶对背根神经节细胞是有损伤的。要慎用胶原酶的用量和浓度,使胶原酶作用于精确合适的部位,提高胶原酶应用安全性。     

Regional patterns of compensation following genetic deletion of either 5-hydroxytryptamine(1A) or 5-hydroxytryptamine(1B) receptor in the mouse

Plasticity in serotonergic transmission in serotonin or 5-hydroxytryptamine (5-HT) receptor mutants was examined by measuring the regulation of extracellular 5-HT levels in the striatum and ventral hippocampus of 5-HT(1A) and 5-HT(1B) receptor knockout mice using in vivo microdialysis. The efficacy of genetic deletion was verified by showing blunted regulation of extracellular 5-HT with selective 5-HT receptor agonists. 5-HT(1A) receptor knockout mice failed to demonstrate reduction of extracellular 5-HT in response to systemic administration of the 5-HT(1A) receptor agonist R-8-hydroxydipropylaminotetralin (R-8-OH-DPAT) and 5-HT(1B) receptor knockout mice failed to demonstrate reduction of extracellular 5-HT in response to systemic administration of the 5-HT(1B) receptor agonist CP 94,253. Plasticity also developed to deletion of the complementary autoreceptor. 5-HT(1A) receptor knockout mice demonstrated a significantly greater response to CP 94,253 in the striatum, but not the ventral hippocampus, suggesting the development of enhanced sensitivity of striatal 5-HT(1B) receptors. In 5-HT(1B) receptor knockout mice, R-8-OH-DPAT evoked a significantly diminished response in the ventral hippocampus, but not the striatum, suggesting the potential desensitization of 5-HT(1A) receptors in the median raphe nucleus. The pattern of regional compensations between somatodendritic and terminal autoreceptors was confirmed by pharmacological challenges using the selective serotonin reuptake inhibitor fluoxetine combined with either a 5-HT(1A) (WAY 100635) or a 5-HT(1B/1D) (GR 127935) receptor antagonist. The regional pattern of compensation may be determined by the preferential role of 5-HT(1A) or 5-HT(1B) receptors in regulating 5-HT release. Taken together, these results demonstrate the development of regional plasticity between complementary somatodendritic and terminal autoreceptors after the genetic deletion of 5-HT(1A) or 5-HT(1B) receptors.