Neighboring glycine residues are essential for P2X2 receptor/channel function

The roles of a glycine-rich region in the cloned P2X₂ receptor/channel were evaluated by site-directed mutagenesis. Responsiveness to ATP was lost when Gly²⁴⁷ was replaced by alanine. The sensitivity to ATP was reduced when Gly²⁴⁸ was replaced by alanine, and the responsiveness to ATP was lost when Gly²⁴⁸ was replaced by valine. The results suggest that the neighboring glycine residues are essential for P2X₂ receptor/channel function.     

大黄素对P2X_(2/3)受体介导的三叉神经痛的作用研究

目的探究大黄素(emodin)对P2X2/3受体介导的三叉神经痛的作用影响。方法慢性压迫性损伤大鼠眶下神经(CCI-ION)建立大鼠三叉神经痛动物模型,应用von Frey细丝法测试机械痛敏阈值,观察大黄素对三叉神经痛(TN)大鼠机械痛敏阈值的影响。结合原位杂交、PT-PCR、免疫组织化学、免疫荧光、蛋白印迹等方法观察大鼠三叉神经节(TG)中P2X2/3受体的表达变化。结果术后14 d,Ⅲ组(TN模型组)和Ⅰ组(生理盐水对照组)、Ⅱ组(假手术组)、Ⅳ组(TN模型+大黄素干预组)相比较,大鼠手术侧眶下神经面部感觉区域机械痛敏阈值明显降低(P<0.01),大鼠TG中P2X2/3受体表达明显升高(P<0.01);Ⅰ、Ⅱ、Ⅳ组之间相比,大鼠手术侧眶下神经面部感觉区域机械痛敏阈值差异没有显著性(P>0.05);Ⅳ组TG中P2X2/3受体的表达较Ⅲ组低(P<0.01)。结论 P2X2/3受体涉及到三叉神经痛,大黄素对P2X2/3受体介导的三叉神经痛有一定抑制作用。     

Neighboring glycine residues are essential for P2X2 receptor/channel function.

The roles of a glycine-rich region in the cloned P2X₂ receptor/channel were evaluated by site-directed mutagenesis. Responsiveness to ATP was lost when Gly²⁴⁷ was replaced by alanine. The sensitivity to ATP was reduced when Gly²⁴⁸ was replaced by alanine, and the responsiveness to ATP was lost when Gly²⁴⁸ was replaced by valine. The results suggest that the neighboring glycine residues are essential for P2X₂ receptor/channel function.     

Peripheral adenosine 5′-triphosphate enhances nociception in the formalin test via activation of a purinergic p2X receptor

The pronociceptive effects of adenosine 5′-triphosphate (ATP) were examined in the low concentration formalin model (0.5%) by coadministration of ATP, ATP analogs (,β-methylene-ATP and 2-methylthio-ATP) and antagonists (suramin, pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid) with formalin and determining effects on the expression of flinching behaviours. Coadministration of ATP (5–500 nmol) with formalin enhanced phase 2 (12–60 min after injection) but not phase 1 (0–10 min after injection) responses. ,β-methylene-ATP (0.5–50 nmol) but not 2-methylthio-ATP (50–500 nmol) produced a similar enhancement of activity, generating an order of potency of ,β-methylene-ATP, ATP2-methylthio-ATP. This enhancement was primarily expressed in the latter part of phase 2, 30–60 min after injection. Coadministration of suramin 50–500 nmol, a non-selective P_2X and P_2Y purinoceptor antagonist and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid 5–500 nmol, a selective P_2X purinoceptor antagonist, dose-dependently inhibited the augmentation of the formalin response by ATP 50 nmol, but did not reduce the response to formalin itself. Pretreatment for 30 min with higher doses of suramin inhibited the response to formalin (0.5%, 1.5%) and this appeared to be by a systemically mediated action as it was seen following administration into the contralateral paw. The results of this study provide evidence in support of a P_2X purinoceptor mediated augmentation of the pain signal by ATP. The delayed time-course of the effect suggests that it may occur in concert with other mediators that are recruited by the inflammatory process, rather than reflecting a direct depolarization of sensory nerves. Other behavioural paradigms may be required to examine the fast onset, direct effect. Suramin appears to exert both local and systemic effects on the expression of pain behaviours in response to formalin.     

Anxiolytic-like actions of BW 723C86 in the rat Vogel conflict test are 5-HT2B receptor mediated

The 5-HT_2B receptor agonist, BW 723C86 (10, 30 mg/kg i.p. 30 min pre-test), increased the number of punishments accepted in a rat Vogel drinking conflict paradigm over 3 min, as did the benzodiazepine anxiolytics, chlordiazepoxide (2.5–10 mg/kg p.o. 1 h pre-test) and alprazolam (0.2–5 mg/kg p.o. 1 h pre-test), but not the 5-HT_2C/2B receptor agonist, m-chlorophenylpiperazine (mCPP, 0.3–3 mg/kg i.p) or the 5-HT_1A receptor agonist, buspirone (5–20 mg/kg p.o. 1 h pre-test). The effect of BW 723C86 was unlikely to be secondary to enhanced thirst, as BW 723C86 did not increase the time that rats with free access to water spent drinking, nor did it reduce sensitivity to shock in the apparatus. The anti-punishment effect of BW 723C86 was opposed by prior treatment with the 5-HT_2C/2B receptor antagonist, SB-206553 (10 and 20 mg/kg p.o. 1 h pre-test), and the selective 5-HT_2B receptor antagonist, SB-215505 (1 and 3 mg/kg p.o. 1 h pre-test), but not by the selective 5-HT_2C receptor antagonist, SB-242084 (5 mg/kg p.o.), or the 5-HT_1A receptor antagonist, WAY 100635 (0.1 or 0.3 mg/kg s.c. 30 min pre-test). Thus, the anti-punishment action of BW 723C86 is likely to be 5-HT_2B receptor mediated. This is consistent with previous reports that BW 723C86 exhibited anxiolytic-like properties in both the social interaction and Geller-Seifter conflict tests.     

P2X7R 拮抗剂对小鼠慢性胰腺炎的作用及机制研究

目的 探讨嘌呤能 P2X7 受体（P2X7R）拮抗剂——氧化三磷酸腺苷（OxATP）和亮蓝 G（BBG）在抑制下游靶蛋白——核苷酸结合寡聚化结构域样受体 3（NLRP3）炎性体活化和慢性胰腺炎（CP）胰腺纤维化进程中的作用及可能机制。 方法 40 只 C57BL/6 小鼠随机均分为正常对照组、CP 模型组、OxATP 组和 BBG 组。 除正常对照组腹腔注射与处理组等体积的生理盐水外, 其余组均通过腹腔注射雨蛙素法制作 CP 小鼠模型（6 周）。 造模结束后, CP 模型组、 OxATP 组和 BBG 组分别予以腹腔注射生理盐水、OxATP（20 μL, 300 μmol/L）和 BBG（20 μL, 10 μmol/L）处理, 连续注射 2 周。 然后处死各组小鼠并取胰腺组织, 行病理学检查, 以 HE 染色对胰腺病理组织学（炎性细胞浸润、腺泡萎缩及纤维化程度）进行评分, 天狼星红染色和 α-平滑肌肌动蛋白（α-SMA）免疫组化染色分别对胰腺纤维化程度进行评估;免疫组化法检测胰腺 P2X7R、NLRP3 和半胱氨酸天冬氨酸蛋白酶 1（Caspase-1）中的累积光密度（IOD）值。 结果 与正常对照组比, CP 模型组炎症损伤组织学评分增加, 胰腺纤维化程度显著增加, P2X7R、NLRP3 和 Caspase-1 免疫组化 IOD 值显著升高（P < 0.05）; 与 CP 模型组相比, OxATP 和 BBG 组炎症损伤组织学评分降低, HE 染色纤维化评分、天狼星红染色和 α-SMA 免疫组化染色均显著减轻（P < 0.05）, 胰腺 P2X7R、NLRP3 和 Caspase-1 IOD 值均明显降低（P < 0.05）。 结论 P2X7R 拮抗剂 OxATP 和 BBG 能够显著减轻 CP 小鼠模型的慢性炎症和纤维化程度, 阻断 P2X7R-NLRP3 炎性体信号通路, 这有望成为治疗 CP 及其纤维化进程的一种潜在新策略。     

Nitric oxide modulates cardiovascular function in the rat by activating adenosine A2A receptors and inhibiting acetylcholine release in the rostral ventrolateral medulla

1. Nitric oxide (NO), a gas transmitter, modulates many physiological processes, including the central regulation of cardiovascular activity. However, the mechanisms underlying the regulation of cardiovascular activity remain relatively unexplored. In the present study, we hypothesized that central NO-dependent sympathetic inhibition is mediated by activation of adenosine A(2A) receptors (A(2A)R) and inhibition of acetylcholine (ACh) release in the rostral ventrolateral medulla (RVLM). 2. L-Arginine (L-Arg; an NO donor; 100 nmol/100 nL) was microinjected into the RVLM of male Sprague-Dawley rats and heart rate variability (HRV) was assessed as an index of cardiac sympathovagal balance. Following microinjection of L-Arg, decreases were seen in mean arterial pressure (MAP), heart rate (HR) and the ratio of the low- to high-frequency components (LF/HF) of HRV. Pretreatment of rats with SCH58261 (40 pmol/60 nL into the RVLM), a competitive antagonist of the A(2A) R, attenuated these effects. 3. Western blot analysis and ELISA revealed that adenosine and A(2A)R levels increased in the RVLM following L-Arg microinjection, whereas ACh and muscarinic M(1) receptor levels decreased significantly, in parallel with the cardiovascular responses to L-Arg microinjection. The decrease in ACh levels was abolished by SCH58261 pretreatment. 4. Microinjection of N(G)-nitro-L-arginine methyl ester (a non-selective inhibitor of NO synthase; 15 nmol/100 nL) into the RVLM significantly increased MAP, HR and sympathetic activity, as evidenced by HRV (LF, HF and the LF/HF ratio were all increased). 5. The results indicate that the central NO/NO synthase system in the RVLM may modulate cardiovascular activity by activating the A(2A)R, which subsequently inhibits activation of the muscarinic M(1) receptor.     

基因检测指导P2Y12受体拮抗药个体化治疗的研究现状

本文综述基因检测指导P2Y₁₂受体拮抗药个体化治疗的研究进展,对细胞色素P450 2C19代谢型、药品说明书、临床指南、随机对照试验等相关资料进行综述,为读者了解相关领域提供参考。     

M3受体对体外H2O2诱导大鼠心肌细胞凋亡的保护作用

目的探讨M₃受体激动对H₂O₂诱导的大鼠培养心肌细胞凋亡的作用，进一步阐明其机制。方法末端标记法 (TUNEL)进行细胞凋亡检测；免疫组化方法检测Bcl-2和Fas的表达；共聚焦显微镜观察［Ca²⁺］_i荧光强度变化。结果M₃受体激动剂胆碱(10 mmol·L^-1)可减少H₂O₂诱导的心肌细胞凋亡的数量，并可增加心肌Bcl-2的表达，减少Fas表达，抑制H₂O₂诱导的［Ca²⁺］_i荧光强度的升高。但预先应用4DAMP (10 nmol·L^-1)阻断M₃受体可逆转胆碱作用。结论激动M₃受体对H₂O₂诱导的心肌细胞凋亡有保护作用，其机制可能与Bcl-2和Fas表达以及下调［Ca²⁺］_i有关。     

Anxiolytic activity of tachykinin NK2 receptor antagonists in the mouse light-dark box

The tachykinin NK₂ receptor antagonists, GR100679 (0.02–200 μg/kg s.c.) and (±)-SR48968 (0.05–5.0 μg/kg s.c.), dose-dependently increased the time which mice spent in the light side of the light-dark box. There was no evidence of sedation or other overt behaviours. The amplitudes of these effects were similar to that evoked by diazepam (1.75 mg/kg s.c.). These results indicate a disinhibitory action of NK₂ antagonists on suppressed behaviours in a novel aversive environment. This suggests an involvement of NK₂ receptors in anxiety-related behaviours.     

The receptor mechanisms underlying the disruptive effects of haloperidol and clozapine on rat maternal behavior: A double dissociation between dopamine D2 and 5-HT2A/2C receptors

Many antipsychotic drugs disrupt active components of maternal behavior such as pup approach, pup retrieval and nest building at clinically relevant doses in postpartum female rats. However, the neurochemical mechanisms underlying such a disruptive effect remain to be determined. This study examined the neurochemical mechanisms that mediate the disruptive effects of haloperidol (a typical antipsychotic) and clozapine (an atypical antipsychotic) on rat maternal behavior. Postpartum rats were administered with haloperidol (0.2 mg/kg, sc) or clozapine (10.0 mg/kg, sc) together with either vehicle (saline or water), quinpirole (a selective dopamine D₂/D₃ agonist, 0.5 or 1.0 mg/kg, sc), or 2,5-dimethoxy-4-iodo-amphetamine (DOI, a selective 5-HT_2A/2C agonist, 1.0 or 2.5 mg/kg, sc), and their maternal behaviors were tested at different time points before and after drug administration. Haloperidol and clozapine treatment disrupted pup approach, pup retrieval, pup licking and nest building. Pretreatment of quinpirole, but not DOI, dose-dependently reversed the haloperidol-induced disruptions. In contrast, pretreatment of DOI, but not quinpirole, dose-dependently reversed the clozapine-induced disruptions. Quinpirole pretreatment even exacerbated the clozapine-induced disruption of pup retrieval and nest building. These findings suggest a double dissociation mechanism underlying the disruption of haloperidol and clozapine on rat maternal behavior. Specifically, haloperidol disrupts maternal behavior primarily by blocking dopamine D₂ receptors, whereas clozapine exerts its disruptive effect primarily by blocking the 5-HT_2A/2C receptors. Our findings also suggest that 5-HT receptors are involved in the mediation of rat maternal behavior.     

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site

Experimental probing of the model of the muscarinic M₂ receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8–15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [³H] N-methylscopolamine and [³H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [³H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [³H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (<10-fold) to the binding site of the M₂ receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M₂ receptor binding site but W155 is important for proper ligand binding on the muscarinic M₂ receptor, as predicted by the proposed model.     

腺苷A1受体阻断剂对学习记忆的影响及机制分析腺苷A1受体阻断剂对学习记忆的影响及机制分析

目的探讨腺苷A₁受体阻断剂对学习记忆的影响及其与胆碱能、氨基酸能神经的关系。方法采用避暗实验、分光光度法和HPLC法,观察腺苷A₁受体特异性阻断剂8-环戊-1,3-二丙基黄嘌呤(DPCPX)对东莨菪碱(Scop)、2-氨基-5-磷戊酸(AP₅)致小鼠记忆障碍及脑胆碱酯酶(AChE)活性、氨基酸水平的影响。结果DPCPX可显著改善Scop致记忆障碍,但对AP₅致记忆障碍无影响；在体内外高剂量DPCPX可显著抑制小鼠脑AChE活性；DPCPX icv可显著升高小鼠脑Glu和Asp含量,降低GABA含量,使脑内Glu/GABA比值显著升高。结论腺苷A₁受体特异性阻断剂DPCPX可显著改善Scop而不能改善AP₅致记忆障碍,在高剂量时可影响脑AChE活性和脑氨基酸水平、升高脑内Glu/GABA比值。     

The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A3 adenosine receptor

Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A₃ (hA₃R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([¹²⁵I]N⁶-(4-amino-3-iodobenzyl) adenosine-5′-(N-methyluronamide) - [¹²⁵I] AB MECA) binding at the hA₃R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [¹²⁵I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA₃R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA₃R and the basal signalling of this receptor. Since the hA₃R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.     

Characterizing the effects of 5-HT2C receptor ligands on motor activity and feeding behaviour in 5-HT2C receptor knockout mice

5-HT_2C receptor agonists have considerable therapeutic potential, however there is little in vivo data to compare the potency and selectivity of 5-HT_2C receptor agonists. Since 5-HT_2C receptor agonists reduce locomotor activity and food intake, changes in these drug-induced behaviours in 5-HT_2C receptor knockout mice could provide a means to examine receptor selectivity in-vivo. Initially this study compared older 5-HT_2C agonists mCPP and MK212, to newer, apparently more selective compounds: Ro 60-0175, WAY161503, CP809,101 and lorcaserin (APD356) on motor activity in wild-type, and 5-HT_2C receptor knockout mice. Two 5-HT_2C receptor antagonists SB242084 and SDZ SER 082 were also examined. mCPP did not significantly alter activity in wild-type mice, but enhanced activity in knockout animals. MK212 (3 and 10 mg/kg) and Ro 60-0175 (1 and 3 mg/kg) reduced activity in wild-type but not knockout animals. At 10 mg/kg, Ro 60-0175 reduced activity in knockout animals, suggesting loss of 5-HT_2C receptor selectivity. CP809,101 and lorcaserin reduced activity in wild-type but not knockout mice. In subsequent feeding studies, Ro 60-0175 and lorcaserin reduced food intake in wild-type animals only. Selectivity of effect for mCPP was marginal. The antagonist SB242084 increased activity in wild-type animals but not in knockout mice; SB242084 did not alter feeding in either genotype. SDZ SER 082 reduced activity in both genotypes implying poor selectivity for 5-HT_2C receptors. The data demonstrate that studying food intake, and particularly motor behaviour, in the 5-HT_2C receptor knockout mouse is a useful and relatively simple approach for screening 5-HT_2C receptor ligands in vivo.     

肿瘤免疫治疗新靶标PGE2受体EP4的研究进展

肿瘤免疫治疗已成为人们对抗癌症的重要手段,但响应率低仍是目前亟需解决的关键问题。大量研究表明,逆转肿瘤免疫抑制是阻断肿瘤免疫逃逸、增强和扩大免疫疗法疗效的重要策略。前列腺素E₂(PGE₂)是肿瘤微环境中的强效免疫介质,可特异性结合细胞膜上的七次跨膜蛋白EP4受体,诱导肿瘤微环境免疫抑制,驱动肿瘤免疫逃逸。特异性阻断PGE₂/EP4信号通路可有效解除肿瘤微环境免疫抑制,增强抗肿瘤免疫反应,促进肿瘤消退。本文从EP4受体的结构、信号转导、调控机制及其拮抗剂开发现状等方面阐述了EP4受体在肿瘤免疫治疗领域的新进展和新发现,并展望了新的发展方向。     

P2Y1 receptors inhibit long-term depression in the prefrontal cortex

Long-term depression (LTD) is a form of synaptic plasticity that may contribute to information storage in the central nervous system. Here we report that LTD can be elicited in layer 5 pyramidal neurons of the rat prefrontal cortex by pairing low frequency stimulation with a modest postsynaptic depolarization. The induction of LTD required the activation of both metabotropic glutamate receptors of the mGlu1 subtype and voltage-sensitive Ca²⁺ channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca²⁺. The subsequent release of Ca²⁺ from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y₁ receptors blocked LTD. This effect was prevented by P2Y₁ receptor antagonists and was absent in mice lacking P2Y₁ but not P2Y₂ receptors. We also found that activation of P2Y₁ receptors inhibits Ca²⁺ transients via VSCCs in the apical dendrites and spines of pyramidal neurons. In addition, we show that the release of ATP under hypoxia is able to inhibit LTD by acting on postsynaptic P2Y₁ receptors. In conclusion, these data suggest that the reduction of Ca²⁺ influx via VSCCs caused by the activation of P2Y₁ receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex.