Targeting endothelin ETA and ETB receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ET(A) and ET(B) receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ET(B) receptor antagonism, but rather by endothelin ET(A) receptor antagonism. Furthermore, endothelin ET(A), but not ET(B), receptor antagonism inhibited antigen-induced PGE(2) production, whereas either selective or combined blockade of endothelin ET(A) and/or ET(B) receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.     

mGlu and NMDA receptor contributions to capsaicin-induced thermal and mechanical hypersensitivity

Metabotropic glutamate (mGlu) receptors are G protein-coupled receptors, some of which are localized in the spinal cord dorsal horn, and are involved with pain perception. The anti-nociceptive effects of intrathecal (i.t.) pretreatment with various mGlu receptor agonists and antagonists were assessed in Long Evans rats with mechanical and thermal hypersensitivity after sub-dermal injection of capsaicin in the hindpaw. Selective group II (aminopyrrolidine-2R,4R-dicarboxylate, APDC) and group III (l-2-amino-4-phosphonobutyrate, L-AP4) agonists, as well as selective mGlu(1) (1-aminoindan-1,5(R,S)-dicarboxylic acid, AIDA) and mGlu(5) (2-methyl-6-(phenylethynyl)-pyridine, MPEP) receptor subtype antagonists were compared with that of an NMDA receptor antagonist (dizocilipine maleate, MK-801). The rats were observed for signs of capsaicin-induced mechanical and thermal hypersensitivity 15 min after capsaicin injection, and 20 min following i.t. drug administration. Results indicate there was a dose-dependent reduction in capsaicin-induced mechanical hypersensitivity for all mGlu receptor agents; with maximal increases in mechanical thresholds that were 7-fold for AIDA and APDC, 7.5-fold for L-AP4 and 5.6-fold for MPEP. However, only a weak reduction (often non-significant) in thermal hypersensitivity was observed with each of the mGlu receptor drugs; thermal latencies were maximally increased by 125% (AIDA), 0% (MPEP), 8% APDC and 205% (L-AP4). By contrast, the highest dose of MK-801 was able to significantly reduce both mechanical (maximal 6.67-fold increase in threshold) and thermal (maximal 3-fold increase in latencies) hyperalgesia. We conclude that mGlu receptors contribute to the development of mechanical allodynia, but not thermal hyperalgesia, following capsaicin injury; while iGluRs may contribute to both thermal and mechanical hypersensitivity.     

硫化氢在大鼠肺纤维化中的表达

目的：研究内源性硫化氢（H2S）在大鼠肺纤维化中的表达。方法：①实验分组：将健康Wistar大鼠30只随机分为对照组、模型组（肺纤维化组）。②模型制备：采用气管内注射博来霉素（BLM—A5）建立肺纤维化模型。③评价：通过对大鼠肺组织形态学,病理学观察,评价模型成功与否。④观察指标：于试验的第7、14、28天时每组分别处死5只大鼠。采用去蛋白分析方法测量血浆中H2S含量．取肺组织行HE染色和Mallory三色染色。结果：①模型组大鼠血浆中H2S含量第7天时较对照组降低50％．第14天时降低32％．第28天时降低21％（P〈0．01）。②造模后首先出现肺泡炎。随着时间的延长,炎性反应逐渐减轻,肺纤维化程度逐渐加重。结论：①经过观察．采用气管内注射博来霉素（BLM—A5）的方法成功的制备了大鼠肺纤维化模型。②正常大鼠体内存在一定量的硫化氢。③硫化氢参与了大鼠肺纤维化发展的病理生理过程。     

硫化氢抗炎及免疫调节作用研究进展

硫化氢是一种气体信号分子,具有多种生物活性,如抗氧化、调节细胞周期、促凋亡、抗炎和免疫调节作用等.其供体可直接或间接释放硫化氢,对心血管系统疾病、自身免疫性疾病、神经退行性疾病、糖尿病和肿瘤等多种疾病具有防治作用.本文综述硫化氢及其供体在炎症和免疫调节方面的研究进展.     

阵发性心房颤动与血浆硫化氢水平的相关性研究

目的：观察阵发性心房颤动患者血浆硫化氢(H2S)水平变化,研究其与阵发性心房颤动的相关性。方法50例阵发性心房颤动患者作为病例组,50例健康受试者作为对照组,采用去蛋白法测定血清H2S水平。结果病例组血浆H2S水平(28.63±12.74)μmol/L显著低于对照组(49.21±10.95)μmol/L (P<0.01)。结论阵发性心房颤动的发生可能与患者血浆H2S水平的降低有关。     

H2S促进神经胶质瘤细胞增殖作用的机制探讨

目的为了明确硫化氢(H2S)是否能够促进人胶质瘤U251细胞的增长,探讨Survivin和Sirt1在U251细胞中是否有表达及H2S促进U251细胞增长机制。方法 CCK8(Cell Counting Kit-8)法测细胞活力,Western Blot检测U251细胞内Survivin和Sirt1表达。结果 100-400μmol/L的H2S呈浓度依赖性地促进了U251细胞的增长;100-400μmol/L的H2S处理U251细胞24 h后,细胞内Survivin和Sirt1呈H2S浓度依赖性地增加,与空白对照比较,差异有统计学意义(P<0.01);用100μmol/L Sirt1抑制剂,Sirtinol预处理U251细胞0.5 h后,在继续用200μmol/L H2S继续培养细胞4 h后,预处理组和单独的硫化氢组相比,细胞的活力显著下降,差异有统计学意义(P<0.001),但是单独Sirtinol组对细胞活力影响不大,说明H2S促进细胞增殖功能可能是通过促进Sirt1的表达来实现的。结论 H2S促进了U251细胞的增长,H2S促进了U251细胞内Survivin和Sirt1表达,Sirt1抑制剂抑制了H2S对细胞的促进增长作用。     

冠心病患者血浆硫化氢含量变化

目的观察冠心病患者血浆硫化氢水平变化,探讨硫化氢在冠心病发病中的临床意义。方法选取冠心病患者45例,将其分为稳定型心绞痛组、不稳定型心绞痛组和心肌梗死组,各15例;选择健康体检者15名为对照组。血浆硫化氢检测采用分光光度法检测。结果冠心病患者组血浆硫化氢水平为(37．08+10．36)μmol/L低于对照组的(50．98+6．23)μmol/L(P＜0．01);冠心病稳定型心绞痛、不稳定型心绞痛组和心肌梗死组血浆硫化氢水平分别为(47．73±3．87)μmol/L、(37．38±4．60)μmol/L、(26．87±7．56)μmol/L,各组间均有显著性差异(P＜0．01)。结论冠心病患者组血浆硫化氢水平与健康人对比相对较低,并与病变程度相关,而提示内源性硫化氢不足可能参与冠心病的病理过程。     

HYP-1, a novel diamide compound, relieves inflammatory and neuropathic pain in rats

In the present study, we investigated whether a novel compound, 2-(2-(4-((4-chlorophenyl)(phenyl)methyl)piperazin-1-yl)-2-oxoethylamino)-N-(3,4,5-trimethoxybenzyl)acetamide (HYP-1), is capable of binding to voltage-gated sodium channels (VGSCs) and evaluated both its inhibitory effect on Na⁺ currents of the rat dorsal root ganglia (DRG) sensory neuron and its in vivo analgesic activity using rat models of inflammatory and neuropathic pain. HYP-1 showed not only high affinity for rat sodium channel (site 2), but also potent inhibitory activity against the TTX-R Na⁺ currents of the rat DRG sensory neuron. HYP-1 co-injected with formalin (5%, 50 μl) under the plantar surface of rat hind paw dose-dependently reduced spontaneous pain behaviors during both the early and late phases. This result was confirmed by c-Fos immunofluorescence in the L4-5 spinal segments. A large number of c-Fos-positive neurons were observed in rat injected with a mixture of formalin and vehicle, but not in rat treated with a mixture of formalin and HYP-1. In addition, the effectiveness of HYP-1 (6 and 60 mg/kg, i.p.) in suppression of neuropathic pain, such as mechanical, cold and warm allodynia, induced by rat tail nerve injury was investigated. HYP-1 showed limited selectivity over hERG, N-type and T-type channels. Our present results indicate that HYP-1, as a VGSC blocker, has potential analgesic activities against nociceptive, inflammatory and neuropathic pain.     

硫化氢对中枢神经系统作用的研究进展

在哺乳类动物中枢神经系统中硫化氢（H2S）主要来源于胱硫醚-β-合成酶（cystanthi-onine-β-synthetase,CBS）的催化作用,并受到细胞内钙浓度的影响与变构激活剂S-腺苷蛋氨酸的调节。已有的研究提示在哺乳动物的大脑中H2S充当气体信号分子,具有维持钙稳态、诱导长时程效应增强（long-term potentiation,LTP）、抑制氧化应激和调节神经信号等生理功能。H2S通过多种方式介导生物效应,除了其强还原性之外还具有促进环磷腺苷（cAMP）的生成、激活ATP依赖性的钾离子通道、增加细胞内钙浓度等作用。H2S也与中枢神经系统的作用病理学相关,比如中风、阿尔兹海默氏症等。由于实验表明控制H2S的含量能够在多种疾病模型起到保护性治疗作用,因此H2S在中枢神经系统疾病领域有良好的治疗学前景。     

硫化氢联合亚低温对肢体缺血再灌注后大鼠心肌损伤的影响

目的评价硫化氢联合亚低温对肢体缺血再灌注后大鼠心肌损伤的影响。方法健康雄性Wistar大鼠60只,体重260³00 g,采用随机数字表法,将其随机分为5组(n=12):假手术组(S组)、肢体缺血再灌注组(IR组)、硫氢化钠组+IR组(H组)、亚低温+IR组(M组)和硫氢化钠联合亚低温+IR组(HM组)。采用双大腿根部止血带制备大鼠双后肢缺血及再灌注模型。光镜下观察心肌组织形态学变化。采用原位末端标记(TUNEL)法观察肺组织细胞凋亡情况;采用蛋白质印迹方法(Western blotting)测定葡萄糖调节蛋白(GRP78)和半胱氨酸蛋白水解酶(Caspase-12)蛋白表达。结果 M组、H组和HM组病理学损伤较IR组减轻,其中HM组减轻最明显。与S组比较,IR组、H组、M组和HM组心肌细胞凋亡指数升高,GRP78和Caspase-12蛋白表达上调(P<0.05);与IR组比较,H组、M组和HM组心肌细胞凋亡指数降低,GRP78和Caspase-12蛋白表达下调(P<0.05);与H组和M组比较,HM组心肌细胞凋亡指数降低,GRP78和Caspase-12蛋白表达下调(P<0.05)。结论硫化氢联合亚低温可减轻肢体缺血再灌注后大鼠心肌损伤,其机制与下调心肌细胞GRP78和Caspase-12蛋白的表达,减轻内质网应激反应有关。     

持续性房颤与血浆硫化氢水平的关系研究

目的：观察持续性房颤患者血浆硫化氢(H2S)水平的变化,研究其与持续性房颤的关系。方法选择本院2013年4月~2014年7月心内科住院的持续性房颤患者60例为病例组,其中男性32例,女性28例,平均年龄(61.07±10.35)岁。选取同期健康人60例为对照组,其中男性30例,女性30例,平均年龄(60.13±9.64)岁。采用敏感硫电极法测定血清H2S水平。结果病例组血浆H2S水平显著低于对照组(P<0.01)。结论持续性房颤的发生可能与血浆H2S水平的降低有关。     

硫化氢对老龄急性脑梗死大鼠海马神经元腺苷酸活化蛋白激酶和星形胶质细胞的影响

目的评价硫化氢(H2S)对老龄急性脑梗死大鼠海马神经元腺苷酸活化蛋白激酶和星形胶质细胞的影响。方法健康雄性老龄SD大鼠96只,体重450⁵50 g,采用随机数字表法,将其随机分为4组:对照组(C组)、模型组(O组)、生理盐水+模型组(S组)、H2S组+模型组(H组),每组24只。采用线栓法致大鼠大脑中动脉阻塞致急性脑梗死模型。H组于制作模型前25 min给予大鼠腹腔注射H2S外源性供体NaHS(14μmol/kg);S组于制作模型前25 min给予大鼠腹腔注射注入等量的生理盐水;C组不行任何处理。采用Morris水迷宫法记录大鼠逃避潜伏期和游泳路径;采用RT-PCR法和Western-blot法分别测定大鼠海马AMPK mRNA和AMPK、p-AMPK的表达。采用免疫组织化学法计数GFAP阳性细胞总个数。结果与C组比较,O组、S组、H组大鼠逃避潜伏期和游泳路径延长,海马AMPK mRNA、AMPK、p-AMPK表达上调,GFAP表达细胞数量增多(P<0.05);与O组、S组比较,H组大鼠逃避潜伏期和游泳路径缩短,海马AMPK mRNA、AMPK、p-AMPK表达下调,GFAP表达细胞数量减少(P<0.05)。结论 H2S可改善老龄急性脑梗死大鼠认知功能,其机制可能与下调海马神经元腺苷酸活化蛋白激酶和抑制星形胶质细胞激活有关。     

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice

Hydrogen sulfide (H₂S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H₂S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H₂S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine β-synthase (CBS) has been shown to catalyze H₂S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H₂S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H₂S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H₂S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H₂S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H₂S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H₂S for the treatment of cardiovascular and other diseases.     

硫化氢对老龄急性脑梗死大鼠认知功能的影响

目的评价硫化氢对老龄急性脑梗死大鼠认知功能的影响。方法选取健康雄性SD大鼠(青岛大学实验动物科学部提供)68只,月龄18²0个月,体重50020个月,体重500⁷00 g,采用随机数字表分为4组(n=17):对照组(C组)、模型组(M组)、Na HS组(N组)和生理盐水组(S组)。N组于制作模型前25 min给予大鼠腹腔注射H2S外源性供体Na HS(14μmol/kg);S组于制作模型前25 min给予大鼠腹腔注入等量的生理盐水;C组不行任何处理。采用线栓法致大鼠大脑中动脉阻塞致急性脑梗死模型。采用Morris水迷宫测试大鼠逃避潜伏期和穿越平台次数。采用ELISA法检测血清S-100β蛋白和NSE浓度。结果与C组比较,M组、N组和S组急性脑梗死后逃避潜伏期延长,穿越平台次数减少,T2、T3、T4各点血清S-100β及NSE浓度明显升高(P<0.05);与M组比较,N组急性脑梗死后逃避潜伏期缩短,穿越平台次数增多,T2、T3、T4各点血清S-100β及NSE浓度明显降低(P<0.05);与N组比较,S组急性脑梗死后逃避潜伏期延长,穿越平台次数减少,T2、T3、T4各点血清S-100β及NSE浓度明显升高(P<0.05),与第1天比较,M组、N组和S组各组第2700 g,采用随机数字表分为4组(n=17):对照组(C组)、模型组(M组)、Na HS组(N组)和生理盐水组(S组)。N组于制作模型前25 min给予大鼠腹腔注射H2S外源性供体Na HS(14μmol/kg);S组于制作模型前25 min给予大鼠腹腔注入等量的生理盐水;C组不行任何处理。采用线栓法致大鼠大脑中动脉阻塞致急性脑梗死模型。采用Morris水迷宫测试大鼠逃避潜伏期和穿越平台次数。采用ELISA法检测血清S-100β蛋白和NSE浓度。结果与C组比较,M组、N组和S组急性脑梗死后逃避潜伏期延长,穿越平台次数减少,T2、T3、T4各点血清S-100β及NSE浓度明显升高(P<0.05);与M组比较,N组急性脑梗死后逃避潜伏期缩短,穿越平台次数增多,T2、T3、T4各点血清S-100β及NSE浓度明显降低(P<0.05);与N组比较,S组急性脑梗死后逃避潜伏期延长,穿越平台次数减少,T2、T3、T4各点血清S-100β及NSE浓度明显升高(P<0.05),与第1天比较,M组、N组和S组各组第2⁵天逃避潜伏期缩短(P<0.05),与T1比较,M组、N组和S组T2、T3、T4各点血清S-100β及NSE浓度升高(P<0.05)。结论硫化氢可减轻急性脑梗死后大鼠认知功能障碍,其机制可能与降低血清S-100β和NSE浓度有关。     

Garlic-derived natural polysulfanes as hydrogen sulfide donors: Friend or foe?

In vitro and in vivo studies reported the anti-cancer potential of organosulfur compounds (OSCs) as they trigger biological effects leading to cell cycle arrest with accumulation of cells in G2/M, alteration of the microtubular network, modulation of Bcl-2 family protein expression patterns and changes of the redox status. Despite these well-described effects, no OSC derivative is yet undergoing clinical trials even though their chemistry is well understood as OSCs act as hydrogen sulfide (H₂S) donors. H₂S is a biological mediator, synthesized through cysteine degradation and modulates vasodilation, cytoprotection, inflammation and angiogenesis. It is well accepted that H₂S plays a biphasic pharmacological role: the inhibition of endogenous synthesis of H₂S and paradoxically also the use of H₂S donors to increase H₂S concentration, induce both anti-cancer effects leading therefore to controversial discussions. Altogether, the role of H₂S in the anti-cancer action of OSCs remains poorly understood. In this review, we hypothesize that OSCs act through H₂S signaling pathways in cancer cells, and that a clearer understanding of the mechanism of action of H₂S in OSC-mediated anti-cancer activity is required for further application of these compounds in translational medicine.     

Role of complement C5a in mechanical inflammatory hypernociception: potential use of C5a receptor antagonists to control inflammatory pain

BACKGROUND AND PURPOSE: C5a, a complement activation product, exhibits a broad spectrum of inflammatory activities particularly neutrophil chemoattraction. Herein, the role of C5a in the genesis of inflammatory hypernociception was investigated in rats and mice using the specific C5a receptor antagonist PMX53 (AcF-[OP(D-Cha)WR]). EXPERIMENTAL APPROACH: Mechanical hypernociception was evaluated with a modification of the Randall-Selitto test in rats and electronic pressure meter paw test in mice. Cytokines were measured by ELISA and neutrophil migration was determined by myeloperoxidase activity. KEY RESULTS: Local pretreatment of rats with PMX53 (60-180 microg per paw) inhibited zymosan-, carrageenan-, lipopolysaccharide (LPS)- and antigen-induced hypernociception. These effects were associated with C5a receptor blockade since PMX53 also inhibited the hypernociception induced by zymosan-activated serum and C5a but not by the direct-acting hypernociceptive mediators, prostaglandin E(2) and dopamine. Underlying the C5a hypernociceptive mechanisms, PMX53 did not alter the cytokine release induced by inflammatory stimuli. However, PMX53 inhibited cytokine-induced hypernociception. PMX53 also inhibited the recruitment of neutrophils induced by zymosan but not by carrageenan or LPS, indicating an involvement of neutrophils in the hypernociceptive effect of C5a. Furthermore, the C5a-induced hypernociception was reduced in neutrophil-depleted rats. Extending these findings in rats, blocking C5a receptors also reduced zymosan-induced joint hypernociception in mice. CONCLUSIONS AND IMPLICATIONS: These results suggest that C5a is an important inflammatory hypernociceptive mediator, acting by a mechanism independent of hypernociceptive cytokine release, but dependent on the presence of neutrophils. Therefore, we suggest that inhibiting the action of C5a has therapeutic potential in the control of inflammatory pain.     

The oxidative damage and inflammatory response induced by lead sulfide nanoparticles in rat lung

Lead sulfide nanoparticles (PbS NPs) are one important nanoparticle materials which is widely used in photoelectric production, but its potential health hazard to respiratory system is not clear. This study aimed to explore the possible mechanism of lung injury induced by PbS NPs. Male SD rats were treated with nanoparticles of 60 nm and 30 nm lead sulfide. The main methods were detecting the vigor of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) and the content of malondialdehyde (MDA) in both blood and lung tissues and observing the pathological changes in lung tissue. PbS NPs suppressed the activity of SOD and T-AOC, and increased serum MDA content (P < 0.05); both effects were observed together in lung tissues of 30-nm group (P < 0.05) accompanied by an obviously inflammatory response. PbS NPs induced oxidative damage and inflammatory response in lung tissue, which may be an underlying mechanism for its pulmonary toxicity. Additionally, the toxicity of PbS NPs was closely related with the size of nanoparticles.     

米力农对老年缺血性心力衰竭患者硫化氢及同型半胱氨酸的影响

目的评价米力农对老年缺血性心力衰竭患者硫化氢(H2S)及同型半胱氨酸(HCY)的影响。方法选择2013年1-11月于我院住院确诊的老年冠状动脉性心脏病心力衰竭患者100例,年龄60⁸0岁,体重6080岁,体重60⁹0 kg,NYHAⅢ90 kg,NYHAⅢ^Ⅳ级。采用随机、双盲、对照法,将其随机分为2组,每组50例:对照组(C组)和米力农组(D组)。米力农组在常规治疗的基础上给予米力农注射液10 mg/d,连续泵入5 d。对照组在常规治疗的基础上泵入等量的生理盐水。于治疗前(T1)、治疗后3 d(T2)、治疗后5 d(T3)采用去蛋白法测定血浆H2S浓度,荧光偏振免疫分析法测定血清HCY浓度,彩色多普勒超声测量患者左室射血分数(LVEF)。结果与C组比较,D组T2、T3时H2S浓度升高、HCY的浓度降低,LVEF升高(P<0.05)。与T1比较,两组T2、T3时H2S浓度升高、HCY的浓度降低,LVEF升高(P<0.05);与T2比较,两组T3时H2S浓度升高、HCY的浓度降低,LVEF升高(P<0.05)。结论米力农可改善老年缺血性心力衰竭患者心脏功能,其机制可能与升高血浆H2S浓度及降低HCY浓度有关。     

Hop Extract Produces Antinociception by Acting on Opioid System in Mice

In the present study, the antinociceptive profiles of hop extract were characterized in ICR mice. Hop extract administered orally (from 25 to 100 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. Antinociceptive action of hop extract was maintained at least for 60 min. Moreover, cumulative response time of nociceptive behaviors induced with intraplantar formalin injection was reduced by hop extract treatment during the 2nd phases. Furthermore, the cumulative nociceptive response time for intrathecal injection of substance P (0.7 µg) or glutamate (20 µg) was diminished by hop extract. Intraperitoneal pretreatment with naloxone (an opioid receptor antagonist) attenuated antinociceptive effect induced by hop extract in the writhing test. However, methysergide (a 5-HT serotonergic receptor antagonist) or yohimbine (an α₂-adrenergic receptor antagonist) did not affect antinociception induced by hop extract in the writhing test. Our results suggest that hop extract shows an antinociceptive property in various pain models. Furthermore, the antinociceptive effect of hop extract may be mediated by opioidergic receptors, but not serotonergic and α₂-adrenergic receptors.