20 http://www.actaps.com.cn Research Paper Morphine treatment enhances extracellular ATP enzymolysis and adenosine generation in rat astrocytes LIU Wei 1,2,* YANG Zhan-Li 2, ZHOU Le-Quan 1, LI Xiao-Ying 1, YAN Fu-Man 1, GUAN Li 1, LIU Hai-Mei 1, FENG Jian-Qiang 2 1 Department of Physiology, College of Fundamental Medical Science, Guangzhou University of Chinese Medicine, Guangzhou 510006, China; 2 Department of Physiology, Zhongshan Medical College, Sun Yat-sen University, Guangzhou 510080, China Abstract: Recent studies have shown that astrocytes play important roles in ATP degradation and adenosine (a well known analgesic molecule) generation, which are closely related to pain signaling pathway. The aim of this study was to investigate whether morphine, a well known analgesic drug, could affect the speeds of ATP enzymolysis and adenosine generation in rat astrocytes. Intracellular calcium concentration ([Ca 2+ ] i ) of astrocyte was measured by flow cytometry, and the time points that morphine exerted notable effects were determined for subsequent experiments. Cultured astrocytes were pre-incubated with morphine (1 μmol/l) and then were incubated with substrates, ATP and AMP, for 30 min. The speeds of ATP enzymolysis and adenosine generation were measured by high performance liquid chromatography (HPLC). The results showed that both 1.5 and 48 h of morphine pre-incubation induced maximal ATP enzymolysis speed in astrocytes among all the time points, and there was no statistical difference of ATP enzymolysis speed between morphine treatments for 1.5 and 48 h. As to adenosine, morphine pre-incubation for 1.5 h statistically increased adenosine generation, which was degraded from AMP, in cultured astrocytes compared with control group. However, no difference of adenosine generation was observed after 48 h of morphine pre-incubation. These results indicate that treatment of morphine in vitro dynamically changes the concentrations of ATP and adenosine in extracellular milieu of astrocytic cells. In addition, astrocyte can be regarded as at least one of the target cells of morphine to induce changes of ATP and adenosine levels in central nervous system. Key words: morphine; ATP; adenosine; astrocytes 吗啡促进大鼠星形胶质细胞外 ATP 的酶解和腺苷的生成 刘微 1,2,*, 杨战利 2, 周乐全 1, 李小英 1, 闫福曼 1, 关莉 1, 刘海梅 1, 冯鉴强 2 1 广州中医药大学基础医学院生理学教研室, 广州 510006; 2 中山大学中山医学院生理学教研室, 广州 510080 摘要 : 近年来的研究表明星形胶质细胞在 ATP 的降解和腺苷的生成方面发挥了重要的作用, 而 ATP 和腺苷与痛觉的形成有着密切关系 本文旨在探讨镇痛药物吗啡是否能够影响星形胶质细胞酶解 ATP 以及生成腺苷 通过检测大鼠脑星形胶质细胞内钙的水平选择合适的吗啡孵育时间, 应用高效液相色谱 (high performance liquid chromatography, HPLC) 检测 ATP 和腺苷的水平 结果显示 :(1) 同其他处理时间点相比,1.5 和 48 h 的吗啡处理对 ATP 酶解的促进作用更为显著, 而二者相比没有统计学差别 (2) 对于镇痛物质腺苷, 同对照组相比,1.5 h 的吗啡处理能够上调腺苷生成速度, 而 48 h 的吗啡处理后腺苷生成速度同对照组没有差异 以上结果提示, 体外应用吗啡能够动态地改变星形胶质细胞外 ATP 和腺苷的水平, 星形胶质细胞可以被认为是吗啡改变中枢神经系统 ATP 和腺苷水平的靶细胞之一 关键词 : 吗啡 ;ATP; 腺苷 ; 星形胶质细胞中图分类号 :R338 Received 2010-09-19 Accepted 2010-11-25 * Corresponding author. Tel: +86-20-39358028; E-mail: weiliu1980@yahoo.com
LIU Wei et al: Morphine Enhances Extracellular ATP Enzymolysis and Adenosine Generation in Rat Astrocytes 21 Enzymes responsible for ATP degradation and adenosine generation have been reported to be expressed in astrocytes [1,2]. These enzymes are called ecto-nucleotidases, by which ATP can be degraded to ADP, AMP and finally to adenosine. Notably, ATP and adenosine have different effects on pain transmission. Exogenous administration of ATP or its receptor agonists caused thermal hyperalgesia and mechanical allodynia [3-5]. On the contrast, both systemic and intrathecal injection of adenosine had antinociceptive effects in various pain models [6-8]. In addition, endogenous adenosine was considered to mediate analgesic effect of some therapies, such as acupuncture [9]. Morphine, a well known analgesic drug, can directly affect astrocytes by binding to μ or δ receptors. Previous studies have shown that morphine modulates chemokine gene regulation in astrocytes [10], alters astrocytes proliferation and differentiation [11], and also regulates intracellular calcium concentration ([Ca 2+ ] i ) [12,13]. Whether morphine treatment could alter the activities of ecto-nucleotidases to degrade ATP and produce adenosine in astrocytes remains unknown. In addition, the activities of ecto-nucleotidases involved in ATP degradation and adenosine generation have been reported to be closely associated with calcium concentration [14]. Therefore measurement of calcium level was included to determine morphine treatment duration. In this study, we investigated the effects of both acute and chronic morphine treatment on the levels of extracellular ATP and adenosine respectively. 1 MATERIALS AND METHODS 1.1 Reagents Dulbecco s Modified Eagle s Medium Nutrient Mixture F-12 HAM (DMEM/F12) and fetal bovine serum (FBS) were both purchased from Gibco Company (USA); Morphine hydrochloride was from Qinghai Pharmaceutical Factory (China); Fluo-3 AM was purchased from Biotium (USA); ATP, AMP and adenosine were all bought from Sigma. 1.2 Astrocytes culture Cultures of rat cortical astrocytes were prepared from newborn (1 2 d) Sprague-Dawley rats according to the method of McCarthy and de Vellis [15] with a modification. Briefly, rat pups were killed by cervical dislocation and whole brains were removed. Cerebral cortices were isolated, mechanically dissociated, trypsinized, triturated and centrifuged at 400 g for 5 min. The resulting pellet was resuspended in DMEM/F12 containing 10% FBS and antibiotics, and then plated in Nunc flasks. At confluence (7 9 d in culture), the flasks were shaken vigorously at 250 r/min for 16 h to remove neurons, microglia and oligodendrocytes. Astrocytes were trypsinized and seeded onto 12- or 24-well plates. 1.3 Calcium measurement [Ca 2+ ] i was measured to determine morphine treatment duration using Fluo-3 AM staining detected by flow cytometer (FCM, BENKMAN-COULTER company, USA). Cultured astrocytes plated on 12-well plates were incubated with morphine (1 μmol/l) for 0, 1.5, 6, 24, 48 and 72 h, respectively. Then they were loaded with Fluo-3 AM (5 μmol/l) at 37 C for 30 min without light, then washed 3 times and centrifuged to get the cell pellet. [Ca 2+ ] i was measured by using FCM (exciting wavelength: 488 nm; emitting wavelength: 525 nm). These measurements were conducted in at least three independent experiments, in triplicates. The data were expressed as the percentage over control level. 1.4 Ecto-nucleotidases assay Cultured astrocytes in 24-well plates were incubated with morphine (1 μmol/l, diluted with culture media) for 1.5 and 48 h, and then washed three times. The reaction was started by the addition of 200 ml incubation medium prepared with HEPES buffer (2 mmol/l CaCl 2, 120 mmol/l NaCl, 5 mmol/l KCl, 10 mmol/l glucose, 20 mmol/l HEPES, and ph 7.4). For AMP incubation, [16] MgCl 2 (2 mmol/l) was used instead of CaCl 2. Final concentrations of ATP and AMP were both 100 mmol/l, ensuring enough substrates to evaluate the enzyme activity. After incubation at 37 C for 30 min, the medium were collected and placed on dry ice instantly, then stored at 80 C until measurement. The chromatographic conditions were as described previously with some modifications [17]. A Hypersil C18 column (4.6 mm 300 mm, 5 μm, Merk) was used, and KH 2 PO 4 (50 mmol/l) solution was used as the mobile phase at a rate of 0.5 ml/min. 20 μl of standards or samples were injected into HP1100 pump (ESA) to measure absorbance at wavelength of 254 nm. Retention times were assessed by using nucleotide standards. The non-enzymatic hydrolysis of ATP and AMP was consistently less than 5%. If cells were incubated without the addition of nucleotides, the culture medium would not present any detectable peaks. The activities were determined in at least three independent experiments, in triplicates. Specific activity was expressed as nmol/min per 1 10 6 cells.
22 1.5 Data analysis and statistics All data were presented as means±sem. Significant differences were analyzed with one-way ANOVA and following LSD test using the software SPSS (version 13.0 for Windows). P<0.05 was defined as statistically significant. 2 RESULTS 2.1 [Ca 2+ ] i remarkably changed under 1.5 and 48 h of morphine incubation In order to determine suitable time points, we used flow cytometry to measure the levels of cytoplastic calcium. Consistent with previous findings [12,13], morphine treatment firstly up-regulated the level of [Ca 2+ ] i, and then downregulated it. As shown in Fig.1, the calcium concentration increased by (15±6)% and decreased by (19±4)% after morphine incubation for 1.5 h and 48 h respectively, and the changes of [Ca 2+ ] i at the time points of 1.5 and 48 h were most significant compared with the other time points (1.5 h vs 6 h, 48 h vs 72 h, n=9, P<0.05). Therefore both the two time points were selected for the following experiments. 2.2 More ATP was degraded under 1.5 and 48 h of morphine incubation As shown in Fig. 2, control group with no morphine incubation degraded ATP at the speed of (8.468±0.143) nmol/min per 1 10 6 cells. After 1.5 h of morphine incubation, the speed of ATP enzymolysis increased to (9.061±0.231) nmol/min per 1 10 6 cells, which was significantly different from that in control group (P<0.05). Moreover, 48 h of morphine pretreatment also increased the speed of ATP enzymolysis to (9.366±0.109) nmol/min per 1 10 6 cells, which was higher than that of control group (P<0.05). However, there was no statistical difference in ATP enzymolysis between 1.5 h group and 48 h group (P>0.05). 2.3 More adenosine was generated under 1.5 and 48 h of morphine incubation Figure 3 is the statistical histograms of adenosine Fig. 2. Effect of morphine treatment on enzymolysis speed of extracellular ATP in rat cortical astrocytes detected by chromatography. Both 1.5 and 48 h of morphine (1 μmol/l) incubation enhanced the speed of ATP enzymolysis. However, there was no statistical difference in ATP enzymolysis between 1.5 and 48 h groups. Means±SEM, n=9. * P<0.05 vs control (0 h). Fig. 1 Effect of morphine treatment on intracellular calcium concentration ([Ca 2+ ] i ) in rat cortical astrocytes detected by flow cytometer. Morphine (1 μmol/l) firstly up-regulated [Ca 2+ ] i and then down-regulated it in cultured astrocytes. Means±SEM, n=9. * P<0.05 vs 6 h of morphine incubation, # P<0.05 vs 72 h of morphine incubation. Fig. 3. Effect of morphine treatment on generation speed of extracellular adenosine in rat cortical astrocytes detected by chromatography. Morphine (1 μmol/l) incubation for 1.5 h enhanced the speed of adenosine generation from AMP, while 48 h of morphine treatment had no effect on adenosine generation compared with control group. Means±SEM, n=9. * P<0.05 vs control (0 h), # P<0.05 vs 48 h of morphine incubation.
LIU Wei et al: Morphine Enhances Extracellular ATP Enzymolysis and Adenosine Generation in Rat Astrocytes 23 generation in different groups. Control group (0 h) generated adenosine at the speed of (1.899±0.249) nmol/min per 1 10 6 cells. Morphine incubation for 1.5 h increased the speed of adenosine production to (2.485±0.188) nmol/min per 1 10 6 cells, which was significantly higher than that of control group (P<0.05). However, the speed of adenosine production after 48 h of morphine incubation was (1.909±0.165) nmol/min per 1 10 6 cells, showing no difference from that of control group (P>0.05). 3 DISCUSSION In the present study, we demonstrated that acute morphine treatment induced adenosine generation in extracellular milieu of astrocytes. This result was consistent with previous reports that the antinociceptive effects of opioids were partly mediated by endogenous adenosine. Morphine acted on opioid receptors to release adenosine which subsequently bound with its receptors to produce analgesia [18]. On the contrary, intrathecal injection of adenosine antagonists inhibited antinociception produced by intrathecal injection of morphine in both hot-plate and tail-flick tests in mice and rats [19,20]. Furthermore, our results showed that both acute and chronic morphine treatment ur-pregulated ATP enzymolysis. The decrease of ATP level in extracellular astrocytes would contribute to the down-regulation of pain transmission to some extent, which may enrich the mechanisms of morphine antinociception. Additionally, all these results were observed on astrocytes, thus astrocytes may be regarded as one of the target cells in morphine-induced changes of ATP and adenosine levels. Importantly, our results showed the time-dependent changes of ATP and adenosine after morphine incubation: more ATP was degraded after 1.5 and 48 h of morphine treatment; on the contrary, the speed of adenosine generation increased after morphine incubation for 1.5 h, while morphine treatment for 48 h did not increase adenosine generation speed at all. In conjunction with our data and previous findings that ATP induced pain whereas adenosine alleviated pain [3,6-8], we assume that increased adenosine level and decreased ATP level might contribute to the analgesic effect of morphine, then adenosine returned to the normal level, and the analgesic effect decreased to some extent. This postulation was consistent with the phenomenon that acute morphine treatment alleviated pain effectively, whereas chronic administration of morphine would produce decreased antinociception effect. Furthermore, the substantial changes of ATP and adenosine concentration indicate that the activities or expressions of ectonucleotidases expressed on astrocytes responsible for ATP enzymolysis and adenosine generation are significantly influenced by morphine treatment. However, the mechanisms of morphine affecting levels of ATP and adenosine need further study. REFERENCES 11 Fields RD, Burnstock G. Purinergic signaling in neuron-glia interactions. Nat Rev Neurosci 2006; 7(6): 423 436. 22 Zimmermann H. Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system. Prog Neurobiol 1996; 49(6): 589 618. 33 Nakagawa T, Wakamatsu K, Zhang N, Maeda S, Minami M, Satoh M, Kaneko S. Intrathecal administration of ATP produces long-lasting allodynia in rats: differential mechanisms in the phase of the induction and maintenance. Neuroscience 2007; 147(2): 445 455. 44 Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 2003; 424(6950): 778 783. 55 Burnstock G. Purinergic P2 receptors as targets for novel analgesics. Pharmacol Ther 2006; 110(3): 433 454. 66 Lee YW, Yaksh TL. Pharmacology of the spinal adenosine receptor which mediates the antiallodynic action of intrathecal adenosine agonists. J Pharmacol Exp Ther 1996; 277(3): 1642 1648. 77 Sawynok J, Liu XJ. Adenosine in the spinal cord and periphery: release and regulation of pain. Prog Neurobiol 2003; 69(5): 313 340. 88 Han BF, Zhang C, Qi JS, Qiao JT. ATP-sensitive potassium channels and endogenous adenosine are involved in spinal antinociception produced by locus coeruleus stimulation. Acta Physiol Sin ( 生理学报 ) 2002; 54(2): 139 144. 99 Goldman N, Chen M, Fujita T, Xu Q, Peng W, Liu W, Jensen TK, Pei Y, Wang F, Han X, Chen J, Schnermann J, Takano T, Bekar L, Tieu K, Nedergaard M. Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture. Nature Neurosci 2010; 13(7): 883 889. 111 Mahajan SD, Schwartz SA, Aalinkeel R, Chawda RP, Sykes DE, Nair MP. Morphine modulates chemokine gene regulation in normal human astrocytes. Clin Immunol 2005; 115(3): 323 332. 111 Stiene-Martin A, Gurwell JA, Hauser KF. Morphine alters astrocyte growth in primary cultures of mouse glial cells: evidence for a direct effect of opiates on neural maturation. Brain Res Dev Brain Res 1991; 60(1): 1 7.
24 111 Hauser KF, Stiene-Martin A, Mattson MP, Elde RP, Ryan SE, Godleske CC. mu-opioid receptor induced Ca 2+ mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca 2+ dependent mechanism. Brain Res 1996; 720(122): 191 203. 111 Xiong JX ( 熊加祥 ), Li HD, Li XC. Effect of morphine on the changes of intracellular free calcium in cultured hippocampal astrocytes in rat. Acta Academiae Medicinae Militaris Tertiae ( 第三军医大学学报 ) 2003; 25(3): 251 254 (Chinese, English abstract). 111 Smith TM, Kirley TL. The calcium activated nucleotidases: A diverse family of soluble and membrane associated nucleotide hydrolyzing enzymes. Purinergic Signal 2006; 2(2): 327 333. 111 McCarthy KD, de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 1980; 85(3): 890 902. 111 Wink M, Lenz G, Braganhol E, Tamajusuku A, Schwarts- mann G, Sarkis J, Battastini A. Altered extracellular ATP, ADP and AMP catabolism in glioma cell lines. Cancer Lett 2003; 198(2): 211 218. 111 Volonté MG, Yuln G, Quiroga P, Consolini AE. Development of an HPLC method for determination of metabolic compounds in myocardial tissue. J Pharm Biomed Anal 2004; 35(3): 647 653. 111 Sweeney MI, White TD, Sawynok J. Involvement of adenosine in the spinal antinociceptive effects of morphine and noradrenaline. J Pharmacol Exp Ther 1987; 243(2): 657 665. 111 Cahill CM, White TD, Sawynok J. Spinal opioid receptors and adenosine release: neurochemical and behavioral characterization of opioid subtypes. J Pharmacol Exp Ther 1995; 275(1): 84 93. 222 Sandner-Kiesling A, Li X, Eisenach JC. Morphine-induced spinal release of adenosine is reduced in neuropathic rats. Anesthesiology 2001; 95(6): 1455 1459.