Hauser K F, Stiene-Martin A, Mattson M P, Elde R P, Ryan S E, Godleske C C
Department of Anatomy and Neurobiology, University of Kentucky Medical Center, Lexington 40536-0084, USA.
Brain Res. 1996 May 13;720(1-2):191-203. doi: 10.1016/0006-8993(96)00103-5.
Morphine, a preferential mu-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca(2+)-dependent mechanism, few studies have examined whether Ca2+ mediates the effect of opioids on cell proliferation, or whether a particular Ca2+ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of mu-opioid receptors and Ca2+ mobilization in morphine-induced astrocyte development. Morphine (1 microM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca(2+)-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca2+ ([Ca2+]o), or in unmodified media containing Ca2+ ionophore (A23187), nifedipine (1 microM), dantrolene (10 microM), thapsigargin (100 nM), or L-glutamate (100 microM) for 0-72 h. mu-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca2+ ([Ca2+]i) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating mu-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective mu agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca2+]i in developing astroglia. At normal [Ca2+]o, morphine attenuated DNA synthesis by increasing [Ca2+]i; low [Ca2+]o (0.3 mM) blocked this effect, while treatment with Ca2+ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca2+]o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca2+]i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca2+ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP3-sensitive Ca2+ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca(2+)-dependent Ca2+ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca(2+)-dependent Ca2+ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.
吗啡是一种选择性μ-阿片受体激动剂，它通过抑制细胞增殖和促进细胞分化来改变星形胶质细胞的发育。尽管吗啡通过一种依赖Ca(2+)的机制影响细胞分化，但很少有研究探讨Ca2+是否介导阿片类药物对细胞增殖的作用，或者是否有特定的Ca2+信号转导途径介导阿片类药物的作用。此外，尚不确定一种或多种阿片受体类型是否介导阿片类药物的发育效应。为了解决这些问题，本研究探讨了μ-阿片受体和Ca2+动员在吗啡诱导的星形胶质细胞发育中的作用。将富含小鼠星形胶质细胞的吗啡处理组和未用吗啡处理的培养物在补充有<0.005、0.3、1.0或3.0 mM Ca2+([Ca2+]o)的无钙培养基中，或在含有Ca2+离子载体(A23187)、硝苯地平(1 μM)、丹曲林(10 μM)、毒胡萝卜素(100 nM)或L-谷氨酸(100 μM)的未修饰培养基中孵育0至72小时。使用特异性(MOR1)抗体通过免疫细胞化学法检测μ-阿片受体表达。使用fura-2通过微量荧光分析测量细胞内Ca2+( [Ca2+]i)。在胶质纤维酸性蛋白(GFAP)免疫反应性星形胶质细胞中评估星形胶质细胞形态和溴脱氧尿苷(BrdU)掺入(DNA合成)。结果表明，吗啡通过激活μ-阿片受体抑制星形胶质细胞生长。星形胶质细胞表达MOR1免疫反应性，选择性μ激动剂PL017可模拟吗啡的作用。此外，吗啡通过动员发育中星形胶质细胞中的[Ca2+]i抑制DNA合成。在正常[Ca2+]o时，吗啡通过增加[Ca2+]i减弱DNA合成；低[Ca2+]o(0.3 mM)阻断了这种作用，而用Ca2+离子载体或谷氨酸处理可模拟吗啡的作用。在极低的[Ca2+]o(<0.005 mM)时，吗啡反而增加了BrdU掺入。尽管阿片类药物可通过多种途径增加星形胶质细胞中的[Ca2+]i，但并非所有途径都影响DNA合成或细胞形态。硝苯地平(阻断L型Ca2+通道)不能阻止吗啡诱导的BrdU掺入减少或细胞分化，而毒胡萝卜素(耗尽IP3敏感的Ca2+储存)严重影响抑制的DNA合成和细胞分化——与吗啡处理无关。然而，丹曲林(一种Ca(2+)依赖性Ca2+释放抑制剂)选择性地阻断了吗啡的作用。总体而言，这些发现表明阿片类药物通过抑制丹曲林敏感细胞内储存的Ca(2+)依赖性Ca2+释放来抑制星形胶质细胞DNA合成并促进细胞肥大。这暗示了阿片类药物影响中枢神经系统成熟的一种基本机制。
