類腺苷酸受體調控九孔(Haliotis diversicolor)血液細胞 附著之胞內訊息傳導

Adenosine-like receptor mediates the intracellular signaling transduction on hemocyte adhesion in abalones (Haliotis diversicolor)

陳家嬅(Chia-Hua Chen)

指導教授 ： 陳俊宏

國立臺灣大學/生命科學院/動物學研究研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00977

摘要

細胞防禦機制，例如：細胞吞噬(phagocytosis)以及囊包作用(encapsulation)，是先天免疫中主要的一類免疫反應機制，而血球細胞附著是細胞防禦機制的起始步驟。相對於在哺乳動物血球細胞中已有的詳細研究，無脊椎血液細胞的附著機制尚未清楚了解。在這篇研究中，九孔(Haliotis diversicolor)血液細胞處理咖啡因(caffeine)和其他的腺苷酸受體促效劑(adenosine receptor agonists)會影響血液細胞的附著。腺苷酸(adenosine)、咖啡因以及A1和A3腺苷酸受體促效劑會抑制九孔血液細胞附著，在另一方面，CGS21680(A2A腺苷酸受體促效劑)可以回復受到腺苷酸所抑制的血液細胞附著。除此之外，ZM241385(A2A腺苷酸受體對抗劑; Adenosine A2A receptor antagonist)與腺苷酸同時作用能更抑制九孔血液細胞的附著。這些實驗結果顯示，可能有多種不同的類型的腺苷酸受體存在於九孔血液細胞上，且類A1腺苷酸受體活化可能是造成九孔血液細胞附著受抑制的原因。腺苷酸受體是G 蛋白耦合性受體(G-protein coupled receptor)家族的一員，此受體主要的下游訊息傳導路徑為環苷腺酸-蛋白激酶A(cAMP-PKA)和磷脂酶C(PLC)相關的訊息傳導路徑。在這篇研究中，當九孔血液細胞處理R-PIA(A1腺苷酸受體促效劑)時，細胞內的環苷腺酸濃度會下降，但並沒有顯著影響蛋白激酶A的活性。增加細胞內的環苷腺酸濃度並不能回復R-PIA造成的附著受到抑制，顯示環苷腺酸-蛋白激酶A的訊息傳導路徑可能不是調控類A1腺苷酸受體活化後造成九孔血液細胞附著下降的主要原因。但以磷脂酶C的活化劑m-3M3FBS處理九孔血液細胞，可以回復CHA所抑制的九孔血液細胞附著，而且單獨以U73122(磷脂酶C的抑制劑)處理來抑制細胞的磷脂酶C的活性，會降低九孔血液細胞的附著。這些結果指出九孔血液細胞附著需要磷脂酶C的活性，又在九孔血液細胞中，磷脂酶C可能位於活化的類A1腺苷酸受體訊息傳遞的下游。以PMA(蛋白激酶C活化劑)會增進九孔血液細胞的附著，但蛋白激酶C抑制劑並不影響九孔血液細胞的附著。另一方面，用A23187(鈣離子通道)增加胞內鈣離子的濃度卻可以增加九孔血液細胞的附著。又，以wortmannin和LY294002抑制PI3K (phosphoinositide-3-kinase)的活性會降低九孔血液細胞的附著。這些研究結果顯示，類A1腺苷酸受體可能經由抑制磷脂酶C-鈣離子或PI3K的訊息傳導路徑來降低九孔血液細胞的附著。環苷腺酸-蛋白激酶A訊息傳導路徑和蛋白激酶C參與在九孔血液細胞附著的調控中，但可能不是主要影響類A1腺苷酸受體活化後抑制九孔血液細胞附著的原因。在九孔血液細胞中的Focal adhesion蛋白質，例如：paxillin和FAK，可以被西方點墨法(Western blot)、免疫沉澱法(immunoprecipitation)和免疫螢光染色(immunocytochemistry)偵測到。此外，免疫螢光染色的螢光在細胞的邊緣呈現點狀分布，且類似focal adhesion的螢光點也可以在細胞邊緣較靠近細胞中心點的地方被發現。Integrin β1和αVβ3如同focal adhesion蛋白質一樣，可以被免疫螢光染色偵測到，顯示在哺乳動物中調控細胞附著的重要分子也存在於九孔血液細胞中。

關鍵字

先天性免疫反應 ； 九孔 ； 血液細胞 ； 腺苷酸受體 ； 細胞附著 ； 訊息傳遞

並列摘要

Cellular defenses are the principal responses of innate immunity, and blood cell adhesion is the very first step to perform those cellular defenses like phagocytosis and encapsulation. Unlike well-known mechanisms in mammalian blood cells, little is known about hemocyte adhesion in invertebrates. Here, I report that caffeine and other adenosine receptor agonists affected hemocyte adhesion in abalones (Haliotis diversicolor). Adenosine, caffeine and adenosine A1 and A3 receptor agonists all inhibit the hemocyte adhesion. On the other hand, CGS21680, an adenosine A2A receptor agonist, can recover the adenosine inhibited hemocyte adhesion. In addition, ZM241385 (an adenosine A2A receptor antagonist) synergistically inhibited hemocyte adhesion with adenosine. The result suggests that several subtypes of adenosine receptors exist in hemocytes, and adenosine A1-like receptor may be responsible for the adhesion inhibitory effect. Adenosine receptors have been recognized as the members of G-protein coupled receptor family, and the major intracellular signaling transduction pathways downstream of the receptors are cAMP-PKA and PLC related signaling pathways. In this study, the treatment of R-PIA (an adenosine A1 receptor agonist) decreased intracellular cAMP concentration, but not PKA activity. Increasing intracellular cAMP concentration could not overcome the inhibitory effect of R-PIA, which suggests that the cAMP-PKA signaling pathway may not be the key factor that regulates adhesion inhibition upon adenosine A1-like receptor activation. However, treating hemocytes with PLC activator, m-3M3FBS, can increase the CHA-inhibited hemocyte adhesion, and inhibition of PLC activity alone decreased hemocyte adhesion. These data indicate the requirement of PLC activity in hemocyte adhesion, and PLC might locate downstream of adenosine A1-like receptor activation. While PMA (a PKC activator) increased hemocyte adhesion, the PKC inhibitors treatment left hemocyte adhesion unaffected. On the other hand, increasing intracellular Ca2+ by A23187 (a Ca2+ ionophore) improved hemocyte adhesion. Moreover, inhibiting PI3K activity by wortmannin or LY294002 decreased hemocyte adhesion. Together, hemocyte adhesion that inhibited by activation of adenosine A1-like receptor should be regulated by signaling pathways like PLC-Ca2+ pathway or PI3K. The cAMP-PKA pathway and PKC are involved, but may not be the major signaling pathways that control adhesion inhibitory effect of adenosine A1-like receptor activation. Focal adhesion proteins such as paxillin and FAK could be detected by Western blotting, immunoprecipitation (IP), or immunocytochemistry (ICC) in abalone hemocytes. Furthermore, ICC showed spot-like fluorescence scattering in the edge of cells, and focal adhesion-like spots can also be observed at inner side of cells. As well as focal adhesion proteins, integrin β1 and αVβ3 were also discovered using ICC process, which suggests the critical molecules that are involved in regulation of cell adhesion in mammals are also existence in abalone hemocytes.

並列關鍵字

innate immunity ； abalones ； hemocytes ； adenosine receptor ； cell adhesion ； signaling transduction

參考文獻

Lai, CP. (2004). The cell-matrix adhesion of hemocytes in abalone (Haliotis diversicolor) is regulated by protein kinase a signal transduction pathway. Master thesis, National Taiwan University.

Chen, J.H., Yang, H.Y., Peng, S.W., Chen, Y.J., and Tasi, K.Y. (1996). Characterization of Abalone (Haliotis diversicolor) hemocytes in vitro. Biol. Bull. NTNU. 31(1):31-38.

Asako, H., Wolf, R.E., and Granger, D.N. (1993). Leukocyte adherence in rat mesenteric venules: effects of adenosine and methotrexate. Gastroenterology 104(1):31-7.

Baillie, G.S., Scott, J.D., and Houslay, M.D. (2005). Compartmentalisation of phosphodiesterases and protein kinase A: opposites attract. FEBS Lett. 579:3264–3270.

Bank, I., Koltakov, A., Nir-Glickman, E., Goldstein, I., Li, J., Roitelman, J., and Chess, L. (2003). Lovastatin and phospholipase C regulate constitutive and protein kinase C dependent integrin mediated interactions of human T-cells with collagen. Cell. Immunol. 223:35–45.