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

Abstract

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.