甘草次酸對於新生大鼠心肌細胞中間隙接合蛋白connexin43磷酸化之影響

Abstract

間隙接合提供相鄰心肌細胞間小於1 kDa的分子自由擴散的一個途徑，而心肌細胞間隙接合主要構成的蛋白質為Cx43。Cx43的碳端磷酸化狀態會影響Cx43的結構、分布及更新，進而影響間隙接合的功能。甘草次酸(18beta-GA)是間隙接合的抑制劑，可以調控心肌細胞Cx43上serine和tyrosine的磷酸化狀態，進而影響間隙接合的功能。本研究我們利用可辨識磷酸化蛋白質之專一性抗體，來探討甘草次酸是否會使Cx43的Ser368 (Ser368Cx43)磷酸化，並進一步測試這過程是否有蛋白激酶C (PKC)參與。我們發現甘草次酸的濃度越高，心肌細胞Ser368Cx43磷酸化程度就越高。隨著處理的時間越久，磷酸化的Ser368Cx43及活化的PKCepsilon也越多。我們利用PKC抑制劑chelerythrine和甘草次酸共同處理心肌細胞，發現抑制PKC會降低甘草次酸所引起的Ser368Cx43磷酸化上升，確定Ser368Cx43的磷酸化與PKC活化有關。雙重免疫螢光染色的結果顯示，甘草次酸處理使間隙接合處的Ser368Cx43及去磷酸化的Cx43-NP都呈明顯的點狀分布，而隨處理的時間延長Cx43-NP的染色點會分布在細胞質。免疫螢光染色的結果進一步顯示，甘草次酸使細胞接合處PKCepsilon的染色增強，並和Cx43同位分布。Ser368Cx43染色的變化雖可以透過抑制PKC而回復，然而Cx43-NP則不受影響。先前的研究指出甘草次酸會活化蛋白質水解酶，使心肌細胞的Cx43全面性去磷酸化，並使Cx43-NP上升。我們發現蛋白質水解酶的抑制劑calyculin A，可以防止甘草次酸所引起Cx43-NP及Ser368Cx43的增加。免疫沉澱分析發現甘草次酸使活化態的pPKCepsilon和Cx43作用增加，而共同處理calyculin A可抑制此作用。我們推測甘草次酸經由活化PKCepsilon使Ser368Cx43的磷酸化上升，同時活化蛋白質水解酶去磷酸化Cx43，使Cx43-NP上升，並使間隙接合去組裝，其機制可能是心肌細胞受甘草次酸刺激產生的Cx43-NP可以當成pPKCepsilon的受質，再經由pPKCepsilon磷酸化之後即可使Ser368Cx43的量增加。

Gap junctions are channels between contiguous cardiomyocytes that provide a direct route for cytoplasmic diffusion of small molecules. The predominant gap junction protein expressed in ventricular cardiomyocytes is connexin43 (Cx43). Phosphorylation of the C-terminus of Cx43 has been implicated in regulation of its localization, turnover, and gap junction function. 18beta-Glycyrrhetinic acid (18beta-GA) has been used as a gap junction uncoupler, which regulates gap junction function by affecting phosphorylation of serine and tyrosine residue of Cx43. In this study phosphor-specific antibodies were used to determine whether 18beta-GA induces phosphorylation of Cx43 at serine 368 residue (pSer368Cx43), and whether this phosphorylation involves PKC. 18beta-GA induced a dose-dependent increase in pSer368Cx43 levels and a time-dependent increase in both pSer368Cx43 and pPKCe levels. The increase in pSer368Cx43 levels was inhibited by co-treatment with the PKC inhibitor, chelerythrine, suggesting that phosphorylation of pSer368Cx43 is mediated by PKC. 18beta-GA induced a punctuate pSer368Cx43 immunostaining and a prominent Cx43-NP immunostaining at cell-cell contact sites. Longer incubation of 18beta-GA may cause some cytoplasmic distribution of Cx43-NP. 18beta-GA also induced an increase of PKCepsilon immunoreactivity which colocalized with Cx43 at gap junction plaques. The 18beta-GA-induced change in pSer368Cx43 staining was reserved by co-treatment with chelerythrine, while the 18beta-GA-induced change in Cx43-NP staining were unaffected. However, the 18beta-GA-induced increase in pSer368Cx43 and Cx43-NP levels were prevented by protein phosphatase inhibitor, calyculin A. 18beta-GA also increased the co-immunoprecipitation of total Cx43 and PKCepsilon, and this effect was prevented by calyculin A co-treatment. We conclude that 18beta-GA induces disassembly of gap junction concomitant with Ser368 phosphorylation of Cx43 via PKCepsilon activation. The Cx43-NP induced by 18beta-GA could be substrates for PKCepsilon to increase pSer368Cx43.