對甲酚對於新生大鼠心肌細胞間隙接合的影響

Abstract

對甲酚 (p-cresol)，是一種與蛋白質結合的尿毒素 (uremic toxin)，緣自於酪胺酸 (tyrosine) 經腸道細菌發酵後的代謝產物。慢性腎臟病變 (chronic kidneydisease) 的病患，由於腎絲球過濾率 (glomerular filtration rate) 下降，導致血液中對甲酚的含量增加。近期的研究報告指出對甲酚與心血管疾病具有顯著的相關性。然而至目前為止，對甲酚是否會損傷心肌細胞及其機制仍待研究。所以本研究擬探討對甲酚對於新生大白鼠心肌細胞間隙接合 (gap junction) 的影響及其機轉。心肌細胞以對甲酚處理不影響細胞的存活度，也不會造成細胞的凋亡及壞死，且隨著處理時間增加，心肌細胞自發性收縮速率降低且具有可逆性。利用免疫螢光染色發現connexin43 (Cx43) 於心肌細胞接合處的點狀染色分布減少且變小，顯示Cx43 已去組合，且此現象亦具有可逆性。接著利用scrape loading /dyetransfer 技術評估間隙接合的功能，發現對甲酚處理後dye transfer 的距離會減少，證實對甲酚會降低心肌細胞間隙接合溝通 (GJIC) 的功能。流式細胞儀證明對甲酚會引起心肌細胞內活性氧化物及鈣離子濃度增加；我們也發現PKCα磷酸化(活化)上升，且加入抗氧化劑N-acetylcystein (NAC) 及鈣離子螯合劑BAPTA-AM 可抑制PKCα磷酸化的上升。使用西方墨點分析法證明對甲酚處理造成去磷酸化Cx43 (NP-Cx43 及triton X-100 soluble P0 Cx43) 表現量上升及P2+P1 Cx43 表現量下降，而Cx43 去磷酸化被認為會造成間隙接合去組合及GJIC 下降。加入PKCα/βI 抑制劑Gö6976 可以抑制對甲酚引起的P2+P1 Cx43 磷酸化下降、NP-Cx43 表現量的上升及NP-Cx43 於細胞質中的染色；加入抗氧化劑NAC 及鈣離子螯合劑BAPTA-AM 可抑制NP-Cx43 表現量的上升，上述抑制劑均可抑制對甲酚所造成之Cx43 去組合及GJIC 下降。以上實驗結果顯示對甲酚可能藉由增加細胞內活性氧化物及鈣離子濃度進而活化PKCα訊息傳遞路徑使Cx43 去磷酸化，並造成Cx43 去組合及GJIC 下降。另外，對甲酚處理後ROCK 下游受質MYPT-1 (myosin light chain phosphatase-1)磷酸化程度增加，顯示ROCK 的活化。ROCK (Rho-kinase) 抑制劑Y27632 可抑制對甲酚引起之PKCα磷酸化、NP-Cx43表現量上升、Cx43 去組合及間隙接合功能異常。所以此激酶位於PKCα的上游，也參與了對甲酚影響接隙接合的分佈及功能之訊息傳遞。我們也預先處理去磷酸化酶抑制劑calyculin A 及okadaic acid，發現calyculinA 及okadaic acid 可阻止因對甲酚引起間隙接合的變化，顯示絲胺酸/蘇胺酸去磷酸化酶可能也參與了對甲酚對於間隙接合的影響。

p-cresol, a protein-bound uremic toxin, is a colonic bacterial fermentation metabolite of the amino acid tyrosine. In chronic kidney disease patients, decrease of glomerular filtration rate results in increased plasma levels of p-cresol. Recent studies indicate that p-cresol is significantly related to cardiovascular diseases. However, whether p-cresol directly affects cardiomyocytes and the mechanisms underlying p-cresol-induced damage remain to be investigated. Therefore, the purpose of this study was to investigate the effects of p-cresol on gap junctions in primary cultured neonatal cardiomyocytes. p-cresol did not affect cell viability, cell apoptosis and necrosis. p-cresol reduced the spontaneous contraction rates of cardiomyocytes in atime-dependent manner and this phenomenon was reversible. In immunofluorescence study, Cx43 dots of cardiomyocytes at cell-cell junctions are decreased by p-cresol treatment, suggesting Cx43 disassembly and this phenomenon was also reversible. In functional study of scrape loading dye transfer assay, the distance of dye transfer became shorten after p-cresol treatment, confirming that p-cresol impaired gap junction intercellular communication (GJIC). We demonstrated that reactive oxygen species (ROS) and [Ca2+] i increased after p-cresol treatment using flow cytometry. p-cresol also induced PKCα