Rao Y P, Stravitz R T, Vlahcevic Z R, Gurley E C, Sando J J, Hylemon P B
Department of Microbiology/Immunology, Virginia Commonwealth University/Medical College of Virginia, and McGuire Veterans Affairs Medical Center, Richmond 23298, USA.
J Lipid Res. 1997 Dec;38(12):2446-54.
The feedback repression of cholesterol 7alpha-hydroxylase transcriptional activity and mRNA levels by taurocholate (TCA) occurs via a protein kinase C (PKC)-dependent signal. To determine whether bile acids could activate PKC indirectly via generation of diacylglycerol (DG), their effects on DG levels in primary cultures of rat hepatocytes were determined using a DG kinase assay. To determine whether bile acids might activate PKC isozymes more directly, their effects on PKC alpha and delta purified from baculovirus expression systems were examined in phosphatidylserine/phosphatidylcholine/Triton X-100 (PS/PC/TX) mixed micelles. Addition of tauroursodeoxycholate (TUDCA), taurocholate (TCA), or taurodeoxycholate (TDCA) (50 microM) to the cells rapidly (15 min) increased DG content in cultured rat hepatocytes to 105%, 155%, and 130%, respectively, as compared to untreated control cultures. Addition of TCA increased PKC alpha specific activity with EC50 of approximately 400 nM; maximal activity was observed with 5 microM. Other taurine-conjugated bile acids (5 microM) increased PKC alpha specific activity (pmol/min/microg protein) in proportion to their relative hydrophobicity: PS/PC/TX 17 +/- 2; + TUDCA 29 +/- 18; + TCA 68 +/-13; + TDCA 166 +/- 21; and, taurochenodeoxycholate 178 +/- 20 (P vs. PS/PC/TX = 0.54, 0.019, 0.002, and 0.001, respectively); unconjugated bile acids gave similar results (r2 for activity vs. hydrophobicity index 0.59). Taurine-conjugated bile acid interaction enthalpies, as determined by dimyristoyl-phosphatidylcholine chromatography, were more highly correlated (r2 = 0.96) with PKC alpha activation than with the hydrophobicity index. TCA also stimulated the activity of purified PKCdelta with EC50 of approximately 150 nM and maximally (2.7-fold) at 1 microM. Free and taurine-conjugated bile acids (1 microM) increased PKCdelta activity according to their hydrophobicity index (r2 = 0.89) and interaction enthalpies (r2 = 0.96).
牛磺胆酸盐（TCA）对胆固醇7α-羟化酶转录活性和mRNA水平的反馈抑制是通过蛋白激酶C（PKC）依赖性信号发生的。为了确定胆汁酸是否可通过生成二酰基甘油（DG）间接激活PKC，利用DG激酶测定法测定了它们对大鼠肝细胞原代培养物中DG水平的影响。为了更直接地确定胆汁酸是否可能激活PKC同工酶，在磷脂酰丝氨酸/磷脂酰胆碱/曲拉通X-100（PS/PC/TX）混合胶束中检测了它们对从杆状病毒表达系统纯化的PKCα和δ的影响。与未处理的对照培养物相比，向细胞中添加牛磺熊去氧胆酸盐（TUDCA）、牛磺胆酸盐（TCA）或牛磺去氧胆酸盐（TDCA）（50μM）可迅速（15分钟）将培养的大鼠肝细胞中的DG含量分别提高到105%、155%和130%。添加TCA可增加PKCα的比活性，EC50约为400 nM；在5μM时观察到最大活性。其他牛磺酸结合的胆汁酸（5μM）按其相对疏水性成比例增加PKCα的比活性（pmol/分钟/μg蛋白）：PS/PC/TX为17±2；+TUDCA为29±18；+TCA为68±13；+TDCA为166±21；以及牛磺鹅去氧胆酸盐为178±20（与PS/PC/TX相比，P分别为0.54、0.019、0.002和0.001）；未结合的胆汁酸也得到类似结果（活性与疏水指数的r2为0.59）。通过二肉豆蔻酰磷脂酰胆碱色谱法测定的牛磺酸结合胆汁酸的相互作用焓与PKCα激活的相关性更高（r2 = 0.96），而不是与疏水指数的相关性。TCA还刺激纯化的PKCδ的活性，EC50约为150 nM，在1μM时最大激活（2.7倍）。游离和牛磺酸结合的胆汁酸（1μM）根据其疏水指数（r2 = 0.89）和相互作用焓（r2 = 0.96）增加PKCδ的活性。
