Combettes L, Berthon B, Claret M
Institut National de la Santé et de la Recherche Medicale, U. 274, Bât. 443 Université Paris-Sud, Orsay, France.
Biochem J. 1992 Nov 1;287 ( Pt 3)(Pt 3):891-6. doi: 10.1042/bj2870891.
The monohydroxy bile acid taurolithocholate (TLC) causes a rapid and transient increase in free cytosolic Ca2+ concentration ([Ca2+]i) in suspensions of rat hepatocytes similar to that elicited by the InsP3-dependent hormone vasopressin. The effect of the bile acid is due to a mobilization of Ca2+, independent of InsP3, from the endoplasmic reticulum (ER). Short-term preincubation of cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), which activates protein kinase C (PKC), blocked the increase in [Ca2+]i induced by TLC, but did not alter that mediated by vasopressin. We obtained the following results, indicating that the effect of PMA is mediated by the activation of PKC. (1) Phorbol esters were effective over a concentration range where they activate PKC (IC50 = 0.5 nM); (2) phorbol esters that do not activate PKC did not inhibit the effects of TLC; (3) the permeant analogue oleoylacetylglycerol mimicked the inhibitory effect of PMA; (4) lastly, the inhibition of the TLC-induced Ca2+ mobilization by phorbol esters was partially prevented by preincubating the cells with the PKC inhibitors H7 and AMG-C16. Preincubating hepatocytes with PMA had no effect on the cell uptake of labelled TLC, indicating that the phorbol ester does not interfere with the transport system responsible for the accumulation of bile acids. In saponin-treated liver cells, PMA added before or after permeabilization failed to abolish TLC-induced Ca2+ release from the ER. The possibility is discussed that PMA, via PKC activation, may alter the intracellular binding or the transfer of bile acids in the liver.
单羟基胆汁酸牛磺石胆酸（TLC）可使大鼠肝细胞悬液中的游离胞质钙离子浓度（[Ca2+]i）迅速短暂升高，类似于由依赖肌醇三磷酸（InsP3）的激素血管加压素所引发的升高。胆汁酸的这种作用是由于从内质网（ER）动员钙离子，且不依赖InsP3。用佛波酯4β-佛波醇12β-肉豆蔻酸酯13α-乙酸酯（PMA）对细胞进行短期预孵育，PMA可激活蛋白激酶C（PKC），这阻断了TLC诱导的[Ca2+]i升高，但未改变血管加压素介导的[Ca2+]i升高。我们得到以下结果，表明PMA的作用是由PKC激活介导的。（1）佛波酯在激活PKC的浓度范围内有效（半数抑制浓度[IC50]=0.5 nM）；（2）不激活PKC的佛波酯不抑制TLC的作用；（3）可渗透类似物油酰乙酰甘油模拟了PMA的抑制作用；（4）最后，用PKC抑制剂H7和AMG-C16对细胞进行预孵育，部分阻止了佛波酯对TLC诱导的钙离子动员的抑制作用。用PMA对肝细胞进行预孵育对标记的TLC的细胞摄取没有影响，表明佛波酯不干扰负责胆汁酸积累的转运系统。在皂角苷处理的肝细胞中，在透化前或透化后添加PMA均未能消除TLC诱导的内质网钙离子释放。讨论了PMA通过激活PKC可能改变肝脏中胆汁酸的细胞内结合或转运的可能性。
