Ou X, Ramos K S
Department of Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station 77843.
Chem Biol Interact. 1994 Oct;93(1):29-40. doi: 10.1016/0009-2797(94)90083-3.
Recent studies in this laboratory have shown that benzo[a]pyrene (BaP) interferes with protein kinase C (PKC)-mediated phosphorylation of aortic smooth muscle cell (SMC) proteins in vivo. To evaluate the biochemical basis of this response, the present studies have been conducted to examine the time- and concentration-dependent effects of BaP on PKC activity in vitro. Growth-arrested subcultures of rat aortic SMCs were exposed to 0.3, 3, or 30 microM BaP in the presence of fetal bovine serum for various times and then processed for measurements of exogenous histone Type III-S phosphorylation under PKC-activating conditions. Challenge of SMCs with BaP for 8 h was associated with a concentration-dependent inhibition of PKC activity in both cytosolic and particulate fractions. While no changes of enzymatic activity were observed in either fraction following exposure of SMCs to 0.3 microM BaP, higher concentrations of BaP inhibited PKC in both cytosolic and particulate fractions. A 49% and 68% reduction of cytosolic PKC activity was observed in SMCs treated with 3 and 30 microM BaP, respectively. The inhibitory response elicited by BaP was more pronounced in the particulate fraction where 61% and 89% decreases in PKC activity were observed in cultures treated with 3 and 30 microM BaP, respectively. Time course studies revealed that inhibition of PKC activity by 30 microM BaP occurred as early as 30 min and was sustained for up to 24 h in both fractions. Benzo[a]pyrene (30 microM) did not interfere with the ability of phorbol-12-myristate-13-acetate to induce PKC translocation from the cytosolic to particulate compartment since maximal translocation occurred by 5 min and lasted for up to 60 min in both control and BaP-treated cultures. The inhibitory effects of BaP were independent of new protein or RNA synthesis, but appear to involve oxidative metabolism of the parent compound since 3-hydroxy-BaP, the major P450-derived BaP metabolite in SMCs, also inhibited cytosolic and particulate PKC activity. Collectively, these data demonstrate that BaP and its 3-hydroxy metabolite inhibit PKC activity in rat aortic SMCs and raise the possibility that interference with PKC-mediated protein phosphorylation participates in the deregulation of SMC growth and differentiation induced by BaP.
本实验室最近的研究表明，苯并[a]芘（BaP）在体内会干扰蛋白激酶C（PKC）介导的主动脉平滑肌细胞（SMC）蛋白磷酸化。为评估这种反应的生化基础，本研究检测了BaP在体外对PKC活性的时间和浓度依赖性影响。将生长停滞的大鼠主动脉SMC传代培养物在胎牛血清存在的情况下，分别暴露于0.3、3或30微摩尔/升的BaP中不同时间，然后在PKC激活条件下处理以测量外源性组蛋白III-S的磷酸化。用BaP刺激SMC 8小时与胞质和微粒部分中PKC活性的浓度依赖性抑制相关。当SMC暴露于0.3微摩尔/升BaP后，两个部分的酶活性均未观察到变化，但较高浓度的BaP会抑制胞质和微粒部分中的PKC。在用3和30微摩尔/升BaP处理的SMC中，分别观察到胞质PKC活性降低了49%和68%。BaP引起的抑制反应在微粒部分更为明显，在用3和30微摩尔/升BaP处理的培养物中，PKC活性分别降低了61%和89%。时间进程研究表明，30微摩尔/升BaP对PKC活性的抑制早在30分钟就出现，并在两个部分中持续长达24小时。苯并[a]芘（30微摩尔/升）不干扰佛波醇-12-肉豆蔻酸酯-13-乙酸酯诱导PKC从胞质向微粒区室转位的能力，因为在对照和BaP处理的培养物中，最大转位在5分钟时发生并持续长达60分钟。BaP的抑制作用与新蛋白质或RNA合成无关，但似乎涉及母体化合物的氧化代谢，因为3-羟基-BaP（SMC中主要的细胞色素P450衍生的BaP代谢物）也抑制胞质和微粒PKC活性。总体而言，这些数据表明BaP及其3-羟基代谢物抑制大鼠主动脉SMC中的PKC活性，并增加了干扰PKC介导的蛋白质磷酸化参与BaP诱导的SMC生长和分化失调的可能性。
