Lahaye P, Tazi K A, Rona J P, Dellis O, Lebrec D, Moreau R
Laboratoire d'Hémodynamique Splanchnique et de Biologie Vasculaire, INSERM, Hôpital Beaujon, Clichy, France.
Hepatology. 1998 Sep;28(3):663-9. doi: 10.1002/hep.510280310.
Protein kinase C (PKC) modulates the activity and phosphorylation of the catalytic alpha-subunit of sodium-potassium-adenosine triphosphatase (Na+/K+ ATPase) in normal arteries. Because PKC is altered in cirrhotic aortae, Na+/K+ ATPase may also be altered in these arteries. The aim of the present study was to investigate alpha-subunit activity and phosphorylation in aortae from normal and cirrhotic rats, under baseline conditions and during exposure to PKC modulators. Alpha-subunit activity was assessed by measuring the amount of 32P released by hydrolysis of [gamma-32P]ATP in freshly isolated cell membranes (in the absence of PKC modulators only) and membrane depolarization caused by ouabain-induced alpha-subunit inhibition in isolated aortae (in the absence and presence of PKC modulators). Alpha-subunit phosphorylation was assessed by incorporation of 32P into alpha-subunits. Staurosporine, a PKC inhibitor, and phorbol 12,13-dibutyrate (PDBU), a PKC activator, were used. In addition, alpha-subunit expression was studied by Western blot analysis. In the absence of PKC modulators, the amount of 32P released by hydrolysis of [gamma-32P]ATP and ouabain-induced membrane depolarization were significantly lower in cirrhotic than in normal aortae. Staurosporine suppressed ouabain-induced membrane depolarization in cirrhotic and normal arteries. Ouabain-induced membrane depolarization was similar in cirrhotic aortae exposed to PDBU and in normal arteries studied under baseline conditions. Alpha-subunit phosphorylation was significantly lower in cirrhotic than in normal aortae, in aortae under baseline conditions, and in arteries exposed to staurosporine. Phosphorylation of the alpha-subunit was similar in cirrhotic aortae exposed to PDBU and in normal arteries under baseline conditions. Western blot analysis showed that the amount of alpha-subunit did not significantly differ between cirrhotic and normal aortae. In conclusion, a decrease in baseline Na+/K+ ATPase alpha-subunit activity occurs in aortae from cirrhotic rats as a result of reduced basal PKC activity. This PKC-dependent decreased alpha-subunit activity may be caused by a reduction in PKC-induced alpha-subunit phosphorylation.
蛋白激酶C（PKC）可调节正常动脉中钠钾-三磷酸腺苷酶（Na+/K+ ATP酶）催化性α亚基的活性和磷酸化。由于PKC在肝硬化主动脉中发生改变，这些动脉中的Na+/K+ ATP酶可能也会改变。本研究的目的是在基线条件下以及暴露于PKC调节剂期间，研究正常和肝硬化大鼠主动脉中α亚基的活性和磷酸化。通过测量新鲜分离细胞膜中[γ-32P]ATP水解释放的32P量（仅在不存在PKC调节剂的情况下）以及在分离的主动脉中哇巴因诱导的α亚基抑制引起的膜去极化（在不存在和存在PKC调节剂的情况下）来评估α亚基活性。通过将32P掺入α亚基来评估α亚基磷酸化。使用了PKC抑制剂星形孢菌素和PKC激活剂佛波醇12,13-二丁酸酯（PDBU）。此外，通过蛋白质印迹分析研究α亚基表达。在不存在PKC调节剂的情况下，肝硬化主动脉中[γ-32P]ATP水解释放的32P量和哇巴因诱导的膜去极化明显低于正常主动脉。星形孢菌素抑制了肝硬化和正常动脉中哇巴因诱导的膜去极化。在暴露于PDBU的肝硬化主动脉和基线条件下研究的正常动脉中，哇巴因诱导的膜去极化相似。在肝硬化主动脉中，α亚基磷酸化在基线条件下以及暴露于星形孢菌素的动脉中明显低于正常主动脉。在暴露于PDBU的肝硬化主动脉和基线条件下的正常动脉中，α亚基的磷酸化相似。蛋白质印迹分析表明，肝硬化和正常主动脉中α亚基的量没有显著差异。总之，由于基础PKC活性降低，肝硬化大鼠主动脉中基线Na+/K+ ATP酶α亚基活性降低。这种PKC依赖性的α亚基活性降低可能是由于PKC诱导的α亚基磷酸化减少所致。
