Eble D M, Strait J B, Govindarajan G, Lou J, Byron K L, Samarel A M
Cardiovascular Institute and Departments of Medicine and Physiology, Loyola University Chicago, Maywood, Illinois 60153, USA.
Am J Physiol Heart Circ Physiol. 2000 May;278(5):H1695-707. doi: 10.1152/ajpheart.2000.278.5.H1695.
Endothelin-1 (ET) produces neonatal rat ventricular myocyte (NRVM) hypertrophy and activates focal adhesion kinase (FAK) in other cell types. In the present study, we examined whether ET activated FAK in NRVM and whether FAK was necessary and/or sufficient for ET-induced NRVM hypertrophy. Chronic ET-1 stimulation (100 nM, 48 h) increased protein-to-DNA and myosin heavy chain (MHC)-to-DNA ratios and stimulated the assembly of newly synthesized MHC into sarcomeres. ET-1 also induced the assembly of focal adhesions and costameres, as evidenced by increased phosphotyrosine, FAK, and paxillin immunostaining. Acutely, ET treatment rapidly increased tyrosine phosphorylation of FAK and paxillin. FAK was also activated by phorbol 12-myristate 13-acetate (2 microM, 5 min). Pretreatment with chelerythrine (5 microM) or rottlerin (10 microM) completely blocked ET-induced FAK phosphorylation, indicating that protein kinase C activation was upstream of ET-induced FAK activation. In contrast, ET-induced FAK activation was not affected by blocking calcium influx via L-type voltage-gated calcium channels. Adenoviruses (Adv) containing FAK and FAK-related nonkinase (FRNK) were used to specifically define the role of FAK in ET-induced hypertrophy. ET stimulation failed to increase total protein-to-DNA or MHC-to-DNA ratios or to stimulate sarcomeric assembly in myocytes infected with Adv-FRNK. However, Adv-FAK alone did not increase total protein-to-DNA or MHC-to-DNA ratios and failed to increase the number or size of myofibrils as evidenced by double immunofluorescence labeling for MHC and FAK. Thus, although FAK is necessary for ET-induced NRVM hypertrophy, other ET-generated signals are also required to elicit the hypertrophic phenotype.
内皮素-1（ET）可导致新生大鼠心室肌细胞（NRVM）肥大，并在其他细胞类型中激活粘着斑激酶（FAK）。在本研究中，我们检测了ET是否在NRVM中激活FAK，以及FAK对于ET诱导的NRVM肥大是否必要和/或充分。慢性ET-1刺激（100 nM，48小时）增加了蛋白质与DNA以及肌球蛋白重链（MHC）与DNA的比率，并刺激新合成的MHC组装到肌节中。ET-1还诱导了粘着斑和肌动蛋白重复序列的组装，磷酸酪氨酸、FAK和桩蛋白免疫染色增加证明了这一点。急性情况下，ET处理迅速增加了FAK和桩蛋白的酪氨酸磷酸化。佛波酯12-肉豆蔻酸酯13-乙酸酯（2 microM，5分钟）也激活了FAK。用白屈菜红碱（5 microM）或罗勒菌素（10 microM）预处理可完全阻断ET诱导的FAK磷酸化，表明蛋白激酶C激活在ET诱导的FAK激活上游。相反，通过L型电压门控钙通道阻断钙内流并不影响ET诱导的FAK激活。含有FAK和FAK相关非激酶（FRNK）的腺病毒（Adv）用于明确FAK在ET诱导的肥大中的作用。ET刺激未能增加感染Adv-FRNK的心肌细胞中总蛋白质与DNA或MHC与DNA的比率，也未能刺激肌节组装。然而，单独的Adv-FAK并未增加总蛋白质与DNA或MHC与DNA的比率，并且如通过MHC和FAK的双重免疫荧光标记所示，未能增加肌原纤维的数量或大小。因此，尽管FAK对于ET诱导的NRVM肥大是必要的，但还需要其他ET产生的信号来引发肥大表型。
