Chu Miensheng, Koshman Yevgeniya, Iyengar Rekha, Kim Taehoon, Russell Brenda, Samarel Allen M
Cardiovascular Institute, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, USA.
J Signal Transduct. 2012;2012:473410. doi: 10.1155/2012/473410. Epub 2012 Jul 26.
Intracellular nitric oxide (NO(i)) is a physiological regulator of excitation-contraction coupling, but is also involved in the development of cardiac dysfunction during hypertrophy and heart failure. To determine whether contractile activity regulates nitric oxide synthase (NOS) expression, spontaneously contracting, neonatal rat ventricular myocytes (NRVM) were treat with L-type calcium channel blockers (nifedipine and verapamil) or myosin II ATPase inhibitors (butanedione monoxime (BDM) and blebbistatin) to produce contractile arrest. Both types of inhibitors significantly reduced iNOS but not eNOS expression, and also reduced NO(i) production. Inhibiting contractile activity also reduced focal adhesion kinase (FAK) and AKT phosphorylation. Contraction-induced iNOS expression required FAK and phosphatidylinositol 3-kinase (PI(3)K), as both PF573228 and LY294002 (10 μM, 24 h) eliminated contraction-induced iNOS expression. Similarly, shRNAs specific for FAK (shFAK) caused FAK knockdown, reduced AKT phosphorylation at T308 and S473, and reduced iNOS expression. In contrast, shRNA-mediated knockdown of PYK2, the other member of the FAK-family of protein tyrosine kinases, had much less of an effect. Conversely, overexpression of a constitutively active form of FAK (CD2-FAK) or AKT (Myr-AKT) reversed the inhibitory effect of BDM on iNOS expression and NO(i) production. Thus, contraction-induced iNOS expression and NO(i) production in NRVM are mediated via a FAK-PI(3)K-AKT signaling pathway.
细胞内一氧化氮（NO(i)）是兴奋-收缩偶联的生理调节因子，但也参与肥大和心力衰竭期间心脏功能障碍的发展。为了确定收缩活动是否调节一氧化氮合酶（NOS）的表达，用L型钙通道阻滞剂（硝苯地平和维拉帕米）或肌球蛋白II ATP酶抑制剂（丁二酮一肟（BDM）和blebbistatin）处理自发收缩的新生大鼠心室肌细胞（NRVM）以产生收缩停滞。这两种类型的抑制剂均显著降低诱导型一氧化氮合酶（iNOS）而非内皮型一氧化氮合酶（eNOS）的表达，并且还降低了NO(i)的产生。抑制收缩活动也降低了粘着斑激酶（FAK）和AKT的磷酸化。收缩诱导的iNOS表达需要FAK和磷脂酰肌醇3激酶（PI(3)K），因为PF573228和LY294002（10 μM，24小时）均可消除收缩诱导的iNOS表达。同样，针对FAK的特异性短发夹RNA（shFAK）导致FAK敲低，降低T308和S473处的AKT磷酸化，并降低iNOS表达。相比之下，蛋白酪氨酸激酶FAK家族的另一个成员PYK2的短发夹RNA介导的敲低作用要小得多。相反，组成型活性形式的FAK（CD2-FAK）或AKT（Myr-AKT）的过表达逆转了BDM对iNOS表达和NO(i)产生的抑制作用。因此，NRVM中收缩诱导的iNOS表达和NO(i)产生是通过FAK-PI(3)K-AKT信号通路介导的。
