蛋白酪氨酸激酶和蛋白激酶C对培养的大鼠和人类视网膜色素上皮细胞中L型钙通道的调节作用

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells.

作者信息

Strauss O, Mergler S, Wiederholt M

机构信息

Institut für Klinische Physiologie, Universitätsklinikum Benjamin Franklin der Freien Universität Berlin, Germany.

出版信息

FASEB J. 1997 Sep;11(11):859-67. doi: 10.1096/fasebj.11.11.9285484.

DOI:10.1096/fasebj.11.11.9285484

PMID:9285484

Abstract

The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.

摘要

在培养的大鼠和人类视网膜色素上皮细胞中研究了蛋白酪氨酸激酶（PTK）对L型钙通道电流的影响。采用穿孔膜片钳技术测量通过L型通道的钡电流，并使用L型钙通道开放剂Bay K8644（10⁻⁶ M）进行鉴定。应用PTK阻滞剂染料木黄酮（5×10⁻⁶ M）或拉文达ustin A（Lavendustin A，5×10⁻⁶ M）导致L型电流降低。无活性的染料木黄酮类似物大豆苷元（10⁻⁵ M）对钙通道无影响。在传统全细胞模式下，通过膜片吸管向细胞内施加pp60（c-src）（30 U/ml）导致L型电流增加。蛋白激酶A和蛋白激酶G阻滞剂H9（10⁻⁶ M）对L型电流无影响；染料木黄酮在存在H9的情况下降低了电流。蛋白激酶C（PKC）阻滞剂白屈菜红碱（10⁻⁵ M）降低了L型电流；拉文达ustin对PTK的额外抑制导致电流进一步降低。向细胞内施加肉豆蔻酰化的PKC底物（5×10⁻⁵ M）以抑制PKC导致L型电流幅度快速衰减。向细胞内施加肉豆蔻酰化的PKC底物（10⁻⁴ M）与pp60（c-src）一起对L型电流无影响。用10⁻⁶ M佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）上调PKC对L型电流幅度无影响。然而，在经PMA预处理的细胞中，染料木黄酮导致L型电流增加。在经PMA处理的细胞中向细胞内施加pp60（c-src）导致L型电流降低。我们得出结论，在静息细胞中，PTK和PKC以累加方式调节L型钙通道。L型通道似乎是PTK激活和PKC依赖性途径整合的位点。PKC的活性决定了PTK是降低还是增加L型通道活性。