Hirsch Jochen, Schlatter Eberhard
Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Münster.
Cell Physiol Biochem. 2003;13(4):223-8. doi: 10.1159/000072425.
HEK-293 cells are known to reflect many features of the late distal tubule. Furthermore, they have the ability to release urodilatin, the structural analog to ANP. RT-PCR was performed to test for the expression of natriuretic peptide receptors. While the mRNA for the human ANP receptor (NPR-A, GC-A) could be amplified, the CNP-specific receptor NPR-B (GC-B) and the receptor specific for guanylins, GC-C, could not be detected. In patch clamp experiments the effects of ANP (10 nM) on membrane voltage (V(m)) were monitored and HEK-293 cells depolarized by 2.3 +/- 0.5 mV (n=14). In the presence of the EGF receptor blocker genistein (10 microM) the effect of ANP was increased by 65% to 3.9 +/- 0.8 mV (n=14). After removal of genistein the ANP-mediated depolarization further increased by 147% to 5.7 +/- 1.0 mV (n=14). ANP given repetitively without genistein had no increasing depolarizing effect in HEK-293 cells with time. The ANP effect could be fully blocked by 1 mM Ba(2+) and by 1 microM of the specific PKG inhibitor KT5823 indicating that ANP inhibits a K(+)-conductance via a cGMP-dependent protein kinase. Genistein itself hyperpolarized the membrane voltage of HEK-293 cells by -3.9 +/- 0.6 mV (n=11) and this effect could also be fully blocked by Ba(2+) (-0.3 +/- 0.1 mV, n=5), indicating that genistein activates a K(+)-conductance which contributes significantly to the membrane potential of HEK-293 cells.
已知HEK - 293细胞反映了远曲小管晚期的许多特征。此外，它们能够释放尿钠素，即心房钠尿肽的结构类似物。进行逆转录聚合酶链反应（RT-PCR）以检测利钠肽受体的表达。虽然可以扩增出人心房钠尿肽受体（NPR - A，GC - A）的信使核糖核酸（mRNA），但未检测到C型利钠肽特异性受体NPR - B（GC - B）和鸟苷素特异性受体GC - C。在膜片钳实验中，监测了心房钠尿肽（10 nM）对膜电压（V(m)）的影响，HEK - 293细胞去极化了2.3±0.5毫伏（n = 14）。在存在表皮生长因子（EGF）受体阻滞剂染料木黄酮（10 microM）的情况下，心房钠尿肽的作用增加了65%，达到3.9±0.8毫伏（n = 14）。去除染料木黄酮后，心房钠尿肽介导的去极化进一步增加了147%，达到5.7±1.0毫伏（n = 14）。在没有染料木黄酮的情况下重复给予心房钠尿肽，在HEK - 293细胞中随着时间的推移没有增加去极化作用。心房钠尿肽的作用可被1毫摩尔/升的钡离子（Ba(2+)）和1 microM的特异性蛋白激酶G（PKG）抑制剂KT5823完全阻断，这表明心房钠尿肽通过环磷酸鸟苷（cGMP）依赖性蛋白激酶抑制钾离子（K(+)）电导。染料木黄酮本身使HEK - 293细胞的膜电压超极化了-3.9±0.6毫伏（n = 11），并且这种作用也可被钡离子（Ba(2+)）完全阻断（-0.3±0.1毫伏，n = 5），这表明染料木黄酮激活了一种对HEK - 293细胞的膜电位有显著贡献的钾离子（K(+)）电导。
