佛波醇酯敏感蛋白激酶对犬肾Madin-Darby细胞中钠、钾、氯协同转运的完全抑制作用

Complete inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells by PMA-sensitive protein kinase.

作者信息

Gagnon F, Orlov S N, Tremblay J, Hamet P

机构信息

Centre de Recherche du CHUM, Pavillon Hôtel-Dieu, Laboratory of Molecular Pathophysiology, 3850 St. Urbain St., Montréal, Qué., Canada.

出版信息

Biochim Biophys Acta. 1998 Mar 2;1369(2):233-9. doi: 10.1016/s0005-2736(97)00225-3.

DOI:10.1016/s0005-2736(97)00225-3

PMID:9518630

Abstract

This study examines the involvement of hormones and neuromediators in the regulation of Na+, K+, Cl- cotransport in renal epithelial cells using Madin-Darby canine kidney cells with low transepithelial electrical resistance (194+/-47 Omega/cm2). In this cell line, Na+, K+, Cl- cotransport measured as bumetanide-sensitive 86Rb influx was inhibited up to 50-60% with agonists of P2-purinoceptors (ATP approximately ADP>UTP>AMP), slightly (15-30%) increased by activators of cAMP signaling (forskolin, 8-Br-cAMP) and was insensitive to activators of cGMP signaling (8-Br-cGMP, nitroprusside), EGF, angiotensin II, bradykinin, methacholine, propranolol, vasopressin, adenosine, dopamine and histamine. Thirty min of preincubation of MDCK cells with 0.1 microM PMA completely blocked the activity of Na+, K+, Cl- cotransport whereas down-regulation of this enzyme by 24 h of preincubation with 1 microM PMA activated Na+, K+, Cl- cotransport by 60% and abolished the effect of short-term treatment with PMA. Regulation of Na+, K+, Cl- cotransport by ATP was insensitive to down-regulation of PMA-sensitive isoforms of protein kinase C. In addition, an inhibitor of protein kinase activity, staurosporine, abolished the effect of 0.1 microM PMA but did not change inhibition of this carrier by ATP. Thus, these results show for the first time that P2-purinoceptors and PMA-sensitive isoforms of protein kinase C play a key role in the regulation of Na+, K+, Cl- cotransport in MDCK cells. These results also show that neither PMA- nor staurosporine-sensitive forms of protein kinase are involved in the inhibition of Na+, K+, Cl- cotransport by activators of P2-purinoceptors.

摘要

本研究利用跨上皮电阻较低（194±47Ω/cm²）的Madin-Darby犬肾细胞，研究激素和神经介质在肾上皮细胞中对Na⁺、K⁺、Cl⁻协同转运的调节作用。在该细胞系中，以布美他尼敏感的⁸⁶Rb内流来衡量的Na⁺、K⁺、Cl⁻协同转运，被P2嘌呤受体激动剂（ATP≈ADP＞UTP＞AMP）抑制高达50 - 60%，被cAMP信号激活剂（福斯高林、8-溴-cAMP）轻微增加（15 - 30%），且对cGMP信号激活剂（8-溴-cGMP、硝普钠）、表皮生长因子、血管紧张素II、缓激肽、乙酰甲胆碱、普萘洛尔、血管加压素、腺苷、多巴胺和组胺不敏感。用0.1μM佛波酯预孵育MDCK细胞30分钟可完全阻断Na⁺、K⁺、Cl⁻协同转运的活性，而用1μM佛波酯预孵育24小时使该酶下调则使Na⁺、K⁺、Cl⁻协同转运激活60%，并消除了短期用佛波酯处理的效应。ATP对Na⁺、K⁺、Cl⁻协同转运的调节对蛋白激酶C的佛波酯敏感同工型的下调不敏感。此外，蛋白激酶活性抑制剂星形孢菌素消除了0.1μM佛波酯的效应，但未改变ATP对该载体的抑制作用。因此，这些结果首次表明P2嘌呤受体和蛋白激酶C的佛波酯敏感同工型在MDCK细胞中对Na⁺、K⁺、Cl⁻协同转运的调节中起关键作用。这些结果还表明，P2嘌呤受体激活剂对Na⁺、K⁺、Cl⁻协同转运的抑制作用既不涉及蛋白激酶的佛波酯敏感形式，也不涉及星形孢菌素敏感形式。