环磷酸腺苷（cAMP）对大鼠髓袢升支粗段钠钾ATP酶活性及磷酸化作用的影响
Effect of cAMP on the activity and the phosphorylation of Na+,K(+)-ATPase in rat thick ascending limb of Henle.
作者信息
Kiroytcheva M, Cheval L, Carranza M L, Martin P Y, Favre H, Doucet A, Féraille E
机构信息
Laboratoire de Néphrologie, Fondation pour Recherches Médicales, Genève, Switzerland.
出版信息
Kidney Int. 1999 May;55(5):1819-31. doi: 10.1046/j.1523-1755.1999.00414.x.
BACKGROUND
In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL.
METHODS
Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators.
RESULTS
cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit.
CONCLUSION
In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage.
背景
先前有报道称，在大鼠肾髓质亨氏袢升支粗段（MTAL）中，蛋白激酶A（PKA）的激活会抑制Na⁺,K⁺-ATP酶活性。这与已知的环磷酸腺苷（cAMP）对钠重吸收的刺激作用相矛盾。由于这种抑制作用是由磷脂酶A2（PLA2）激活介导的，而PLA2是一种受缺氧刺激的途径，因此我们评估了氧供应对cAMP作用于MTAL中Na⁺,K⁺-ATP酶的影响。
方法
在分离的MTAL中测量哇巴因敏感的⁸⁶Rb摄取和Na⁺,K⁺-ATP酶活性。在外髓质肾小管悬浮液中测定细胞内ATP含量和Na⁺,K⁺-ATP酶的磷酸化水平。实验在无氧或有氧条件下，在不存在或存在PKA激活剂的情况下进行。
结果
cAMP类似物或与3-异丁基-1-甲基黄嘌呤（IBMX）联合使用的福斯可林抑制了无氧MTAL中哇巴因敏感的⁸⁶Rb摄取。相反，当氧供应增加时，cAMP刺激了哇巴因敏感的⁸⁶Rb摄取和Na⁺,K⁺-ATP酶活性。氧供应改善与细胞内ATP含量增加有关。在有氧和无氧肾小管中，cAMP类似物或与IBMX联合使用的福斯可林均可增加Na⁺,K⁺-ATP酶α亚基的磷酸化水平。在无氧条件下，Na⁺,K⁺-ATP酶的抑制作用与其cAMP依赖性磷酸化作用分离，而在有氧条件下，cAMP类似物对哇巴因敏感的⁸⁶Rb摄取的刺激作用与Na⁺,K⁺-ATP酶α亚基的磷酸化水平呈线性相关且共同饱和。
结论
在有氧的MTAL中，PKA介导的对Na⁺,K⁺-ATP酶的刺激作用可能参与了cAMP依赖性的钠重吸收刺激过程。在无氧条件下，这种刺激途径可能被PLA2介导的抑制途径所取代，这可能是一种保护细胞免受缺氧损伤的适应性反应。
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