Benharouga M, Fritsch J, Banting G, Edelman A
INSERM U.467, CHU Necker, Paris, France.
Eur J Biochem. 1997 Jun 1;246(2):367-72. doi: 10.1111/j.1432-1033.1997.00367.x.
The activity of the Cl(-)-conductive pathways, their regulation by protein kinase A (PKA) and their relationship to the cystic fibrosis transmembrane regulator (CFTR) protein were assessed in rat kidney cortical brush-border-membrane vesicles (cBBMV) and outer medullary vesicles (OMV) by measuring the rate of valinomycin-induced microsomal swelling by light scattering in the presence of an inward Cl- gradient. Valinomycin increased the rate of swelling of cBBMV and OMV, which is consistent with the presence of a Cl(-)-conductive pathway. PKA further increased these rates. This effect was blocked by the inhibitor of protein kinase A, suggesting that phosphorylation by PKA activates these pathways. Four anion-transport inhibitors were tested ¿N-phenylanthranilic acid (PhNHPhCOOH), 5-nitro-2-(3-phenylpropylamino)benzoic acid [N(PhPrNH2)BzOH], glybenclamide and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid¿. Ph2COOH and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid inhibited the basal Cl(-)-conductive pathways, while PKA-treated microsomes were sensitive also to N(PhPrNH2)BzOH and glybenclamide, suggesting that additional Cl- pathways were activated by phosphorylation. The pharmacological properties of these pathways were similar to those of the CFTR Cl- channel. Two anti-CFTR mAbs inhibited PKA-activated valinomycin-induced swelling in cBBMV and OMV, while immunoblot analysis of the corresponding proteins with the same antibodies indicated the presence of a 170-kDa protein. The results thus indicate the presence of a PKA-activated Cl(-)-conductive pathway in cBBMV and OMV, and suggest that CFTR protein is involved in PKA-activated Cl- fluxes in these vesicles.
通过在存在内向Cl⁻梯度的情况下，利用光散射测量缬氨霉素诱导的微粒体肿胀速率，评估了大鼠肾皮质刷状缘膜囊泡（cBBMV）和外髓质囊泡（OMV）中Cl⁻传导途径的活性、其受蛋白激酶A（PKA）的调节及其与囊性纤维化跨膜调节因子（CFTR）蛋白的关系。缬氨霉素增加了cBBMV和OMV的肿胀速率，这与Cl⁻传导途径的存在一致。PKA进一步提高了这些速率。蛋白激酶A抑制剂可阻断此效应，表明PKA磷酸化激活了这些途径。测试了四种阴离子转运抑制剂——N-苯基邻氨基苯甲酸（PhNHPhCOOH）、5-硝基-2-（3-苯基丙氨基）苯甲酸[N(PhPrNH2)BzOH]、格列本脲和4-乙酰氨基-4'-异硫氰酸根合芪-2,2'-二磺酸。Ph2COOH和4-乙酰氨基-4'-异硫氰酸根合芪-2,2'-二磺酸抑制基础Cl⁻传导途径，而PKA处理的微粒体对N(PhPrNH2)BzOH和格列本脲也敏感，表明磷酸化激活了额外的Cl⁻途径。这些途径的药理学特性与CFTR Cl⁻通道相似。两种抗CFTR单克隆抗体抑制了PKA激活的缬氨霉素诱导的cBBMV和OMV肿胀，而用相同抗体对相应蛋白进行免疫印迹分析表明存在一种170 kDa的蛋白。因此，结果表明在cBBMV和OMV中存在PKA激活的Cl⁻传导途径，并提示CFTR蛋白参与了这些囊泡中PKA激活的Cl⁻通量。
