Amlal H, Legoff C, Vernimmen C, Paillard M, Bichara M
Institut National de la Santé et de la Recherche Médicale Unité 356, Université Pierre et Marie Curie, Paris, France.
Am J Physiol. 1996 Aug;271(2 Pt 1):C455-63. doi: 10.1152/ajpcell.1996.271.2.C455.
Cell pH was monitored in suspensions of medullary thick ascending limbs (MTALs) of rat kidney to determine possible effects of various transduction pathways on apical Na(+)-K+ (NH4+)-2Cl- cotransport, the activity of which was measured as the bumetanide-sensitive component of cell acidification caused by abrupt exposure to 4 mM NH4Cl. 8-Bromoadenosine 3',5'-cyclic monophosphate stimulated cotransport activity through activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), since the cAMP effect was abolished by N-[2-(p- bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89); stimulation by cAMP (P < 0.02) was observed even when other Na+, Cl-, and K+ carriers were blocked by ouabain, diphenylamine-2-carboxylate, and barium, which indicates that cotransport was directly affected by PKA. Phorbol 12,13-dibutyrate also stimulated cotransport activity (P < 0.03), which was abolished by protein kinase C (PKC) blockade by staurosporine. In contrast, cotransport activity was reduced (P < 0.001) by arachidonic acid or 20-hydroxyeicosatetraenoic acid (20-HETE), as well as by an ionomycin-induced rise in cytosolic Ca2+ ([Ca2+]i). Inhibition by arachidonic acid or ionomycin was abolished by econazole and SKF-525A that inhibit cytochrome P-450-dependent monoxygenase, which produces 20-HETE from arachidonic acid in the MTAL, and the ionomycin effect was prevented when phospholipase A2 (PLA2) was blocked by 4-bromophenacyl bromide or oleyloxyethyl phosphorylcholine. The results demonstrate that MTAL apical Na(+)-K+(NH4+)-2Cl- cotransport is stimulated by PKA and PKC and inhibited by 20-HETE that may be produced after a rise in [Ca2+]i through PLA2 activation.
监测大鼠肾髓质厚升支（MTALs）悬浮液中的细胞pH值，以确定各种转导途径对顶端Na(+)-K+(NH4+)-2Cl-共转运的可能影响，该共转运活性通过突然暴露于4 mM NH4Cl引起的细胞酸化中布美他尼敏感成分来测量。8-溴腺苷3',5'-环磷酸刺激共转运活性是通过激活腺苷3',5'-环磷酸（cAMP）依赖性蛋白激酶（PKA），因为N-[2-(对溴肉桂氨基)乙基]-5-异喹啉磺酰胺（H-89）消除了cAMP的作用；即使其他Na+、Cl-和K+载体被哇巴因、二苯胺-2-羧酸盐和钡阻断时，仍观察到cAMP的刺激作用（P < 0.02），这表明共转运直接受PKA影响。佛波醇12,13-二丁酸酯也刺激共转运活性（P < 0.03），该作用被星形孢菌素对蛋白激酶C（PKC）的阻断所消除。相反，花生四烯酸或20-羟基二十碳四烯酸（20-HETE）以及离子霉素诱导的胞质Ca2+（[Ca2+]i）升高会降低共转运活性（P < 0.001）。酮康唑和SKF-525A抑制细胞色素P-450依赖性单加氧酶，该酶在MTAL中由花生四烯酸产生20-HETE，从而消除了花生四烯酸或离子霉素的抑制作用，并且当磷脂酶A2（PLA2）被4-溴苯甲酰溴或油酰氧基乙基磷酰胆碱阻断时，离子霉素的作用被阻止。结果表明，MTAL顶端Na(+)-K+(NH4+)-2Cl-共转运受PKA和PKC刺激，并受20-HETE抑制，20-HETE可能在通过PLA2激活导致[Ca2+]i升高后产生。
