Slotki I N, Schwartz J H, Alexander E A
Thorndike Memorial Laboratory, Boston City Hospital, Massachusetts.
Am J Physiol. 1990 Oct;259(4 Pt 2):F666-71. doi: 10.1152/ajprenal.1990.259.4.F666.
In this study we investigated the role of protein kinases in activation of the Na(+)-H+ exchanger in inner medullary collecting duct (IMCD) cells. Monolayers, 24-48 h after achieving confluence, were made quiescent by 24 h incubation in 0.1% serum before study. Changes in pHi were measured with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Phorbol myristate acetate (PMA), a synthetic analogue of diacylglycerol (DAG), was used to stimulate protein kinase C (PKC). In nominally HCO3(-)-free media containing 110 mM Na+ and 1 mM Ca2+, PMA addition increased pHi from 7.29 +/- 0.08 to 7.54 +/- 0.07 after 20 min. The increment in pHi was completely inhibited by 1 mM amiloride or by replacement of extracellular Na+ with choline but not inhibited by 1 mM N-ethylmaleimide, an inhibitor of active proton transport. Downregulation of PKC by overnight incubation of monolayers with PMA also prevented the rise in pHi upon subsequent challenge with PMA. Another active analogue of DAG, 1,2-dioleoyl-rac-glycerol, caused an increment in pHi similar to that produced by PMA, whereas 4 alpha-phorbol, an inactive analogue, did not stimulate Na(+)-H+ exchange. Bradykinin (10(-6) M), a phospholipase C-activating hormone, also induces alkalinization of IMCD cells similar to that produced by phorbol esters. Neither vasopressin (10(-7) M), which induces cellular accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) and activation of protein kinase A (PKA), nor 8-bromo-cAMP (1 mM) changed pHi. Therefore in the IMCD cell activation of PKC but not PKA stimulates a rise in pHi via the Na(+)-H+ exchanger.
在本研究中，我们调查了蛋白激酶在内髓集合管（IMCD）细胞中激活钠氢交换体的作用。在研究前，将融合24 - 48小时后的单层细胞在0.1%血清中孵育24小时使其静止。用2',7'-双（羧乙基）-5（6）-羧基荧光素测量细胞内pH值（pHi）的变化。佛波醇肉豆蔻酸酯乙酸盐（PMA），一种二酰基甘油（DAG）的合成类似物，用于刺激蛋白激酶C（PKC）。在含有110 mM Na⁺和1 mM Ca²⁺的名义上无HCO₃⁻的培养基中，添加PMA 20分钟后，pHi从7.29±0.08升高到7.54±0.07。pHi的升高被1 mM氨氯吡咪完全抑制，或用胆碱替代细胞外Na⁺也可抑制，但不被1 mM N - 乙基马来酰亚胺抑制，后者是一种活性质子转运抑制剂。通过将单层细胞与PMA过夜孵育来下调PKC，也可防止随后用PMA刺激时pHi的升高。另一种DAG的活性类似物1,2 - 二油酰 - rac - 甘油引起的pHi升高与PMA产生的相似，而无活性类似物4α - 佛波醇则不刺激钠氢交换。缓激肽（10⁻⁶ M），一种激活磷脂酶C的激素，也诱导IMCD细胞碱化，类似于佛波酯产生的碱化。诱导细胞内3',5'-环磷酸腺苷（cAMP）积累并激活蛋白激酶A（PKA）的血管加压素（10⁻⁷ M）和8 - 溴 - cAMP（1 mM）均未改变pHi。因此，在IMCD细胞中，PKC而非PKA的激活通过钠氢交换体刺激pHi升高。
