Nath S K, Hang C Y, Levine S A, Yun C H, Montrose M H, Donowitz M, Tse C M
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Am J Physiol. 1996 Mar;270(3 Pt 1):G431-41. doi: 10.1152/ajpgi.1996.270.3.G431.
The effect of hyperosmolarity on cloned Na+/H+ exchanger (NHE) isoforms NHE2 and NHE3 was studied in stably transfected PS120 fibroblasts. Na+/H+ exchanger activity was determined spectrofluorometrically in acidified cells that were exposed to isosmolar (300 mosmol/kg) or hyperosmolar (450 mosmol/kg) media, in which the only difference is the presence or absence of 150 mM mannitol. Hyperosmolar solution reversibly inhibited NHE2 and NHE3 with a delay of approximately 15 s. Hyperosmolarity significantly reduced their maximal reaction velocity compared with isosmolar medium but did not alter their Michaelis-Menten constant for intracellular H+. The Michaelis-Menten constant of the exchangers for extracellular Na+ in hyperosmolar medium was not different from that in isosmolar medium. Pretreatment of PS120/NHE3 cells with the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, the tyrosine kinase inhibitor genistein, and the serine/threonine protein phosphatase inhibitor okadaic acid did not affect the hyperosmolar inhibition of NHE3. Hyperosmolar inhibition of Na+/H+ exchanger activity was also observed in PS120 cells transfected with truncated NHE3 cDNAs (E3/585, E3/543, E3509, and E3/475) and NHE2 cDNA (E2/499). We conclude that 1) hyperosmolarity inhibits NHE2 and NHE3, in contrast to the stimulatory effect on the housekeeping isoform NHE1, 2) this inhibition is reversible, and 3) the COOH termini of NHE2 and NHE3 are not necessary for hyperosmolar inhibition of NHE2 and NHE3.
在稳定转染的PS120成纤维细胞中研究了高渗对克隆的钠氢交换体（NHE）亚型NHE2和NHE3的影响。通过荧光分光光度法测定酸化细胞中的钠氢交换体活性，这些细胞暴露于等渗（300 mosmol/kg）或高渗（450 mosmol/kg）培养基中，两者唯一的区别在于是否存在150 mM甘露醇。高渗溶液可逆性抑制NHE2和NHE3，延迟约15秒。与等渗培养基相比，高渗显著降低了它们的最大反应速度，但没有改变它们对细胞内H⁺的米氏常数。高渗培养基中交换体对细胞外Na⁺的米氏常数与等渗培养基中的没有差异。用蛋白激酶C抑制剂1-(5-异喹啉磺酰基)-2-甲基哌嗪、酪氨酸激酶抑制剂染料木黄酮和丝氨酸/苏氨酸蛋白磷酸酶抑制剂冈田酸预处理PS120/NHE3细胞，并不影响高渗对NHE3的抑制作用。在转染了截短的NHE3 cDNA（E3/585、E3/543、E3509和E3/475）和NHE2 cDNA（E2/499）的PS120细胞中也观察到了高渗对钠氢交换体活性的抑制作用。我们得出以下结论：1）与对管家亚型NHE1的刺激作用相反，高渗抑制NHE2和NHE3；2）这种抑制是可逆的；3）NHE2和NHE3的COOH末端对于高渗抑制NHE2和NHE3不是必需的。
