1. The mechanisms of K+ secretion into endolymph were studied on a preparation of isolated semicircular canal with different pharmacological inhibitors. Three periods of 5 or 30 min were performed, the first as control, the second in the presence of the drugs added to the apical or the basolateral bathing solution, and the third as recovery. Apical fluid was sampled at the beginning and the end of each period, transepithelial potential was recorded, Na+, K+, and Cl- concentrations, and K+ efflux, with 86Rb+ as a tracer, were measured and K+ fluxes were calculated. 2. When both sides of the epithelium were bathed with perilymph-like solution, the epithelium absorbed Na+, secreted K+, and generated a lumen positive potential. 3. The ATPases inhibitors, ouabain (10(-5) and 10(-3) M) and N-ethylmaleimide (10(-4) and 10(-3) M) inhibited the electrogenic K+ secretion when added to the basolateral fluid. N-ethylmaleimide (10(-3) M) applied to the apical fluid during a 5 min period decreased the K+ influx by 43% and the transepithelial potential by 66%. Other ATPase inhibitors, harmaline (10(-3) M), omeprazole (10(-4) M), vanadate (10(-4) M and 10(-3) M), N,N'-dicyclohexylcarbodiimide (DCC, 10(-5) M), 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl, 5 x 10(-6) M and 5 x 10(-5) M), and bafilomycin (10(-7) M) did not affect the K+ transport nor the transepithelial potential when they were added to the apical fluid. 4. The Na(+)-K(+)-Cl- co-transporter inhibitor, bumetanide, decreased both the transepithelial potential and the K+ transport when added to the basolateral solution but not to the apical one. At 10(-6) M, bumetanide maximally decreased the K+ influx by about 60%. 5. K+ channel blockers, quinine (10(-4) M), TEA (5 x 10(-3) M), added to the apical solution and barium (2 x 10(-3) M) added to either the apical or the basolateral solutions, did not affect the K+ transport and the transepithelial potential. 6. The carbonic anhydrase inhibitor acetazolamide (10(-3) M) added to both apical and basolateral solutions did not affect the K+ transport and the transepithelial potential. 7. It is concluded that, in the ampulla of the semicircular canal, a basolateral Na(+)-K(+)-Cl- co-transporter energized by the Na+, K(+)-ATPase was involved for 60% in the K+ secretion into endolymph. The electrogenic K+ transport would partly depend on a N-ethylmaleimide-sensitive protein possibly located at the apical plasma membrane or intracellularly.
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机译:1.研究了分离的具有不同药理抑制剂的半规管的制备方法,研究了钾离子向内淋巴分泌的机制。进行3个5或30分钟的周期,第一个为对照组,第二个为存在于顶端或基底外侧沐浴液中的药物,第三个为恢复期。在每个周期的开始和结束时取样顶液,记录跨上皮电位,以86Rb +作为示踪剂,测量Na +,K +和Cl-浓度和K +外排并计算K +通量。 2.当上皮的两侧用类淋巴液浸浴时,上皮吸收Na +,分泌K +,并产生管腔正电位。 3. ATPases抑制剂哇巴因(10(-5)和10(-3)M)和N-乙基马来酰亚胺(10(-4)和10(-3)M)添加到基底外侧液中后会抑制肌电K +分泌。在5分钟内将N-乙基马来酰亚胺(10(-3)M)应用于根尖液可使K +内流减少43%,跨上皮电位减少66%。其他ATPase抑制剂,harmaline(10(-3)M),奥美拉唑(10(-4)M),钒酸盐(10(-4)M和10(-3)M),N,N'-二环己基碳二亚胺(DCC, 10(-5)M),7-氯-4-硝基苯-2-氧杂-1,3-二唑(NBD-Cl,5 x 10(-6)M和5 x 10(-5)M),和将巴非霉素(10(-7)M)添加到顶端液中时,不影响K +转运或经上皮电位。 4.将Na(+)-K(+)-Cl-共转运蛋白抑制剂布美他尼加到基底外侧溶液中而不是根尖溶液中时,既降低了上皮电位,又降低了K +转运。在10(-6)M时,布美他尼使K +流入量最大程度减少了约60%。 5.将K +通道阻滞剂,奎宁(10(-4)M),TEA(5 x 10(-3)M)添加到顶端溶液中,并将钡(2 x 10(-3)M)添加到顶端溶液中或基底外侧溶液,均不影响K +转运和跨上皮电位。 6.碳酸酐酶抑制剂乙酰唑胺(10(-3)M)加入到顶端和基底外侧溶液中均不影响K +转运和跨上皮电位。 7.结论是,在半规管的壶腹中,由Na +激发的基底外侧Na(+)-K(+)-Cl-共转运子,其中K(+)-ATPase参与其中的60%。 K +分泌进入内淋巴。电动K +转运将部分取决于可能位于顶端质膜或细胞内的N-乙基马来酰亚胺敏感蛋白。
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