Kleinman J G, Harig J M, Barry J A, Ramaswamy K
Nephrology Section, Zablocki Veterans Administration Medical Center, Milwaukee, Wisconsin.
Am J Physiol. 1988 Aug;255(2 Pt 1):G206-11. doi: 10.1152/ajpgi.1988.255.2.G206.
We have examined pH gradient-driven Na+ uptake and Na+-driven H+ transport in brush-border membrane vesicles prepared from jejunal tissue obtained from organ donors by measuring the influx of 22Na and the fluorescence quenching of acridine orange (AO). Vesicle preparation by either Ca2+ or Mg2+ precipitation showed no difference in 22Na uptake or AO fluorescence quenching and dissipation. An outward H+ gradient [intravesicular pH (pHi) 5.5; extravesicular pH (pHo) 7.5] induced a Na+ uptake "overshoot" of threefold over equilibrium, whereas the absence of an H+ gradient (at either pH 5.5 or 7.5) did not produce an overshoot. Voltage clamping by Ki+ = Ko+ plus valinomycin reduced the overshoot by 50%. The initial rate of pH-driven Na+ uptake in voltage-clamped vesicles was related to [Nao+] (Km = 29 mM and Vmax = 9.5 nmol.mg protein-1.3 s-1). Amiloride inhibited this uptake in voltage-clamped vesicles (Ki = 99 microM). Dissipation of AO fluorescence quench in vesicles with a preformed internal acid gradient was hastened by Nao+ as well as voltage clamping in the absence of Na+. In vesicles without a pH gradient, internal Na+, as well as a diffusion potential (Ki+ 100; Ko+ 0 plus valinomycin) in the absence of Na+, induced AO quenching. External Na+ and Li+, but not choline, acted to dissipate AO quenching induced by a diffusion potential, and the rate of dissipation was unaffected by the presence of Cl-.Li+ and NH4+, but not Cs+, K+, Rb+, or choline+, inhibited pH gradient-driven 22Na uptake. We conclude that human jejunal brush-border membrane vesicles contain conductive pathways for both Na+ and H+ and an Na+-H+ exchanger.
我们通过测量²²Na的流入量和吖啶橙（AO）的荧光猝灭，研究了从器官捐献者获得的空肠组织制备的刷状缘膜囊泡中pH梯度驱动的Na⁺摄取和Na⁺驱动的H⁺转运。通过Ca²⁺或Mg²⁺沉淀制备囊泡，在²²Na摄取或AO荧光猝灭及消散方面未显示出差异。向外的H⁺梯度[囊泡内pH（pHi）5.5；囊泡外pH（pHo）7.5]诱导Na⁺摄取“过冲”，超过平衡值三倍，而不存在H⁺梯度（在pH 5.5或7.5时）则不会产生过冲。用Ki⁺ = Ko⁺加缬氨霉素进行电压钳制可使过冲降低50%。电压钳制囊泡中pH驱动的Na⁺摄取的初始速率与[Nao⁺]相关（Km = 29 mM，Vmax = 9.5 nmol·mg蛋白⁻¹·3 s⁻¹）。氨氯吡咪抑制电压钳制囊泡中的这种摄取（Ki = 99 μM）。在没有Na⁺的情况下，预先形成内部酸梯度的囊泡中AO荧光猝灭的消散会因Nao⁺以及电压钳制而加速。在没有pH梯度的囊泡中，内部Na⁺以及在没有Na⁺时的扩散电位（Ki⁺ 一百；Ko⁺ 零加缬氨霉素）会诱导AO猝灭。外部Na⁺和Li⁺，但不是胆碱，会使由扩散电位诱导的AO猝灭消散，并且消散速率不受Cl⁻存在的影响。Li⁺和NH₄⁺，但不是Cs⁺、K⁺、Rb⁺或胆碱⁺，抑制pH梯度驱动的²²Na摄取。我们得出结论，人空肠刷状缘膜囊泡含有Na⁺和H⁺的传导途径以及一种Na⁺ - H⁺交换体。
