布美他尼在离体鳐鱼肝细胞中的转运特性

Blumrich M, Petzinger E, Boyer J L

Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672.

Am J Physiol. 1993 Nov;265(5 Pt 1):G926-33. doi: 10.1152/ajpgi.1993.265.5.G926.

The uptake of [3H]bumetanide was studied in isolated skate hepatocytes in an albumin-free elasmobranch Ringer solution and compared with the uptake of bile acids in the presence of other cholephilic organic anions. [3H]bumetanide uptake was energy dependent, temperature sensitive, and exhibited saturation kinetics. In contrast to taurocholate and cholate, which are transported only by Na(+)-independent mechanisms, removal of Na+ reduced the maximal uptake rate (Vmax) for bumetanide from 404 +/- 80 to 230 +/- 47 pmol.mg-1 x min-1 without a change in the apparent Michaelis constant (Km). The apparent Km for the Na(+)-dependent portion of bumetanide uptake was 58 +/- 24 microM, and Vmax was 151 +/- 38 pmol.min-1 x mg-1. Taurocholate (100 and 200 microM) inhibited Na(+)-independent bumetanide transport competitively but was a noncompetitive inhibitor for Na(+)-dependent bumetanide uptake. Furosemide (100 microM) and two bumetanide analogues, PF-3034 (500 microM) and PF-2203 (500 microM), preferentially inhibited the Na(+)-dependent bumetanide uptake system, whereas cholate (100 microM) and probenecid (100 microM) preferentially inhibited Na(+)-independent bumetanide transport. The sulfhydryl (SH) reagents N-ethylmaleimide, 2,2'-dithio-bis(5-nitropyridine), and p-chloromercuribenzenesulfonic acid (PCMBS) inhibited both bile acid and bumetanide uptake. Dithiothreitol (500 microM) completely reversed the PCMBS-induced inhibition of bumetanide uptake. These results indicate that bumetanide is transported into hepatocytes of the small skate, Raja erinacea, by both Na(+)-dependent and Na(+)-independent mechanisms; the latter is shared by bile acids and probably sulfobromophthalein and other organic anions. Their uptake requires free SH groups.

在无白蛋白的板鳃亚纲林格氏液中，对分离出的鳐鱼肝细胞摄取[³H]布美他尼的情况进行了研究，并与在其他亲胆有机阴离子存在下胆汁酸的摄取情况进行了比较。[³H]布美他尼的摄取是能量依赖性的、对温度敏感的，并且呈现出饱和动力学。与仅通过非Na⁺依赖性机制转运的牛磺胆酸盐和胆酸盐不同，去除Na⁺可使布美他尼的最大摄取速率（Vmax）从404±80降至230±47 pmol·mg⁻¹·min⁻¹，而表观米氏常数（Km）不变。布美他尼摄取的Na⁺依赖性部分的表观Km为58±24 μM，Vmax为151±38 pmol·min⁻¹·mg⁻¹。牛磺胆酸盐（100和200 μM）竞争性抑制非Na⁺依赖性布美他尼转运，但对Na⁺依赖性布美他尼摄取是一种非竞争性抑制剂。呋塞米（100 μM）以及两种布美他尼类似物PF - 3034（500 μM）和PF - 2203（500 μM）优先抑制Na⁺依赖性布美他尼摄取系统，而胆酸盐（100 μM）和丙磺舒（100 μM）优先抑制非Na⁺依赖性布美他尼转运。巯基（SH）试剂N - 乙基马来酰亚胺、2,2'-二硫代双（5 - 硝基吡啶）和对氯汞苯磺酸盐（PCMBS）抑制胆汁酸和布美他尼摄取。二硫苏糖醇（500 μM）完全逆转了PCMBS诱导的布美他尼摄取抑制。这些结果表明，布美他尼通过Na⁺依赖性和非Na⁺依赖性机制转运到小鳐鱼（Raja erinacea）的肝细胞中；后者为胆汁酸所共有，可能还有磺溴酞钠和其他有机阴离子。它们的摄取需要游离的SH基团。