Layden T J, Agnone L M, Schmidt L N, Hakim B, Goldstein J L
Department of Medicine, University of Illinois, Chicago.
Gastroenterology. 1990 Oct;99(4):909-17. doi: 10.1016/0016-5085(90)90606-2.
The development of esophagitis is the result of hydrogen ion diffusion into the mucosa leading to cellular acidification and necrosis. In these studies, whether esophageal cells possess transport system(s) that can respond to cytoplasmic acidification was assessed; specifically, whether esophageal cells possess an Na+,H+ antiport was determined. Nucleated esophageal cells were isolated from rabbit esophagi using a trypsin-digestion technique that yielded 5-8 x 10(6) cells per esophagus, of which 74% +/- 3% were basal and 26% +/- 8% were squamous. Trypan blue was excluded by 95% +/- 2% of the cells. Cytoplasmic pH (pHi) was measured using the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6) carboxyfluorescein acetoxymethyl ester. Cells were acidified to the desired pHi by suspension in solutions with varying external pH (pHo) in the presence of nigericin. When cells acidified to pHi 6.3 were suspended in a choline chloride solution (pHo 7.4), cytoplasmic pHi did not increase. In contrast, Nao+ caused a concentration-dependent increase in the rate of cytoplasmic alkalinization with saturation occurring above 50 mmol/L Nao+. The transporter behaved according to first-order Michaelis-Menten type kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to external Na+ and had an apparent Km for Nao+ of 38.4 mmol/L. In contrast, the transporter behaved with greater than first-order kinetics with respect to cytoplasmic hydrogen ion concentration. Amiloride (10(-4) mol/L) caused a reversible inhibition of Na(+)-dependent alkalinization. Amiloride-sensitive cytoplasmic alkalinization was not observed when either cholineo or Ko+ was substituted for Nao+, while Lio+ resulted in alkalinization that was 60% +/- 8% of that seen with equimolar concentrations of Nao+. The basal pHi of cells suspended in a bicarbonate-free 130 mmol/L NaCl solution (pHo 7.4) averaged 7.42 +/- 0.03 (n = 10); amiloride (10(-4) mmol/L caused the basal pHi to decrease to 7.26 +/- 0.05 (n = 10; P less than 0.0025). When cells were suspended in a choline chloride (pHo 7.4) solution, pHi averaged 7.29 +/- 0.06 (n = 10) (P less than 0.0025 compared with Nao+). These studies indicate that nucleated esophageal cells obtained from rabbits possess an amiloride-sensitive Na+,H+ antiport that functions to regulate basal pHi and responds to intracellular acidification.
食管炎的发生是氢离子扩散进入黏膜导致细胞酸化和坏死的结果。在这些研究中，评估了食管细胞是否拥有能够对细胞质酸化作出反应的转运系统；具体而言，确定了食管细胞是否拥有Na⁺,H⁺反向转运体。使用胰蛋白酶消化技术从兔食管中分离出有核食管细胞，每个食管可获得5 - 8×10⁶个细胞，其中74%±3%为基底细胞，26%±8%为鳞状细胞。95%±2%的细胞排斥台盼蓝。使用pH敏感荧光染料2',7'-双(2-羧乙基)-5（和-6）羧基荧光素乙酰甲酯测量细胞质pH（pHi）。在尼日利亚菌素存在的情况下，将细胞悬浮于具有不同外部pH（pHo）的溶液中，使其酸化至所需的pHi。当酸化至pHi 6.3的细胞悬浮于氯化胆碱溶液（pHo 7.4）中时，细胞质pHi没有升高。相反，Na⁺导致细胞质碱化速率呈浓度依赖性增加，在Na⁺浓度高于50 mmol/L时出现饱和。该转运体对外部Na⁺表现出一级米氏动力学行为，其对Na⁺的表观Km为38.4 mmol/L。相比之下，该转运体对外部Na⁺表现出大于一级的动力学行为，其对Na⁺的表观Km为38.4 mmol/L。相比之下，该转运体对细胞质氢离子浓度表现出大于一级的动力学行为。氨氯吡脒（10⁻⁴ mol/L）引起对Na⁺依赖性碱化的可逆抑制。当用胆碱⁺或K⁺替代Na⁺时，未观察到氨氯吡脒敏感的细胞质碱化，而Li⁺导致的碱化是等摩尔浓度Na⁺时碱化的60%±8%。悬浮于无碳酸氢盐的130 mmol/L NaCl溶液（pHo 7.4）中的细胞的基础pHi平均为7.42±0.03（n = 10）；氨氯吡脒（10⁻⁴ mmol/L）使基础pHi降至7.26±0.05（n = 10；P＜0.0025）。当细胞悬浮于氯化胆碱（pHo 7.4）溶液中时，pHi平均为7.29±0.06（n = 10）（与Na⁺相比，P＜0.0025）。这些研究表明，从兔获得的有核食管细胞拥有一种氨氯吡脒敏感的Na⁺,H⁺反向转运体，其功能是调节基础pHi并对细胞内酸化作出反应。
