Bonanno J A
Morton D. Sarver Center for Cornea and Contact Lens Research, University of California, School of Optometry, Berkeley 94720.
Curr Eye Res. 1990 Jul;9(7):707-12. doi: 10.3109/02713689008999587.
The presence of the membrane transport mechanism, lactate-H+ cotransport, was tested in explants of rabbit corneal epithelium. Basal corneal epithelial cells were loaded with the pH sensitive fluorescent dye BCECF. Intracellular pH (pHi) was measured by rationing the fluorescence emission output following excitation at 490 and 440 nm. Perfusion of explants in lactate-containing Ringer's, pH 7.40, produced a reversible decrease in pHi. The lactate induced proton influx (mM/min) followed saturating kinetics, Km = 10.7 mM lactate, Vmax = 10.2 mM/min. Proton influx following addition of 10 mM lactate was inhibited 36, 60 and 47% by pre-perfusion in 1 mM CHC (cyano-hydroxycinammic acid), 500 microM H2DIDS (4,4'-diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid) and 1 mM LAIE (lactic acid isobutylester), respectively. These inhibitors of lactate-H+ cotransport were reversible. Mersalyl acid (500 microM) inhibited proton flux from 10 mM lactate addition by nearly 100%, but was irreversible. Stimulation of lactate production by perfusion in N2 equilibrated Ringer's (hypoxia) or the addition of 1 mM NaCN led to a slow alkalinization (0.1 pH unit in 10 min). Pre-perfusion with the reversible inhibitors slowed the hypoxic alkalinization by approximately 40%. It is concluded that lactate-H+ cotransport is present in the corneal epithelium and that it contributes to pHi regulation during hypoxia.
在兔角膜上皮外植体中检测了膜转运机制——乳酸-H⁺协同转运的存在情况。用对pH敏感的荧光染料BCECF加载角膜基底上皮细胞。通过测量在490和440nm激发后荧光发射输出的比值来测定细胞内pH（pHi）。用pH 7.40的含乳酸林格氏液灌注外植体，可使pHi发生可逆性降低。乳酸诱导的质子内流（毫摩尔/分钟）呈现饱和动力学，Km = 10.7 mM乳酸，Vmax = 10.2 mM/分钟。在1 mM CHC（氰基-羟基肉桂酸）、500 microM H2DIDS（4,4'-二异硫氰酸根合-二氢芪-2,2'-二磺酸）和1 mM LAIE（乳酸异丁酯）中预灌注，分别使加入10 mM乳酸后的质子内流抑制36%、60%和47%。这些乳酸-H⁺协同转运的抑制剂是可逆的。汞撒利酸（500 microM）使加入10 mM乳酸后的质子通量抑制近100%，但不可逆。在氮气平衡的林格氏液中灌注（缺氧）或加入1 mM NaCN刺激乳酸生成，导致缓慢碱化（10分钟内0.1个pH单位）。用可逆抑制剂预灌注可使缺氧碱化减慢约40%。结论是角膜上皮中存在乳酸-H⁺协同转运，并且它在缺氧期间有助于pHi的调节。
