Marcus Daniel C, Liu Jianzhong, Shiga Nobuyuki, Wangemann Philine
Biophysics Laboratory, and Cell Physiology Laboratory, Boys Town National Research Hospital, Omaha, NE 68132.
Audit Neurosci. 1994;1:101-109.
Vestibular dark cell epithelium was isolated from the semicircular canal of gerbils to test the proposal that the sulfhydryl alkylating agent N-ethylmaleimide (NEM) inhibits K(+) secretion by this tissue and does so by reacting with a site in or near the apical membrane. Dark cell epithelium was mounted in a micro-Ussing chamber for measurements of transepithelial voltage (V(t)) and resistance (R(t)) or in a perfused bath on the stage of a microscope for measurement of cell height as an index of cell volume. Perfusion of the apical or basolateral side with 10(-3) M NEM caused an increase in V(t) superimposed upon a slower decrease of V(t), resulting in a triphasic response. There were only small changes in R(t). Under this condition, V(t) is proportional to short circuit current and to K(+) secretion. Both the stimulatory and the inhibitory responses of V(t) were dose-dependent between 10(-6) and 10(-3) M NEM and the inhibition was irreversible. The specificity of the reaction of NEM with sulfhydryl groups was confirmed by the use of the reducing agent dithiothreitol (DTT). Perfusion of 5×10(-4) M DTT on the apical side caused no significant changes in V(t) but completely prevented both stimulation and inhibition of V(t) by NEM (10(-3) M). The amplitudes of the stimulation and the inhibition of V(t) were greater for basolateral than for apical perfusion of NEM. Similarly, the response times for each effect were faster from the basolateral side, suggesting that the primary sites of action are at or near the basolateral membrane. The site of action of NEM was further explored by subjecting the tissue to a membrane-impermeant sulfhydryl reagent, stilbenedisulfonate maleimide (SDM). Apical perfusion of 10(-3) M SDM had no effect on V(t) or R(t), whereas basolateral perfusion caused a reversible increase of V(t) (5.2 ± 0.5 to initially 6.8 ± 0.5 mV which relaxed after 60 s to 5.8 ± 0.5 mV) and to an initial decrease in R(t) by 4%. No inhibitory phase was observed. Elevation of basolateral [K(+)] from 3.6 to 25 mM is known to increase V(t) and reduce R(t) via direct stimulation of basolateral K(+) uptake and indirect stimulation of the apical membrane conductance. Basolateral perfusion of 10(-3) M NEM fully inhibited the increase of V(t) due to 25 mM K(+). Elevation of basolateral [K(+)] from 3.6 to 25 mM is known to increase reversibly cell volume. NEM was found to inhibit cell swelling in a dose-dependent manner but did not initially affect the rate of shrinking after K(+)-induced swelling, pointing to action only on basolateral transport pathways. The effects of NEM on K(+)-induced cell swelling were completely prevented by 5×10(-4) M DTT, demonstrating that the inhibitory effect of NEM was on sulfhydryl groups. In contrast to interpretations of NEM action in frog semicircular canal, we have found that NEM appears to stimulate an ion transport process in mammalian dark cells at an extracellular site in the basolateral membrane and inhibits another ion transport process in the basolateral membrane at another site. Inhibition by NEM from the apical side occurs most likely by diffusion of the agent to a site at or near the cytosolic side of the basolateral membrane.
从沙鼠的半规管中分离出前庭暗细胞上皮，以验证巯基烷基化剂N - 乙基马来酰亚胺（NEM）抑制该组织钾离子分泌这一假说，且其作用机制是与顶端膜或其附近的位点发生反应。将暗细胞上皮安装在微尤斯灌流小室中，用于测量跨上皮电压（V(t)）和电阻（R(t)）；或将其置于显微镜载物台上的灌注浴中，测量细胞高度作为细胞体积的指标。用10⁻³ M NEM灌注顶端或基底外侧，会使V(t)升高，叠加在V(t)的缓慢下降之上，产生三相反应。R(t)仅有微小变化。在此条件下，V(t)与短路电流及钾离子分泌成正比。在10⁻⁶至10⁻³ M NEM之间，V(t)的刺激和抑制反应均呈剂量依赖性，且抑制作用不可逆。使用还原剂二硫苏糖醇（DTT）证实了NEM与巯基反应的特异性。用5×10⁻⁴ M DTT灌注顶端，V(t)无显著变化，但完全阻止了NEM（10⁻³ M）对V(t)的刺激和抑制。NEM基底外侧灌注时V(t)的刺激和抑制幅度大于顶端灌注。同样，从基底外侧引发每种效应的反应时间更快，表明主要作用位点在基底外侧膜或其附近。通过使组织接触膜不通透的巯基试剂，即芪二磺酸盐马来酰亚胺（SDM），进一步探究NEM的作用位点。用10⁻³ M SDM灌注顶端对V(t)或R(t)无影响，而灌注基底外侧则导致V(t)可逆性升高（从5.2±0.5 mV升至最初的6.8±0.5 mV，60秒后松弛至5.8±0.5 mV），且R(t)最初降低4%。未观察到抑制相。已知将基底外侧[K⁺]从3.6 mM升高到25 mM会通过直接刺激基底外侧钾离子摄取和间接刺激顶端膜电导来增加V(t)并降低R(t)。用10⁻³ M NEM灌注基底外侧完全抑制了由于25 mM K⁺引起的V(t)升高。已知将基底外侧[K⁺]从3.6 mM升高到25 mM会使细胞体积可逆性增加。发现NEM以剂量依赖性方式抑制细胞肿胀，但最初并不影响钾离子诱导肿胀后的收缩速率，表明其仅作用于基底外侧转运途径。5×10⁻⁴ M DTT完全阻止了NEM对钾离子诱导细胞肿胀的影响，表明NEM的抑制作用是针对巯基的。与对青蛙半规管中NEM作用的解释不同，我们发现NEM似乎在基底外侧膜的细胞外位点刺激哺乳动物暗细胞中的离子转运过程，并在基底外侧膜的另一位点抑制另一个离子转运过程。从顶端侧施加NEM的抑制作用最可能是由于该试剂扩散到基底外侧膜胞质侧或其附近的位点。
