Brodin B, Rytved K A, Nielsen R
Biochemical Department, August Krogh Institute, University of Copenhagen, Universitetsparken 13, DK-2100, Denmark.
Pflugers Arch. 1996 Nov-Dec;433(1-2):16-25. doi: 10.1007/s004240050243.
The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.
本研究的目的是探究游离胞质钙浓度（[Ca2+]i）升高导致蛙皮上皮主细胞跨细胞钠吸收减少的机制。在落射荧光显微镜装置中用fura-2测量[Ca2+]i，采用电压钳技术测量钠吸收，并用微电极测量细胞电位。内质网钙ATP酶抑制剂毒胡萝卜素（0.4微摩尔）使[Ca2+]i从66±9纳摩尔升至137±19纳摩尔（n = 13，P = 0.002）。毒胡萝卜素使氨氯地平敏感的短路电流（Isc）从26.4微安·厘米-2降至10.6微安·厘米-2（n = 19，P<0.001），同时细胞从-79±1毫伏去极化至-31±2毫伏（n = 18，P<0.001）。根据氨氯地平敏感电流与顶膜电位之间的电流/电压（I/V）关系估算顶膜钠通透性（PaNa）。[Ca2+]i升高后，PaNa从8.0×10-7厘米·秒-1降至3.7×10-7厘米·秒-1（n = 7，P = 0.04）。仅顶膜钠通透性降低往往会使Isc降低，并导致细胞电位超极化，而非如观察到的去极化。在浆膜侧添加氯通道抑制剂4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）、二苯胺-2-羧酸盐（DPC）、茚满氧基乙酸94（IAA-94）和呋塞米可逆转毒胡萝卜素诱导的去极化，表明氯通道被[Ca2+]i升高激活。这在36Cl洗脱实验中得到证实，实验表明毒胡萝卜素使氯外流从32.49±5.01纳摩尔·分钟-1·厘米-2增加至62.63±13.3纳摩尔·分钟-1·厘米-2（n = 5，P = 0.04）。我们得出结论，[Ca2+]i的小幅升高激活氯通透性并抑制顶膜钠通透性。氯通道的激活和顶膜钠通道的关闭将倾向于降低宏观钠吸收。
