APICAL AND BASAL MEMBRANE ION-TRANSPORT MECHANISMS IN BOVINE RETINAL-PIGMENT EPITHELIUM
APICAL AND BASAL MEMBRANE ION-TRANSPORT MECHANISMS IN BOVINE RETINAL-PIGMENT EPITHELIUM
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DOI：
10.1113/jphysiol.1991.sp018518
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发表时间：
1991-04-01
影响因子：
5.5
通讯作者：
MILLER, SS
中科院分区：
文献类型：
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作者：
JOSEPH, DP;MILLER, SS
1. 使用常规和双管氯离子选择性微电极的细胞内电压记录已被用于鉴定分离的牛视网膜色素上皮(RPE)-脉络膜的顶端和基底侧膜的几种运输机制。细胞内记录来自两个细胞群，黑色素（有色素）和无色素（无色素）。这两个种群的电学性质实际上是相同的。对于黑色素细胞，平均顶端静息膜电位(V(A)为-61 +/- 2 mV（平均+/- S.E.M, n = 49个细胞，33只眼）。这些细胞的顶膜阻力与基底膜阻力之比(a)为0.22 +/- 0.02。平均上皮电压和电阻分别为6 +/- 1 mV和138 +/- 7 OMEGA cm2。顶膜面向远端视网膜，包含一个Ba2+抑制的K+传导和一个瓦巴因抑制的电致Na+-K+泵。此外，它还含有一种布美他尼敏感机制，即假定的Na+- k +- cl -共转运体。基底侧膜含有一个DIDS（4,4'-二异硫氰二苯乙烯-2,2'-二磺酸）抑制氯离子通道。根尖膜和基底膜对K+和Cl-的相对电导分别为T(K)几乎等于0.9和T（Cl）几乎等于0.7。利用瓦巴因诱导的根尖膜去极化快速期（0-30 s）计算根尖（R(A)）和基底外侧（R(B)）细胞膜的等效电阻，以及细胞旁或分流电阻（R(s)）。它们分别是：3190 +/- 400,17920 +/- 2730和2550 +/- 200 OMEGA（平均+/- s.e.m., n = 9个组织）。根据这些数据，还计算了顶端（E(A)）和基底外侧（E(B)）膜的等效电动势（EMF）。它们分别是：-69 +/- 5.0 mV和-24 +/- 5.0 mV。用双管离子选择微电极测定细胞内Cl-活性（a(Cl)i）。稳态a(Cl)i = 61 +/- 4.0 mM，能势E(Cl) = - 13.5 +/- 1.5 mV（平均+/- S.E.M, n = 4）。在完整的眼睛或视网膜中，RPE-脉络膜的制备表明，光和暗之间的转换改变了光感受器和RPE顶端膜之间的细胞外（或视网膜下）空间中的K+浓度。这些光诱导的视网膜下[K+]o的变化在体外通过改变5到2 mM的顶点K+来定性模拟。这在顶点和基底膜上产生了一系列电压变化，这些变化具有三个不同的操作阶段。第一阶段是由顶膜K+扩散电位和电致Na+-K+泵的抑制共同产生的。第二阶段是基底外侧膜的延迟超极化。第3阶段很可能是由[K+]i降低和根尖膜K+电导率降低引起的。这些电压变化伴随着a(Cl)i单调下降26 +/- 3.0 mM （n = 4）。在根尖K+从5毫米减少到2毫米后产生的三个阶段中的第二个阶段，其特征是基底外侧膜的额外或延迟超极化。在这一阶段，a(Cl)i下降了几乎等于14 mM。延迟的超极化被顶端布美他尼或基部DIDS阻断，表明氯离子运输途径是这一反应的主要决定因素。这表明，在第2阶段，在基底膜Cl-电导上表达的a(Cl)i的下降，产生了临床测量的整个人眼的直流记录视网膜电图或眼电图的“快速振荡”成分。
1. Intracellular voltage recordings using conventional and double-barrelled chloride-selective microelectrodes have been used to identify several transport mechanisms at the apical and basolateral membranes of the isolated bovine retinal pigment epithelium (RPE)-choroid preparation. Intracellular recordings were obtained from two cell populations, melanotic (pigmented) and amelanotic (non-pigmented). The electrical properties of these two populations are practically identical. For melanotic cells the average apical resting membrane potential (V(A) is -61 +/- 2 mV (mean +/- S.E.M., n = 49 cells, thirty-three eyes). For these cells the ratio of apical to basolateral membrane resistance (a) was 0.22 +/- 0.02. The mean transepithelial voltage and resistance were 6 +/- 1 mV and 138 +/- 7 OMEGA cm2, respectively.2. The apical membrane, which faces the distal retina, contains a Ba2+-inhibitable K+ conductance and a ouabain-inhibitable, electrogenic Na+-K+ pump. In addition it contains a bumetanide-sensitive mechanism, the putative Na+-K+-Cl- cotransporter. The basolateral membrane contains a DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid)-inhibitable chloride channel. The relative conductances of the apical and basolateral membranes to K+ and Cl- are T(K) almost-equal-to 0.9 and T(Cl) almost-equal-to 0.7, respectively.3. The ouabain-induced fast phase of apical membrane depolarization (0-30 s) was used to calculate the equivalent resistances of the apical (R(A)) and basolateral (R(B)) cell membranes, as well as the paracellular or shunt resistance (R(S)). They are: 3190 +/- 400, 17920 +/- 2730 and 2550 +/- 200 OMEGA (mean +/- S.E.M., n = 9 tissues), respectively. From these data the equivalent electromotive forces (EMF) at the apical (E(A)) and basolateral (E(B)) membranes were also calculated. They are: -69 +/- 5.0 and -24 +/- 5.0 mV, respectively.4. Intracellular Cl- activity (a(cl)i) was measured using double-barrelled ion-selective microelectrodes. In the steady state a(Cl)i = 61 +/- 4.0 mM and the Nernst potential E(Cl) = - 13.5 +/- 1.5 mV (mean +/- S.E.M., n = 4).5. In the intact eye or in retina, RPE-choroid preparations it has been shown that the transition between light and dark alters the K+ concentration in the extracellular (or subretinal) space between the photoreceptors and the apical membrane of the RPE. These light-induced changes in subretinal [K+]o were qualitatively simulated in vitro by altering apical K+ between 5 and 2 mM. This produced a sequence of voltage changes at the apical and basolateral membranes that had three operationally distinct phases. Phase 1 is generated by the combination of an apical membrane K+ diffusion potential and inhibition of the electrogenic Na+-K+ pump. Phase 2 is a delayed hyperpolarization of the basolateral membrane. Phase 3 is most probably caused by a decrease in [K+]i and a decrease in apical membrane K+ conductance. These voltage changes were accompanied by a monotonic decrease in an a(Cl)i of 26 +/- 3.0 mM (n = 4).6. The second of the three phases that is produced following apical K+ reduction from 5 to 2 mM is characterized by an extra or delayed hyperpolarization at the basolateral membrane. During this phase a(Cl)i decreased by almost-equal-to 14 mM. The delayed hyperpolarization was blocked by apical bumetanide or basal DIDS, indicating that the chloride transport pathway is a primary determinant of this response. It is suggested that the drop in a(Cl)i, expressed at the basal membrane Cl- conductance during phase 2, generates the 'fast oscillation' component of the DC-recorded electroretinogram or electro-oculogram that is measured clinically across the human eye.