Newman E A
Department of Physiology, University of Minnesota, Minneapolis 55455.
J Neurosci. 1991 Dec;11(12):3972-83. doi: 10.1523/JNEUROSCI.11-12-03972.1991.
An electrogenic Na+/HCO3- cotransport system was studied in freshly dissociated Müller cells of the salamander retina. Cotransporter currents were recorded from isolated cells using the whole-cell, voltage-clamp technique following the block of K+ conductance with external Ba2+ and internal Cs+. At constant pHo, an outward current was evoked when extracellular HCO3- concentration was raised by pressure ejecting a HCO3(-)-buffered solution onto the surface of cells bathed in nominally HCO3(-)-free solution. The HCO3(-)-evoked outward current was reduced to 4.4% of control by 0.5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate), to 28.8% of control by 2 mM DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), and to 28.4% of control by 2 mM harmaline. Substitution of choline for Na+ in bath and ejection solutions reduced the response to 1.3% of control. Bicarbonate-evoked currents of normal magnitude were recorded when methane sulfonate was substituted for Cl- in bath, ejection, and intracellular solutions. Similarly, an outward current was evoked when extracellular Na+ concentration was raised in the presence of HCO3-. The Na(+)-evoked response was reduced to 16.2% of control by 2 mM DNDS and was abolished by removal of HCO3- from bath and ejection solutions. Taken together, these results (block by stilbenes and harmaline, HCO3- and Na+ dependence, Cl- independence) indicate that salamander Müller cells possess an electrogenic Na+/HCO3- cotransport system. Na+/HCO3- cotransporter sites were localized primarily at the endfoot region of Müller cells. Ejection of HCO3- onto the endfoot evoked outward currents 10 times larger than currents evoked by ejections onto the opposite (distal) end of the cell. The reversal potential of the cotransporter was determined by DNDS block of cotransport current. In the absence of a transmembrane HCO3- gradient, the reversal potential varied systematically as a function of the transmembrane Na+ gradient. The reversal potential was -0.1 mV for a [Na+]o:[Na+]i ratio of 1:1 and -25.2 mV for a Na+ gradient ratio of 7.4:1. Based on these values, the estimated stoichiometry of the cotransporter was 2.80 +/- 0.13:1 (HCO3-:Na+). Possible functions of the glial cell Na+/HCO3- cotransporter, including the regulation of CO2 in the retina and the regulation of cerebral blood flow, are discussed.
在蝾螈视网膜新鲜分离的米勒细胞中研究了一种生电Na⁺/HCO₃⁻共转运系统。在细胞外Ba²⁺和细胞内Cs⁺阻断K⁺电导后，使用全细胞膜片钳技术记录分离细胞的共转运电流。在恒定的细胞外pH值下，当通过压力将HCO₃⁻缓冲溶液喷射到浸泡在名义上无HCO₃⁻的溶液中的细胞表面，从而提高细胞外HCO₃⁻浓度时，会诱发外向电流。0.5 mM DIDS（4,4'-二异硫氰酸根合芪-2,2'-二磺酸盐）将HCO₃⁻诱发的外向电流降低至对照的4.4%，2 mM DNDS（4,4'-二硝基芪-2,2'-二磺酸盐）将其降低至对照的28.8%，2 mM哈尔满将其降低至对照的28.4%。在浴液和喷射溶液中用胆碱替代Na⁺可将反应降低至对照的1.3%。当在浴液、喷射溶液和细胞内溶液中用甲磺酸盐替代Cl⁻时，记录到正常幅度的碳酸氢盐诱发电流。同样，当在HCO₃⁻存在的情况下提高细胞外Na⁺浓度时，会诱发外向电流。2 mM DNDS将Na⁺诱发的反应降低至对照的16.2%，并且通过从浴液和喷射溶液中去除HCO₃⁻可消除该反应。综上所述，这些结果（被芪类化合物和哈尔满阻断、对HCO₃⁻和Na⁺的依赖性、对Cl⁻的非依赖性）表明蝾螈米勒细胞拥有一种生电Na⁺/HCO₃⁻共转运系统。Na⁺/HCO₃⁻共转运体位点主要定位于米勒细胞的终足区域。将HCO₃⁻喷射到终足上诱发的外向电流比喷射到细胞相对（远端）末端诱发的电流大10倍。共转运体的反转电位通过DNDS阻断共转运电流来确定。在不存在跨膜HCO₃⁻梯度的情况下，反转电位随跨膜Na⁺梯度系统地变化。对于[Na⁺]ₒ:[Na⁺]ᵢ比值为1:1，反转电位为 -0.1 mV，对于Na⁺梯度比值为7.4:1，反转电位为 -25.2 mV。基于这些值，估计共转运体的化学计量比为2.80±0.13:1（HCO₃⁻:Na⁺）。讨论了神经胶质细胞Na⁺/HCO₃⁻共转运体的可能功能，包括视网膜中CO₂的调节和脑血流量的调节。
