EXTRACELLULAR PH GRADIENTS MEASURED FROM ISOLATED RETINAL CELLS

脊椎动物视网膜内的神经元信号是高度 取决于细胞外液的pH值。例如， 外界酸碱值从7.8到7.0的温和变化已显示出 几乎完全抑制二阶光诱导反应 虎蜥蜴视网膜中的神经元(1-3)。此外， 光对视网膜的刺激可以诱导显著的变化 细胞外pH(4，5)，增加了正常的可能性 光诱导的胞外质子浓度的变化 可能在调节视觉的流动方面起着重要作用 视网膜内的信息。比率成像技术使用 像BCECF这样的荧光pH指示剂已经开始扩大我们的 视网膜神经元内pH控制的知识(参看。6、7)。 然而，这种方法不容易允许直接检查 穿过神经元和神经胶质细胞膜的质子通量。这 局限性，加上脊椎动物细胞的异质性 Ret ina，使细胞和分子研究变得困难。 细胞外pH及其维持机制的研究进展 光刺激过程中的变化。这项研究的目的是 确定是否可以使用不同的方法来检查质子通量 直接来自分离的视网膜神经元，为未来铺平道路 旨在从分子水平上理解这种机制的实验 这可能是外部PH值变化的原因。我们使用了一个 自参考格式的电极，允许检查 预计在等离子体中产生的小离子梯度 膜-盐水界面(见Smith，Sanger&Jaffe(8)复习)。 分离的冰鱼视网膜神经细胞是 使用所述的酶解离方案制备 Malchow等人。(9)。我们的结果表明，自我参照 PH选择微电极确实可以成功地用于检测 分离的视网膜神经元膜上的质子通量。我们是 目前使用已知的特定药理探针 选择性地抑制各种pH转运机制，努力 阐明造成我们所拥有的通量的分子机制 检测到。参考文献1.Kleinschmidt，J.1990。SoC。神经科。 (节文)16：465。2.克莱因施密特，J.1991。安·纽约·阿卡德SCI。 635：468-470。3.Barnes，S.，Merchant，V.&Mahmad，1993年成立。程序 娜塔莉。阿卡德。SCI 90：10081-10085。4.Oakley，B.II&wen，R.。 J.Physiol.(长)419：353-378。5.博古拉，G.A.，卡尔沃斯基， C.J.和Steinberg，R.H.1989。异象解答29：1069-1077。6. 张晓明，李晓波，1998。实验眼科研究66：449-464。 7.Saarikoski，J.，Ruusuvuori，E.，Koskelainen，A.&Donner，K.1997。 J.Physiol.(长)498：61-72。8.史密斯，P.J.S.，桑格，R.H. 谢菲，L.F.，1994。冰毒。细胞生物。40：115-134。9.马尔乔， 黄瑞平，钱，H.，Ripps，H.&Dowling，J.E.1990。J.Gen.Physiol.95： 177-198。

Neuronal signaling within the vertebrate retina is highly dependent upon the pH of the extracellular fluid. For example, the modest alteration of external pH from 7.8 to 7.0 has been shown to suppress almost completely the light-induced responses of second order neurons in the retina of the tiger salamander (1-3). Further, stimulation of the retina by light can induce significant changes in extracellular pH (4, 5), raising the possibility that normal light-induced alterations in the extracellular proton concentration might play an important role in modulating the flow of visual information within the retina. Ratiometric imaging techniques using fluorescent pH indicator dyes such as BCECF have begun to expand our knowledge of the control of pH within retinal neurons (cf. 6, 7). However, this method does not readily permit direct examination of fluxes of protons across the membranes of neurons and glia. This limitation, coupled withthe cellular heterogeneity of the vertebrate ret ina,has made it difficult to study the cellular and molecular mechanisms responsible for the maintenance of extracellular pH and its alteration during light stimulation. The purpose of this study was to determine if a different method could be used to examine proton flux directly from isolated retinal neurons, setting the stage for future experiments designed to understand at a molecular level the mechanism that might be responsible for changes in external pH. We used an electrode in the self-referencing format that permits examination of the small ionic gradients expected to be generated at the plasma membrane-saline interface (see Smith, Sanger & Jaffe (8) for review). Isolated retinal neurons from skate (Raja erinacea/R. ocellata) were prepared using the enzymatic dissociation protocol described by Malchow et al. (9). Our results demonstrate that self-referencing pH-selective microelectrode can indeed be used successfully to examine proton flux across the membranes of isolated retinal neurons. We are currently employing specific pharmacological probes known to selectively inhibit various pH transport mechanisms in an effort to clarify the molecular mechanisms responsible for the fluxes we have detected. References 1. Kleinschmidt, J. 1990. Soc. Neurosci. (Abstr.) 16: 465. 2. Kleinschmidt, J. 1991. Ann N.Y. Acad. Sci. 635: 468-470. 3. Barnes, S., Merchant, V. & Mahmud, F. 1993. Proc. Natl. Acad. Sci 90: 10081-10085. 4. Oakley, B. II & Wen, R. 1989. J. Physiol. (Lond.) 419: 353-378. 5. Borgula, G.A., Karwoski, C.J., &Steinberg, R.H. 1989. Vision Res. 29: 1069-1077. 6. Haugh-Scheidt, L. & Ripps, H. 1998. Exp. Eye Research 66: 449-464. 7. Saarikoski, J., Ruusuvuori, E., Koskelainen, A. & Donner, K. 1997. J. Physiol. (Lond.) 498: 61-72. 8. Smith, P.J.S., Sanger, R.H. & Jaffe, L. F. 1994. Meth. Cell Biol. 40: 115-134. 9 .Malchow, R.P., Qian, H., Ripps, H. & Dowling, J.E. 1990. J. Gen. Physiol. 95: 177-198.