van Hooijdonk C A, Colbers R M, Piek J, van Erp P E
Department of Dermatology, University Hospital Nijmegen, The Netherlands.
Cell Prolif. 1997 Aug-Sep;30(8-9):351-63. doi: 10.1046/j.1365-2184.1997.00098.x.
In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na+/H+ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na+/H+ exchanger nor a proliferation-associated increase in intracellular pH (pHi) has been demonstrated. The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na+/H+ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pHi measurements in BALB/MK cells. Initial measurements of the pHi using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes. Using SNARF-calcein we were able to show proliferation to be associated with increased pHi. However, culture conditions were critical for these measurements. Our data indicate that the Na+/H+ exchanger is involved in this process, since acid load and pHi-recovery experiments showed the alkalization to be amiloride-sensitive.
在许多细胞类型中，细胞质碱化是细胞激活的早期标志。一种对氨氯地平敏感的Na⁺/H⁺交换体是这一过程的重要调节因子。然而，在角质形成细胞中，尚未证实存在Na⁺/H⁺交换体，也未发现细胞内pH值（pHi）与增殖相关的增加。本研究的目的是调查源自BALB/MK细胞系的角质形成细胞是否含有Na⁺/H⁺交换体，以及这些细胞中的细胞质碱化是否与增殖相关。在特定培养条件下，这种小鼠角质形成细胞系能够很容易地在增殖状态和静止状态之间转换。新型pH敏感染料半萘罗丹氟（SNARF）-钙黄绿素被证明非常适合用于BALB/MK细胞的流式细胞术pHi测量。最初使用已确立的荧光染料2',7'-双（羧乙基）-5,6-羧基荧光素（BCECF）和SNARF-1的混合物测量pHi未成功，因为这些pH敏感染料的摄取和结合动力学存在差异。使用SNARF-钙黄绿素，我们能够证明增殖与pHi升高相关。然而，培养条件对这些测量至关重要。我们的数据表明，Na⁺/H⁺交换体参与了这一过程，因为酸负荷和pHi恢复实验表明碱化对氨氯地平敏感。
