Open AccessDemonstration of an Na + /H + exchanger in mouse keratinocytes measured by the novel pH‐sensitive fluorochrome SNARF‐calcein
Author(s) -
Hooijdonk C. A. E. M.,
Colbers R. M. L.,
Piek J.,
Erp P. E. J.
Publication year - 1997
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/j.1365-2184.1997.tb00947.x
Subject(s) - amiloride , calcein , intracellular ph , cytoplasm , sodium–hydrogen antiporter , cell culture , biophysics , intracellular , cell growth , chemistry , microbiology and biotechnology , biochemistry , biology , membrane , sodium , organic chemistry , genetics
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 (pH i ) 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 pH i measurements in BALB/MK cells. Initial measurements of the pH i 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 pH i . 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 pH i ‐recovery experiments showed the alkalization to be amiloride‐sensitive.