Demonstration of an Na+/H+ exchanger in mouse keratinocytes measured by the novel pH-sensitive fluorochrome SNARF-calcein.
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.
van Hooijdonk
C A
CA
Department of Dermatology, University Hospital Nijmegen, The Netherlands.
Colbers
R M
RM
Piek
J
J
van Erp
P E
PE
D000818:Animals; D001578:Benzopyrans; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004338:Drug Combinations; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009284:Naphthols / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D001578:Benzopyrans; D004338:Drug Combinations; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009284:Naphthols; D012235:Rhodamines; D017923:Sodium-Hydrogen Exchangers; C091666:seminaphthorhodaminefluoride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; C007740:fluorexon