Molecular and functional characterization of sodium-hydrogen exchanger in skin as well as cultured keratinocytes and melanocytes

The sodium-hydrogen (Na+/H+) exchanger is one of the few transporter protei ns involved in the regulation and maintenance of intracellular pH and cell volume in most eukaryotic cell types. The current study investigates the ex pression of isoforms of the Na+/H+ exchanger (NHE) in human skin and in cul tured keratinocytes, melanocytes, and melanoma cells by reverse transcripti on-polymerase chain reaction (RT-PCR), immunohistochemical analysis and fun ctional studies. Neonatal foreskins were used to isolate RNA from epidermis and dermis, and to initiate cultures of keratinocytes and melanocytes. RT- PCR on RNA isolated from epidermis, dermis, keratinocytes, melanocytes and melanoma cells using PCR primers specific for NHE-1 yielded a 463 bp PCR pr oduct. RT-PCR performed using primers specific for NHE isoforms 2, 3, 4 and 5 did not yield any products. Western blotting analysis (of keratinocyte a nd melanocyte cell cultures) and indirect immunohistochemistry on neonatal foreskin, keratinocytes, melanocytes and melanoma cells using a NHE-1-speci fic polyclonal antibody demonstrated NHE-1 expression at the protein level. Physiological regulation of intracellular pH using a pH-sensitive dye, BCE CF, detected an amiloride-sensitive NHE activity in human keratinocyte, mel anocyte and melanoma cell cultures. These results indicate that cultures of human keratinocytes and melanocytes established from human skin and melano ma cells express the NHE-1 isoform of the sodium-hydrogen exchanger. (C) 20 01 Elsevier Science B.V. All rights reserved.