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 proteins involved in the regulation and maintenance of intracellular pH and cell volume in most eukaryotic cell types. The current study investigates the expression of isoforms of the Na(+)/H(+) exchanger (NHE) in human skin and in cultured keratinocytes, melanocytes, and melanoma cells by reverse transcription-polymerase chain reaction (RT--PCR), immunohistochemical analysis and functional 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 product. RT--PCR performed using primers specific for NHE isoforms 2, 3, 4 and 5 did not yield any products. Western blotting analysis (of keratinocyte and melanocyte cell cultures) and indirect immunohistochemistry on neonatal foreskin, keratinocytes, melanocytes and melanoma cells using a NHE-1-specific polyclonal antibody demonstrated NHE-1 expression at the protein level. Physiological regulation of intracellular pH using a pH-sensitive dye, BCECF, detected an amiloride-sensitive NHE activity in human keratinocyte, melanocyte and melanoma cell cultures. These results indicate that cultures of human keratinocytes and melanocytes established from human skin and melanoma cells express the NHE-1 isoform of the sodium--hydrogen exchanger.
Sarangarajan
R
R
Department of Dermatology, University of Cincinnati College of Medicine, OH 45267-0592, USA. sarangr@email.uc.edu
Shumaker
H
H
Soleimani
M
M
Le Poole
C
C
Boissy
R E
RE
D002478:Cells, Cultured; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007231:Infant, Newborn; D015603:Keratinocytes / Q000378:metabolism*; D008544:Melanocytes / Q000378:metabolism*; D020033:Protein Isoforms / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000033:anatomy & histology / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis*
D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers