Relation between alpha, beta, and gamma human amiloride- sensitive epithelial Na+ channel mRNA levels and nasal epithelial potential difference in healthy men.
To analyze messenger RNA (mRNA) levels for the alpha, beta, and gamma subunits of the human amiloride-sensitive epithelial Na+ channel (hENaC) in respiratory epithelia, we developed a competitive quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) assay specific for each subunit, using two human respiratory epithelial-cell lines. We next determined the relation between hENaC mRNA levels and the biologic activity of the hENaC in the respiratory epithelium of eight normal men. The electrical potential difference (PD) between the epithelium of the inferior nasal turbinate and the subcutaneous space was measured, using control and amiloride (100 microM) solutions. QRT-PCR measurement of hENaC-subunit mRNAs and epithelial-specific cytokeratin 18 mRNA allowed us to normalize hENaC expression to epithelial-cell RNA. Respective values for alpha, beta, and gamma hENaC mRNA levels in epithelium obtained at the site of maximal PD were 39 +/- 4.0, 7.5 +/- 0.92, and 1.8 +/- 0.25 attomol/fmol cytokeratin mRNA, respectively. Respiratory epithelial PD exhibited a significant negative correlation with gamma hENaC (r2 = 0.72, p < 0.01), tended to increase with increasing alpha hENaC, and was unaffected by beta hENaC mRNA levels. Our results suggest that hENaC activity in vivo is influenced by expression of the gene for gamma hENaC. The assay used in the study provides a useful tool for evaluating Na+-channel expression in clinically relevant patient populations.
Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada. gotulak@sickkids.on.ca
G
G
Otulakowski
S
S
Flueckiger-Staub
L
L
Ellis
K
K
Ramlall
O
O
Staub
D
D
Smith
P
P
Durie
H
H
O'Brodovich
D000231:Adenocarcinoma, Papillary / Q000235:genetics; D000328:Adult; D000584:Amiloride / Q000494:pharmacology*; D018287:Carcinoma, Large Cell / Q000235:genetics; D004232:Diuretics / Q000494:pharmacology*; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007633:Keratins / Q000032:analysis / Q000235:genetics; D016014:Linear Models; D008175:Lung Neoplasms / Q000235:genetics; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008875:Middle Aged; D009297:Nasal Mucosa / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D012867:Skin / Q000187:drug effects / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014420:Turbinates / Q000187:drug effects / Q000378:metabolism
D004232:Diuretics; D012333:RNA, Messenger; D015222:Sodium Channels; D007633:Keratins; D000584:Amiloride; D012194:RNA-Directed DNA Polymerase