Mineralocorticoid hormone signaling regulates the 'epithelial sodium channel' in fibroblasts from human cornea.
We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na(+)-K(+)-ATPase in the basolateral membrane.
Mirshahi
M
M
Inserm XR-86 et CNRS, Centre des Cordeliers, et Inserm U-9912, Paris, France.
Mirshahi
S
S
Golestaneh
N
N
Nicolas
C
C
Mishal
Z
Z
Lounes
K C
KC
Hecquet
C
C
Dagonet
F
F
Pouliquen
Y
Y
Agarwal
M K
MK
D000450:Aldosterone / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D012313:RNA / Q000032:analysis; D018161:Receptors, Mineralocorticoid / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction / Q000187:drug effects; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*
D017931:DNA Primers; D018161:Receptors, Mineralocorticoid; D015222:Sodium Channels; D000450:Aldosterone; D012313:RNA