Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells.
OBJECTIVE
Bovine corneal epithelial cells (BCEC) were cultured to determine whether endothelin (ET) receptor subtype stimulation affects ET isoform expression (ET-1, ET-2, and ET-3) through capacitative Ca2+ influx. To probe in the isolated bovine corneal epithelium (BCE) for ET isoform and ET (i.e., ETA and ETB) receptor gene expression.


METHODS
[Ca2+]i transients were characterized with microfluorometry. Endothelin isoform and ET receptor gene expression were probed with RNase protection analysis. Enzyme-linked immunosorbent assay was used to measure levels of ET-1-like immunoreactivity (ET-1-LI) in conditioned medium.


RESULTS
ET-1 (10(-6) M) increased [Ca2+]i more than twofold. After treatment with 10(-7) M alltrans retinoic acid (an inducer of differentiation), 10(-6) M sarafotoxin (S-6-c) (a selective ETB agonist), had a similar effect. Preincubation with either 5 microM U73122 (an inhibitor of IP8 formation) or 10 microM cyclopiazonic acid, which depletes intracellular Ca2+ store content, eliminated ET agonist-mediated [Ca2+]i increases. With a nominally Ca(2+)-free solution containing 10 microM cyclopiazonic acid, simultaneous 10(-6) M ET-1 and extracellular Ca2+ additions transiently increased [Ca2+]i twofold, whereas 10(-6) M S-6-c increased it by only 20%. This augmentation was eliminated by preexposure to either BQ123 (10 microM), selective ETA receptor antagonist, U73122 (5 microM), or SKF 96365 (3 x 10(-5) M), an inhibitor of stores-operated channels. ET-1, ET-2 isoforms, and ET receptor mRNAs were identified. S-6-c (10(-6) M) increased the level of ET-1-LI after 12 hours by approximately ninefold.


CONCLUSIONS
In BCEC, capacitative calcium influx is involved in mediating a positive feedback relationship between ETB receptor stimulation and ET protein expression. Identification of ET-1 and ET-2 gene expression in BCE strengthens the notion that this regulation could be autocrine mediated.
Tao
W
W
Department of Pharmacology, University of North Texas Health Science Center, Fort Worth 76107, USA.
Wu
X
X
Liou
G I
GI
Abney
T O
TO
Reinach
P S
PS
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002417:Cattle; D018929:Cell Culture Techniques; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000096:biosynthesis* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017466:Receptors, Endothelin / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction; D014212:Tretinoin / Q000494:pharmacology
D015220:Calcium Channels; D017931:DNA Primers; D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D004791:Enzyme Inhibitors; D017466:Receptors, Endothelin; D014212:Tretinoin; D002118:Calcium