Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells

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.

[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.

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.

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.