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

Purpose. 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 ephithelium (BCE) for ET isoform and ET (i.e., ET(A) and ET(B)) 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 all- trans retinoic acid (an inducer of differentation), 10^-6 M sarafotoxin-6-c (S-6-c) (a selective ET(B) agonist), had a similar effect. Preincubation with either 5 μM U73122 (an inhibitor of IP₃ formation) or 10 μM cyclopiazonic acid, which depletes intracellular Ca²⁺ store content, eliminated ET agonist-mediated [Ca²⁺](i) increases. With a nominally Ca²⁺-free solution containing 10 μM cyclopiazonic acid, simultaneous 10^-6 M ET-1 and extracellular Ca²⁺ additions transiently increased [Ca²⁺]₁ twofold, whereas 10^-6 M S-6-c increased it by only 20%. This augmentation was eliminated by preexposure to either BQ123 (10 μM), selective ET(A) receptor antagonist, U73122 (5 μM), 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 ET(B) 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.