Wh. Tao et al., ENDOTHELIN RECEPTOR-MEDIATED CA2-CELLS( SIGNALING AND ISOFORM EXPRESSION IN BOVINE CORNEAL EPITHELIAL), Investigative ophthalmology & visual science, 38(1), 1997, pp. 130-141
Purpose. Bovine corneal epithelial cells (BCEC) were cultured to deter
mine whether endothelin (ET) receptor subtype stimulation affects ET i
soform expression (ET-I, ET-2, and ET-3) through capacitative Ca2+ inf
lux. To probe in the isolated bovine corneal epithelium (BCE) for ET i
soform and ET (i.e., ET(A) and ET(B)) receptor gene expression. Method
s. [Ca2+](i) transients were characterized with microfluorometry. Endo
thelin isoform and ET receptor gene expression were probed with RNase
protection analysis. Enzyme-linked immunosorbent assay was used to mea
sure levels of ET-l-like immunoreactivity (ET-1-LI) in conditioned med
ium. Results. ET-1 (10(-6) M) increased [Ca2+](i) more than twofold. A
fter treatment with 10(-7) hi all-trans retinoic acid (an inducer of d
ifferentiation), 10(-6) M sarafotoxin-6-c (S-6-c) (a selective ET(B) a
gonist), had a similar effect. Preincubation with either 5 mu M U73122
(an inhibitor of IP3 formation) or 10 mu M cyclopiazonic acid, which
depletes intracellular Ca2+ store content, eliminated ET agonist-media
ted [Ca2+](i) increases. With a nominally Ca2+-free solution containin
g 10 mu M cyclopiazonic acid, simultaneous 10(-6) M ET-I and extracell
ular Ca2+ additions transiently increased [Ca2+](i) twofold, whereas 1
0(-6) M S-6-c increased it by only 20%. This augmentation was eliminat
ed by preexposure to either BQ123 (10 mu M), selective ET(A) receptor
antagonist, U73122 (5 mu M), or SKF 96365 (3 x 10(-5) M), an inhibitor
of stores-sperated channels. ET-1, ET-2 isoforms, and ET receptor mRN
As were identified. S-6-c (10(-6) M) increased the level of ET-1-LI af
ter 12 hours by approximately ninefold. Conclusions. In BCEC, capacita
tive calcium influx is involved in mediating a positive feedback relat
ionship 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.