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

Wenhong Tao, Xiaoping Wu, Gregory I. Liou, Tom O. Abney, Peter S. Reinach

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)130-141
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number1
StatePublished - Feb 6 1997

Fingerprint

Endothelin Receptors
Endothelins
Endothelin-1
Protein Isoforms
Epithelial Cells
Endothelin-2
Endothelin B Receptors
1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole
Gene Expression
Endothelin-3
Cytophotometry
Ribonucleases
Conditioned Culture Medium
Tretinoin
Enzyme-Linked Immunosorbent Assay
Calcium
Messenger RNA

Keywords

  • calcium
  • capacitative Ca influx
  • corneal epithelium
  • endothelin
  • gene expression
  • receptors
  • stores-operated channel

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells. / Tao, Wenhong; Wu, Xiaoping; Liou, Gregory I.; Abney, Tom O.; Reinach, Peter S.

In: Investigative Ophthalmology and Visual Science, Vol. 38, No. 1, 06.02.1997, p. 130-141.

Research output: Contribution to journalArticle

Tao, Wenhong ; Wu, Xiaoping ; Liou, Gregory I. ; Abney, Tom O. ; Reinach, Peter S. / Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells. In: Investigative Ophthalmology and Visual Science. 1997 ; Vol. 38, No. 1. pp. 130-141.
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AU - Tao, Wenhong

AU - Wu, Xiaoping

AU - Liou, Gregory I.

AU - Abney, Tom O.

AU - Reinach, Peter S.

PY - 1997/2/6

Y1 - 1997/2/6

N2 - 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.

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KW - gene expression

KW - receptors

KW - stores-operated channel

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