Endothelin receptor A is expressed and mediates the [Ca2+]i mobilization of cells in human ciliary smooth muscle, ciliary nonpigmented epithelium, and trabecular meshwork.

PURPOSE To identify which endothelin receptor subtype is expressed and is functional in the human ciliary body and trabecular meshwork, tissues that regulate aqueous humor dynamics. METHODS Immunocytochemistry was used to characterize the primary culture cells of normal human ocular cells. Endothelin receptor gene expression was probed with reverse transcription of polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) mobilization was measured with video image microscopy using Fura-2AM as a fluorescent probe. RESULTS Identities of primary cultures, human ciliary smooth muscle (HCSM), ciliary nonpigmented epithelial (HCE), and trabecular meshwork (HTM) cells were confirmed by immunocytochemistry, using cell-specific markers and observing typical cell morphologies. The presence of endothelin receptor A (ETA) was detected with RT-PCR in all three types of cells. The mRNA phenotype was verified with restriction enzyme BamHI digestion. No ETB receptor subtype expression was detected with RT-PCR under the cell culture conditions used. The [Ca2+]i of HCSM cells was increased from 57 +/- 7 nM to 328 +/- 108 nM (n = 23; mean +/- SE; P < 0.05) by 1 nM endothelin-1 (ET-1). In HCE cells, [Ca2+]i increased from 40 +/- 3 nM to 90 +/- 10 nM (n = 55) (P < 0.001) with the same concentration of ET-1. Similarly, ET-1 (1 nM) increased the [Ca2+]i from 51 +/- 6 nM to 185 +/- 47 nM (n = 19) (P < 0.001) in the HTM cells. The agonist for ETB, S6c, had no effect on [Ca2+]i transients in all three cell types. No ETB receptor expression was detected in these cell types under the experimental and culture conditions. CONCLUSION ETA receptor is expressed and is possibly responsible for mediating the signal for [Ca2+]i mobilization by ET-1 in human ciliary smooth muscle, ciliary nonpigmented epithelial cells, and trabecular meshwork cells.

[1]  A. Dibas,et al.  Regulation of endothelin-1 in human non-pigmented ciliary epithelial cells by tumor necrosis factor-alpha. , 1998, Experimental eye research.

[2]  J. Yanni,et al.  Secretion of proinflammatory cytokines by human conjunctival epithelial cells. , 1997, Ocular immunology and inflammation.

[3]  A. Dibas,et al.  Mechanism of vasopressin-induced increase in intracellular Ca2+ in LLC-PK1 porcine kidney cells. , 1997, The American journal of physiology.

[4]  X. Wu,et al.  Endothelin receptor-mediated Ca2+ signaling and isoform expression in bovine corneal epithelial cells. , 1997, Investigative ophthalmology & visual science.

[5]  D. Wilson,et al.  An endothelin-1 induced model of optic nerve ischemia in the rabbit. , 1996, Investigative ophthalmology & visual science.

[6]  S. Tsukahara,et al.  Morphological changes in rabbit ciliary epithelium and blood-aqueous barriers after intravitreal 10(-5) M endothelin-1. , 1996, Experimental eye research.

[7]  T. Yorio,et al.  Endothelin-induced changes of second messengers in cultured human ciliary muscle cells. , 1996, Investigative ophthalmology & visual science.

[8]  Y. Kitazawa,et al.  Effects of BQ-123, an ETA recepter-selective antagonist, on changes of intraocular pressure, blood-aqueous barrier and aqueous prostaglandin concentrations caused by endothelin-1 in rabbit. , 1996, Japanese journal of ophthalmology.

[9]  T. Tokoro,et al.  The effects of endothelin-1 on isolated bovine ciliary muscles. , 1995, Experimental eye research.

[10]  S. Magnuson,et al.  Endothelin receptor in human astrocytoma U373MG cells: binding, dissociation, receptor internalization. , 1995, The Journal of pharmacology and experimental therapeutics.

[11]  Y. Kitazawa,et al.  Intraocular pressure response to intravitreal injection of endothelin-1 and the mediatory role of ETA receptor, ETB receptor, and cyclooxygenase products in rabbits. , 1995, Current eye research.

[12]  M. Wiederholt,et al.  Electrophysiological properties of cultured human trabecular meshwork cells. , 1994, Experimental eye research.

[13]  D. Archer,et al.  Immunoreactive endothelin distribution in ocular tissues. , 1994, Investigative ophthalmology & visual science.

[14]  S. Matsumoto,et al.  Effects of endothelin-1 on [Ca2+]i and pHi in trabecular meshwork cells. , 1994, Current eye research.

[15]  L. Desantis,et al.  Preliminary characterization of a transformed cell strain derived from human trabecular meshwork. , 1994, Current eye research.

[16]  E. Tamm,et al.  Single-cell contraction assay for human ciliary muscle cells. Effect of carbachol. , 1993, Investigative Ophthalmology and Visual Science.

[17]  E. Lütjen-Drecoll,et al.  Distribution of endothelin-like immunoreactivity in the human ciliary epithelium. , 1993, Current eye research.

[18]  M. Wiederholt,et al.  Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells. , 1992, Current eye research.

[19]  M. Wiederholt,et al.  Effects of endothelin and calcium channel blockers on membrane voltage and intracellular calcium in cultured bovine trabecular meshwork cells. , 1992, German journal of ophthalmology.

[20]  K. Huebner,et al.  Cloning and chromosomal localization of a human endothelin ETA receptor. , 1991, Biochemical and biophysical research communications.

[21]  M. Wiederholt,et al.  Endothelin-evoked contractions in bovine ciliary muscle and trabecular meshwork: interaction with calcium, nifedipine and nickel. , 1991, Current eye research.

[22]  E. Tamm,et al.  Cell cultures of human ciliary muscle: growth, ultrastructural and immunocytochemical characteristics. , 1991, Experimental eye research.

[23]  S. Nakanishi,et al.  Molecular cloning of a non-isopeptide-selective human endothelin receptor. , 1991, Biochemical and biophysical research communications.

[24]  H. Jampel,et al.  Ocular effects of the endothelins. Abundant peptides in the eye. , 1991, Archives of ophthalmology.

[25]  J. Nathanson,et al.  Effect of endothelin on outflow facility and accommodation in the monkey eye in vivo. , 1991, Investigative ophthalmology & visual science.

[26]  K. Erickson-Lamy,et al.  Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells. , 1989, Biochemical and biophysical research communications.

[27]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[28]  Benjamin Geiger,et al.  The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells , 1982, Cell.

[29]  S. Tseng,et al.  Correlation of specific keratins with different types of epithelial differentiation: Monoclonal antibody studies , 1982, Cell.