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

Abstract

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 ([Ca 2+ ] 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 immuno-cytochemistry, using cell-specific markers and observing typical cell morphologies. The presence of endothelin receptor A (ET A) was detected with RT-PCR in all three types of cells. The mRNA phenotype was verified with restriction enzyme BamHI digestion. No ET B receptor subtype expression was detected with RT-PCR under the cell culture conditions used. The [Ca 2+ ] 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, [Ca 2+ ] 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 [Ca 2+ ] i from 51 ± 6 nM to 185 ± 47 nM (n = 19) (P < 0.001) in the HTM cells. The agonist for ET B, S6c, had no effect on [Ca 2+ ] i transients in all three cell types. No ET B receptor expression was detected in these cell types under the experimental and culture conditions. CONCLUSION. ET A receptor is expressed and is possibly responsible for mediating the signal for [Ca 2+ ] i mobilization by ET-1 in human ciliary smooth muscle, ciliary nonpigmented epithelial cells, and trabecular meshwork cells.