Rapid Characterization of Bimatoprost Pharmacology in Human Ocular Cells by Using Cellular Dielectric Spectroscopy

Abstract

Purpose: : Bimatoprost is widely used as anti-glaucoma therapy but, to date, the cellular localization and characterization of its effects in individual ocular cell types has not been achieved.

Methods: : Primary cultures of human endothelial cells of Schlemm’s canal (ECSC), trabecular meshwork (TM), and ciliary muscle (CM) cells were prepared from donor eyes. Cellular dielectric spectroscopy permits rapid pharmacological and second messenger characterization in primary cells in a 96 well format. It is a real time, non-invasive, label-free assay, which measures impedance.

Results: : Bimatoprost produced a concentration-dependent change in cell monolayer impedance in ECSC, TM, and CM cells, with EC₅₀ (nM) values of 1.6, 4.3, and 1.4, respectively. These effects were susceptible to the prostamide antagonist AGN 211334. Bimatoprost effects were insensitive to cholera toxin and pertussis toxin but were abolished by phorbol 12-myristate, 13-acetate. AGN 211334 exerted properties consistent with inverse agonism in TM cells but not ECSC cells . The pharmacology of bimatoprost and AGN 211334 in TM cells identified by cell dielectric spectroscopy was confirmed in its entirety by studying hydraulic conductivity in a TM cell monolayer.

Conclusions: : A bimatoprost-sensitive prostamide was identified and rapidly characterized using cellular dielectric spectroscopy. Bimatoprost potently stimulates ECSC, TM, and CM cells and the receptor is G_q coupled. Cellular dielectric spectroscopy represents a new technique for rapid characterization of pharmacology and second messenger signaling in human primary cells .

Keywords: eicosanoids • outflow: trabecular meshwork • outflow: ciliary muscle