Abstract
Purpose: :
Endothelin –1 (ET–1) is a potent vasoactive peptide. The aqueous humor and plasma levels of ET–1 are elevated in glaucomatous patients. Injection of endothelin–1 into rat eyes causes optic nerve damage similar to that seen in glaucoma. Previously our lab has characterized the expression of endothelin A (ETA) and endothelin B (ETB) receptors in human optic nerve head astrocytes. The purpose of this study was to determine the expression of endothelin receptors in GFAP negative Lamina cribrosa cells isolated from lamina cribrosa, the principle site of axonal transport block observed in POAG subjects.
Methods: :
human lamina cribrosa cells were treated with 1nM, 10nM and 100nM of ET–1 for 24 hrs. Total RNA was isolated and cDNA synthesized. ETA and ETB receptor mRNA was analyzed using reverse transcription–polymerase chain reaction (RT–PCR). Total protein lysates of lamina cribrosa cells treated with 1nm, 10nM & 100nM for 24 hrs were separated on a 7.5 % SDS PAGE gel and expression of endothelin A and endothelin B receptors determined by Western blot using anti ETA and anti ETB receptor antibodies. ET –1 mediated intra–cellular calcium changes in the presence of 1, 10 & 100nM of ET–1, 1uM BQ788 an ETB selective antagonist and 1uM BQ123 an ETA selective antagonist were measured using Fura–2 calcium imaging. ET–1 mediated intra cellular calcium levels was also measured following ET–1(100nM) treatment for 24 hrs.
Results: :
Expression of both message and protein of ETA and ETB receptor were detected in human lamina cribrosa cells. The message and protein levels of ETA receptor were down regulated following treatment with ET–1 for 24 hrs. An upregulation of ETB receptor was observed following ET–1 treatment for 24 hrs. Increase in intracellular calcium was observed in a dose dependent manner with a greater increase at 100nM. The increase in intra cellular calcium was blocked by ETA specific inhibitor BQ123 but not by ETB specific inhibitor BQ788. The LC cells treated with ET–1 overnight failed to show an increase in intra cellular calcium levels when retreated with ET–1.
Conclusions: :
human lamina cribrosa cells express functional ETA and ETB receptors and their expression and function can be altered in response to prolong exposure to ET–1. This may have an implication in the normal physiology of Lamina Cribrosa cells in POAG subjects where elevated plasma and aqueous humor levels of endothelin–1 have been detected.
Keywords: lamina cribrosa • receptors: pharmacology/physiology • calcium