Endothelin receptor B in trabecular meshwork.
Endothelin-1 (ET-1), the most potent vasoconstrictor known to date, seems to be involved in the pathogenesis of primary open angle glaucoma. ET-1 was found in different tissues of the eye and in high concentrations in the aqueous humour. The effects of ET-1 are mediated by two receptors, ET-A receptor (ET-AR) and ET-B receptor (ET-BR), which are both expressed in bovine trabecular meshwork (TM). ET-1 induced contraction of TM predominantly by activation of ET-AR. This study analyzes the role of ET-BR in TM function and investigates the synthesis of ET-1 by human TM (HTM) cells. The effect of IRL-1620, a specific ET-BR agonist, on contractility of bovine TM (BTM) was investigated with a force length transducer system. The effect of this agonist on intracellular free Ca(2+) [Ca(2+)](i) was examined using the Ca(2+)-sensitive dye fura-2AM. The expression of the ET-AR and ET-BR was investigated in cultured HTM cells using Western blot and PCR methods. With PCR methods the expression of prepro-endothelin-1 (ppET-1) and isoforms of endothelin-1 converting enzyme (ECE-1) in cultured HTM cells was analyzed. Activation of ET-BR by IRL-1620 (5 x 10(-7)M) results in contraction of native BTM (41% of the carbachol value) and also in a transient increase in [Ca(2+)](i) in cultured BTM and HTM cells (365 and 273% of the basal level, respectively). IRL-1620 also induced contraction in native BTM under intra- and extracellularly Ca(2+)-free conditions. A clear signal for ET-AR at 40 kDa and ET-BR at 35 kDa could be detected in membrane lysates of cultured HTM cells. PCR analysis further confirmed the existence of ET-AR and ET-BR in these cells. Furthermore, RT-PCR revealed that neither ppET-1 nor one of the ECE-1 isoforms was expressed in cultured HTM cells. This study presents evidence for the expression of a functional ET-BR in bovine and human TM. Currently, there is no evidence for ET-1 synthesis in HTM cells. Further investigations are necessary to clarify the physiological function of ET-BR in TM and the source of ET-1 at this tissue.
Rosenthal
Rita
R
Institut für Klinische Physiologie, Charité, Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. rita.rosenthal@charite.de
Choritz
Lars
L
Zorn
Rüdiger
R
Münzer
Galina
G
Fromm
Michael
M
Pfeiffer
Norbert
N
Thieme
Hagen
H
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000494:pharmacology; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D044023:Receptor, Endothelin B / Q000819:agonists / Q000502:physiology*; D017466:Receptors, Endothelin / Q000378:metabolism; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology
D019332:Endothelin-1; D016232:Endothelins; D010446:Peptide Fragments; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; C074308:sovateltide; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D002118:Calcium