Endothelin-1 receptors on cultured rat articular chondrocytes: regulation by age, growth factors, and cytokines, and effect on cAMP production

Abstract

The presence of endothelin-1 receptor proteins and the expression of their specific mRNAs were studied using 1st passage confluent monolayers of articular chondrocytes, isolated from 1-month and 18-month-old rats following 24-h incubation with several growth factors and cytokines. The ET-1- binding sites were predominantly of ETA subtype since BQ123, but not IRL1038 (ETB receptor subtype agonist), effectively blocked 125I-ET-1 binding. The 18-month-old rat cell monolayers bear approximately twice as many 125I-ET-1-binding sites as the 1-month-old rat cells. PDGF, EGF, and IGF-1 increased the number of binding sites in a concentration-dependent manner in both old and young rat cells with PDGF being the most active and EGF more active than IGF-1. IL-1β, more potently than LPS, increased the number of binding sites in young rat cells only, whereas b-FGF, TGF-β and GM-CSF had no effect or decreased slightly 125I-ET-1 binding in both types of cells. TNF-α strongly decreased the number of binding sites on both young and old rat cells, only. RT-PCR showed an increased expression of the specific ETA mRNA with the age of animals and in the presence of 50 ng/ml PDGF BB only. The incubation of the cells with ETs 1–3 for 10 min resulted in a 50% decrease of cellular cAMP but the blocking of the receptors with BQ123 prior to their exposure to ETs had no effect on cAMP production whereas IRL1038 counteracted this effect only marginally. This suggests a receptor-independent mechanism for ETs-induced inhibition of cAMP production. However, a 10-min co-incubation of cells with ET-1 and with one of the following agents: cholera toxin, pertussis toxin, indomethacin, l-NMA, U73122 and calphostin resulted in an almost complete (calphostin) or partial suppression of ET-1-induced inhibition of cAMP production. The significance of these findings is unclear but the increased density of ET-1 binding sites on old rat cells and its regulation by certain growth factors or cytokines suggest the involvement of ET-1 in aging and possibly in age-related diseases.