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

The presence of endothelin-1 receptor proteins and the expression of their specific mRNAs were studied using 1st passage confluent monolayers of artic ular 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 r eceptor subtype agonist). effectively blocked I-125-ET-1 binding. The 18-mo nth-old rat cell monolayers bear approximately twice as many I-125-ET-1-bin ding sires 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 an d young rat cells with PDGF being the most active and EGF more active than IGF-1. IL-1 beta. more potently than LPS, increased the number of binding s ites in young rat cells only, whereas b-FGF. TGF-beta and GM-CSF had no eff ect or decreased slightly I-125-ET-1 binding in both types of cells. TNF-al pha strongly decreased the number of binding sites on both young and old ra t cells, only. RT-PCR showed an increased expression of the specific ETA mR NA with the age of animals and in the presence of 50 ng/ml PDGF BE only. Th e incubation of the cells with ETs 1-3 for 10 min resulted in a 50'%, decre ase 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 coun teracted 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: chol era toxin. pertussis toxin. indomethacin, L-NMA, U73122 and calphostin resu lted in an almost complete (calphostin) or partial suppression of ET-1-indu ced inhibition of cAMP production. The significance of these findings is un clear but the increased density of ET-1 binding sites on old rat cells and its regulation by certain growth factors or cytokines: suggest the involvem ent of ET-1 in aging and possibly in age-related diseases. (C) 2001 Elsevie r Science Ireland Ltd. All rights reserved.