Endothelin-1 in monolayer cultures of articular chondrocytes from young and old rats: regulation by growth factors and cytokines

Abstract

The endothelin-1 (ET-1) concentrations were measured by RIA in the media of confluent monolayer cultures of rat articular chondrocyte (RAC) exposed to fetal calf serum (FCS) and several growth factors and cytokines. The cells were obtained from 1- and 18-month-old rats. First passage cells were starved in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.2% FCS serum for 24 h and then incubated for 48 h in the same fresh medium with each of the following factors: fetal calf serum (FCS), transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), and NO donor, sodium nitroprusside (SNP). The following was found: the cells from 18-month-old animals accumulated about twice as much ET-1 per μg DNA under basal (low serum) and stimulated conditions as cells from young rats. All, but PDGF and SNP produced concentration-dependent rise in ET-1 levels, the most effective being 10% FCS, IL-1β, TNF-α, EGF, IGF-1 and LPS. TGF-β caused the smallest stimulation and PDGF was ineffective or slightly inhibitory at high concentrations. SNP caused concentration-dependent decrease of ET-1 concentrations. ET-1-specific mRNA was identified by RT-PCR in cells incubated with the above factors and its concentration paralleled that of the peptide. This suggests that ET-1 found in the culture media of RAC stems, at least in part, from the synthesis. Increased immunoreactive peptide concentration and mRNA expression with the age of the donor rat and its regulation by several growth factors and cytokines suggest the involvement of ET-1 in chondrocytes’ physiology and possibly pathology.