ENDOTHELIN 1 RECEPTORS, SIGNAL TRANSDUCTION AND EFFECTS ON DNA AND PROTEOGLYCAN SYNTHESIS IN RAT ARTICULAR CHONDROCYTES

Abstract This study showed that endothelins (ETs) stimulate DNA and proteoglycan synthesis in monolayer culture of rat articular chondrocytes (AC) by interacting with specific cell surface receptors. The high affinity receptors bound [ 125 I]ET-1 with a K d of 0.54 nM and B max of 81.4 pM/μg DNA (approximately 40 000 binding sites per cell) was demonstrated. [ 125 I]ET-1 binding was completely inhibited by unlabelled ET-1 or ET-2, and by BQ123 (ET A receptor antagonist), whereas ET-3 and IRL1038 (ET B receptor antagonist) did so only weakly. SDS-PAGE of cell extracts containing [ 125 I]ET-1 cross-linked to the receptors, followed by autoradiography of the gels revealed a single 50-kDa band. These findings indicate that most of the receptors are subtype ET A . Although mRNA transcripts specific for both ET A and ET B receptors were found by RT-PCR, the ET A mRNA was more abundant. ET-1 increased the production of cAMP, cGMP and prostaglandin E 2 (PGE 2 ) and protein kinase C (PKC) activity in a concentration- and time-dependent manner. ET-1, and to a lesser degree ET-2, stimulated DNA synthesis, whereas ET-3 was inactive. Stimulation of DNA synthesis by ET-1 was strongly inhibited in a concentration-dependent manner by BQ123 and, to a much lesser degree, by IRL1038, which is consistent with an ET A receptor. ET-1 also stimulated proteoglycan synthesis and increased the amount of mRNA specific for the aggrecan gene. These findings strongly suggest that ET-1 is involved in regulating chondrocyte proliferation and metabolism in health, and presumably in disease.