Objective: To compare in cell culture endothelin-1 (ET-1) production, receptor density, and effect on macromolecular synthesis by articular chondrocytes (AC).
Methods: AC were isolated from 1-month and 18-month old rats and cultured as monolayers. They were incubated with ET-1 without or with iNOS inhibitors, nitro-L-arginine methyl ester (L-NAME) or guanylate cyclase inhibitor, LY83583 and then [3H]thymidine, 35SO4 and [3H]proline incorporations were measured. The density and affinity for 125I-ET-1 of binding sites, and receptor isotypes were determined. The cells were also treated with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha), and then ET-1 productions measured. As well, the cells were challenged with NOC-5 (nitric oxide donor) or ET-1 and then ET-1 and NO respectively were measured.
Results: A concentration-dependent stimulation of DNA, PG, collagen and NO synthesis was obtained when cells were incubated with ET-1 for 24-h. Eighteen-month old chondrocytes incorporated per microg DNA more [3H]thymidine, 35SO4 and [3H]proline but less NO when challenged with ET-1 than the 1-month old cells. However, strong inhibition of this initial stimulation was seen after 48-h. L-NAME and LY83583 enhanced basal-, and ET-1-induced initial stimulation and completely suppressed late (at 48-h to 72-h) ET-1-induced inhibition, suggesting NO was responsible for this inhibitory effect. Eighteen-month old chondrocytes expressed per mug DNA more high affinity receptors of predominantly ET(A) subtype. They also produced more ET-1 but less NO under basal conditions and more ET-1 when challenged with IL-1beta and TNF-alpha. NOC-5 inhibited the production of ET-1.
Conclusions: Eighteen-month old chondrocytes produce more ET-1, possess more ET-1-specific receptors, and increase more DNA, PG and collagen synthesis when challenged during 24-h with ET-1. NO, which suppresses ET-1 production and the production of which is increased by ET-1, seems to account for the late ET-1-induced inhibition of macromolecular synthesis. The possible implication of ET-1 in aging as related to osteoarthritis is discussed.