Articular chondrocyte aging and endothelin-1.

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.

Basal and induced nitric oxide and cGMP productions are decreased in senescent cultured rat articular chondrocytes.

The effects of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharide (LPS) on the productions of nitric oxide (NO) and cGMP by cultured articular chondrocyte (AC) monolayers from 1-, 8- and 18-month old male Wistar rats were studied. It was found that basal NO and cGMP productions decrease with the age of animals. The productions were more than 2-fold greater in cells from 1-month old rats then in cells from older animals. IL-1, TNF-alpha, and LPS stimulated all three types of cells to produce NO and cGMP in a time- and concentration-dependent manner. Although the cells from young animals produced more NO per microg DNA, the older counterparts were more sensitive to these agents since they produced more NO upon stimulation then the corresponding non-stimulated controls. At the concentration of 10(-3) M, the nitric oxide synthase (NOS) inhibitor, Ng-monomethyl-L-arginine (L-NMA), blocked, although incompletely, both the basal and stimulated NO and cGMP productions in cells from the 1 and 8-month old rats and only induced productions in 18-month old counterparts. These results show a decreased capacity of unstimulated- and stimulated-AC from old rats to produce NO and cGMP in culture, which may affect the ageing cells in some yet unknown way.

Mitogenic and metabolic actions of epidermal growth factor on rat articular chondrocytes: modulation by fetal calf serum, transforming growth factor-beta, and tyrphostin.

The effects of human recombinant epidermal growth factor (EGF) on rat articular chondrocytes from humeral and femoral head cartilage of 21-day-old Wistar rats were analyzed. The cells were cultured under standard conditions as monolayers. Cell proliferation was studied by [3H]thymidine incorporation and determination of DNA content, proteoglycan synthesis by [35S]sulfate incorporation, and collagen synthesis by [3H]proline incorporation. The presence of specific receptors was confirmed by [125I]-EGF binding and that of EGF and EGF-receptor (EGF-R) mRNA by reverse transcription and the polymerase chain reaction. EGF (0.5-2.5 ng/ml) stimulated [3H]thymidine incorporation and increased DNA content of cultures. The effect was strongest when serum concentration was low (< or =1%) and was lost at high (> or =7.5%) serum concentrations. The EGF-induced effect on deoxynucleic acid synthesis was inhibited by transforming growth factor-beta and tyrphostin, a tyrosine kinase inhibitor that blocks the phosphorylation of tyrosine residues on EGF-R. Cultured rat articular chondrocytes possess a single class of high-affinity binding sites (Kd 0.18 nM). There were about 4.5 x 10(9) binding sites per microgram of DNA or about 37,800 binding sites per cell with 8.3 pg DNA per cell. Cultured cells contained EGF mRNA and EGF-R mRNA. Incubation of cells with EGF for 24 h decreased the EGF mRNA transcripts and increased the EGF-R mRNA levels. These findings suggest that EGF probably takes part in the regulation of chondrocyte activity under normal and presumably pathological conditions.