EFFECTS OF ADENOSINE ON CULTURED CHONDROCYTES FROM RABBIT

研究了腺苷受体激动剂对兔关节软骨培养软骨细胞增殖和细胞活力的影响。加入非选择性腺苷受体激动剂cl -腺苷可显著抑制[3H]-胸苷摄取试验检测到的软骨细胞增殖。cl -腺苷的IC50值约为5 μM。加入腺苷也能抑制培养软骨细胞的增殖，EHNA对腺苷脱氨酶的抑制作用进一步增强。MTT法测定暴露于cl -腺苷诱导的细胞死亡呈剂量依赖性。cl -腺苷的EC50值约为30 μM。暴露于100 μM cl -腺苷12小时后，兔关节软骨培养的软骨细胞大部分死亡。为了阐明哪种受体亚型对cl -腺苷诱导的细胞死亡和细胞增殖抑制负责，我们在MTT试验和[3H]-胸腺嘧啶摄取试验中测试了几种选择性腺苷受体激动剂。我们使用的化合物有R-PIA （A1; 200 nM）、CGS-21680 （A2a; 200 nM）、NECA （A2b; 200 nM）、APNEA （A3; 200 nM）和CV-1808 （A4; 2 μM）。所有传统的选择性腺苷受体激动剂对培养软骨细胞的增殖和活力均无显著影响。我们还测试了腺苷受体拮抗剂DPCPX （A1; 100 nM）、DMPX （A2; 10 μM）和硫苯基茶碱（A1/A2; 10 μM）对cl -腺苷的影响。这些拮抗剂在[3H]-胸腺嘧啶摄取试验和MTT试验中均未显示出对30 μM cl -腺苷的抑制作用。这些结果提示腺苷对培养软骨细胞的作用可能通过非典型腺苷受体介导。

Adenosine receptor agonists were tested for cell proliferation and cell viability in cultivated chondrocytes from rabbit joint cartilage. The addition of Cl-adenosine, an unselective adenosine receptor agonist, remarkably inhibited chondrocyte proliferation detected by [3H]-thymidine uptake test. The IC50 value of Cl-adenosine was about 5 μM.Proliferation of cultured chondrocytes were also inhibited by the addition of adenosine which was further enhanced by the inhibition of adenosine deaminase with EHNA.The exposure to Cl-adenosine induced cell death measured by MTT assay in a dose-dependent manner. The EC50 value of Cl-adenosine was about 30 μM.Exposure to 100 μM Cl-adenosine for 12 hours killed most of the chondrocytes cultured from rabbit joint cartilage. To elucidate which receptor subtype was responsible for both the Cl-adenosine-induced cell death and the inhibition of cell proliferation, we tested several selective adenosine receptor agonists for both MTT assay and [3H]-Thymidine uptake test. The compounds we used were R-PIA (A1 ; 200 nM), CGS-21680 (A2a ; 200 nM), NECA (A2b ; 200 nM), APNEA (A3 ; 200 nM) and CV-1808 (A4 ; 2 μM). All the conventional selective adenosine receptor agonists showed no significant effect on both proliferation and viability in cultured chondrocytes. We also tested the adenosine receptor antagonists such as DPCPX (A1 ; 100 nM), DMPX (A2 ; 10 μM) and sulphophenyl-theophilline (A1/A2 ; 10 μM) on the effect of Cl-adenosine. These antagonists did not show any inhibitory action on the effect of 30 μM Cl-adenosine in both [3H]- Thymidine uptake test and MTT assay. These results suggest that the effect of adenosine on cultured chondrocytes may involve the mediation via atypical adenosine receptor.