EFFECTS OF ADENOSINE ON CULTURED CHONDROCYTES FROM RABBIT

腺苷受体激动剂在来自兔关节软骨的培养软骨细胞中测试细胞增殖和细胞活力。加入非选择性腺苷受体激动剂Cl-腺苷，[3 H]-胸苷摄取试验检测到显着抑制软骨细胞增殖。Cl-腺苷的IC_（50）值约为5 μM，腺苷对软骨细胞的增殖也有抑制作用，而EHNA对腺苷脱氨酶的抑制作用则进一步增强腺苷对软骨细胞增殖的抑制作用。Cl-腺苷的EC_（50）值约为30 μM，100 μM的Cl-腺苷作用12小时，可杀死大部分培养的兔关节软骨细胞。为了阐明哪种受体亚型负责Cl-腺苷诱导的细胞死亡和细胞增殖的抑制，我们测试了几种选择性腺苷受体激动剂的MTT测定和[3 H]-胸苷摄取试验。我们使用的化合物是R-PIA（A1 ; 200 nM），CGS-21680（A2 a; 200 nM），NECA（A2 b; 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在培养的软骨细胞的增殖和活力没有显着的影响。我们还测试了腺苷受体拮抗剂如DPCPX（A1 ; 100 nM）、DMPX（A2 ; 10 μM）和磺基苯茶碱（A1/A2 ; 10 μM）对Cl-腺苷的作用。这些拮抗剂在[3 H]-胸苷摄取试验和MTT试验中对30 μM Cl-腺苷的作用均未表现出任何抑制作用。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.