Koolpe M, Benton H P
Department of Veterinary Medicine: Anatomy, Physiology, and Cell Biology; University of California-Davis 95616, USA.
J Orthop Res. 1997 Mar;15(2):204-12. doi: 10.1002/jor.1100150208.
If we are to fully understand mechanisms of cartilage homeostasis, it is essential that we know the full catalogue of receptors present on the surface of a chondrocyte and the pathways regulated by ligands that bind to these receptors. In this study, we describe chondrocyte responses to adenosine 5'-triphosphate and related molecules. Adenosine 5'-triphosphate stimulated a statistically significant, dose-dependent, transient rise in the concentration of calcium ions in Fura 2-loaded, differentiated, primary chondrocytes. The increase occurred in the absence of extracellular calcium, indicating a mobilization from intracellular stores. The increase in concentration of cytoplasmic calcium ions induced by adenosine 5'-triphosphate was mimicked by uridine 5'-triphosphate but not by 2-methylthioadenosine 5'-triphosphate, cytidine 5'-triphosphate, or adenosine. Heterologous desensitization experiments demonstrated that chondrocytes showed no subsequent response to uridine 5'-triphosphate after initial stimulation with adenosine 5'-triphosphate nor did they respond to adenosine 5'-triphosphate in inverse conditions, thereby indicating competition for the same receptor site. Together, these results are consistent with the presence of a P2U receptor on the cell surface of chondrocytes. Purine-induced calcium mobilization in passaged chondrocytes showed the same pharmacological profile with respect to agonist sensitivity, but responses were of greater magnitude than responses in primary differentiated chondrocytes, suggesting upregulation of the receptor with time in culture. Adenosine 5'-triphosphate and uridine 5'-triphosphate (1-100 microM) did not alter cartilage matrix synthesis as measured by rate of incorporation of [35S]sulfate into glycosaminoglycan by cartilage explants or primary chondrocytes. Matrix degradation, measured by release of glycosaminoglycan from cartilage explants, was also unaltered by adenosine 5'-triphosphate or uridine 5'-triphosphate (1-100 microM). Production of prostaglandin E2 was upregulated by incubation with either adenosine 5'-triphosphate or uridine 5'-triphosphate. These data demonstrate the presence of a functional P2U-like purine receptor on the surface of primary articular chondrocytes and support the hypothesis that altered concentrations of extracellular purines may influence chondrocyte metabolism.
如果我们要全面了解软骨稳态的机制，那么至关重要的是，我们要知晓软骨细胞表面存在的所有受体种类以及与这些受体结合的配体所调控的信号通路。在本研究中，我们描述了软骨细胞对腺苷 5'-三磷酸及相关分子的反应。腺苷 5'-三磷酸刺激了负载 Fura 2 的、分化的原代软骨细胞中钙离子浓度出现具有统计学意义的、剂量依赖性的短暂升高。这种升高在细胞外无钙的情况下发生，表明钙离子是从细胞内储存库中动员出来的。腺苷 5'-三磷酸诱导的细胞质钙离子浓度升高可被尿苷 5'-三磷酸模拟，但不能被 2-甲硫基腺苷 5'-三磷酸、胞苷 5'-三磷酸或腺苷模拟。异源脱敏实验表明，软骨细胞在用腺苷 5'-三磷酸初次刺激后，对尿苷 5'-三磷酸无后续反应，在相反条件下对腺苷 5'-三磷酸也无反应，从而表明它们竞争相同的受体位点。综上所述，这些结果与软骨细胞表面存在 P2U 受体一致。传代软骨细胞中嘌呤诱导的钙离子动员在激动剂敏感性方面表现出相同的药理学特征，但反应幅度大于原代分化软骨细胞中的反应，这表明随着培养时间的推移，受体上调。腺苷 5'-三磷酸和尿苷 5'-三磷酸（1 - 100 μM）并未改变软骨基质的合成，这通过软骨外植体或原代软骨细胞将 [35S] 硫酸盐掺入糖胺聚糖的速率来衡量。通过软骨外植体释放糖胺聚糖来衡量的基质降解也不受腺苷 5'-三磷酸或尿苷 5'-三磷酸（1 - 100 μM）的影响。前列腺素 E2 的产生通过与腺苷 5'-三磷酸或尿苷 5'-三磷酸孵育而上调。这些数据证明原代关节软骨细胞表面存在功能性的 P2U 样嘌呤受体，并支持细胞外嘌呤浓度改变可能影响软骨细胞代谢这一假说。
