Töpfer M, Burbiel C E, Müller C E, Knittel J, Verspohl E J
Department of Pharmacology, Institute of Medicinal Chemistry, Münster, Germany.
Cell Biochem Funct. 2008 Dec;26(8):833-43. doi: 10.1002/cbf.1514.
Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.
由于缺乏特异性激动剂和拮抗剂，腺苷受体亚型对胰岛素分泌系统的作用尚未得到充分研究。A1受体可能与不同的第二信使系统相关联，即环磷酸腺苷（cAMP）、钾离子通道和45钙离子通道活性。为了改善糖尿病（提高胰岛素敏感性、降低游离脂肪酸和甘油三酯），将开发部分A1受体激动剂。在本研究中，对新合成的选择性A1受体激动剂和拮抗剂进行了研究，整合了胰岛素分泌细胞系INS-1细胞的三个参数，即胰岛素释放（放射免疫分析）、45钙离子摄取和86铷离子外流（钾离子外流的替代指标）。通过蛋白质印迹法证实了A1受体的存在。高浓度（10微摩尔）的受体非选择性腺苷类似物NECA（5-N-乙基甲酰胺基腺苷）对胰岛素释放和45钙离子摄取没有影响，这可以解释为相互拮抗的腺苷受体亚型介导的效应之和。然而，在10纳摩尔NECA时检测到由A1受体激动介导的抑制作用，并且可以通过添加A1受体拮抗剂PSB-36（1-丁基-8-（3-降冰片基）-3-（3-羟丙基）黄嘌呤）来证实。NECA抑制了86铷离子外流，然而，这与同时抑制胰岛素分泌并不相符。选择性A1受体激动剂CHA（N6-环己基腺苷）抑制胰岛素释放；同时增加的钙离子摄取（依赖硝苯地平）和对86铷离子外流的抑制与胰岛素释放数据不相符。CHA的作用（即使在50微摩尔时的最大作用）可以被10微摩尔NECA增强，这表明CHA和NECA除了它们与A1受体的相互作用外，对86铷离子外流还有非特异性和生理上不相关的作用。由于PSB-36不影响NECA诱导的86铷离子外流抑制，NECA的作用不是由钾离子通道连接的A1受体介导的。非选择性腺苷受体拮抗剂咖啡因增加胰岛素释放，正如假设两者在这种情况下都通过A1受体起作用时所预期的那样，CHA可以逆转这种增加。总之，受体选择性和非选择性化合物对A1受体的刺激减少了胰岛素释放，这与钾离子通道开放（86铷离子外流实验）或钙离子通道抑制（45钙离子摄取实验）无关。可以预期，在所有多效性第二信使中，cAMP系统（此处未测试）对于A1受体的作用占主导地位，而通道系统（钾离子和钙离子）的重要性较小，并且尽管在其他组织中与受体偶联，但对胰岛素释放没有贡献。
