Morin D, Zini R, Querol-Ferrer V, Sapena R, Tillement J P
Département de Pharmacologie, Faculté de Médecine de Paris XII, Creteil, France.
J Neurochem. 1991 Apr;56(4):1114-20. doi: 10.1111/j.1471-4159.1991.tb11400.x.
The simultaneous measurement of the accumulation of cyclic AMP and inositol phosphates in rat cerebral cortical slices is described. After stimulation, the separation of cyclic AMP and inositol phosphates was achieved using ion-exchange chromatography and their concentrations were determined by means of a double-labeling technique, the substrates adenine and inositol being labeled with 14C and 3H, respectively. The recoveries were 70-80% for inositol phosphates and 40-50% for cyclic AMP. To test the applicability of the method, norepinephrine was chosen as an agonist, because it is known to stimulate the production of these two second messengers by interacting with alpha- and beta-adrenergic receptors. This procedure is an improvement over existing methods, because we obtained the simultaneous formation of 3H-inositol phosphates and [14C]cyclic AMP in a concentration-dependent process. EC50 values were similar for the two, 8.5 +/- 3.9 microM for 3H-inositol phosphates and 20.2 +/- 6.3 microM for [14C]cyclic AMP, and close to the values obtained when each process was studied alone. The action of adrenergic antagonists was also tested. Propranolol blocked the norepinephrine stimulation of [14C]cyclic AMP, but did not inhibit the norepinephrine stimulation of 3H-inositol phosphates. The opposite results were observed with prazosin. Our results suggest that this method could be a useful tool to examine the interaction between these two receptor-coupled effectors.
本文描述了在大鼠大脑皮质切片中同时测量环磷酸腺苷（cAMP）和肌醇磷酸积累的方法。刺激后，采用离子交换色谱法分离cAMP和肌醇磷酸，并用双标记技术测定它们的浓度，底物腺嘌呤和肌醇分别用14C和3H标记。肌醇磷酸的回收率为70 - 80%，cAMP的回收率为40 - 50%。为了测试该方法的适用性，选择去甲肾上腺素作为激动剂，因为已知它通过与α和β肾上腺素能受体相互作用来刺激这两种第二信使的产生。该方法是对现有方法的改进，因为我们在浓度依赖性过程中同时获得了3H - 肌醇磷酸和[14C]cAMP的形成。两者的半数有效浓度（EC50）值相似，3H - 肌醇磷酸为8.5±3.9微摩尔/升，[14C]cAMP为20.2±6.3微摩尔/升，且接近单独研究每个过程时获得的值。还测试了肾上腺素能拮抗剂的作用。普萘洛尔阻断了去甲肾上腺素对[14C]cAMP的刺激，但不抑制去甲肾上腺素对3H - 肌醇磷酸的刺激。哌唑嗪则观察到相反的结果。我们的结果表明，该方法可能是研究这两种受体偶联效应器之间相互作用的有用工具。
