Foucart S, Murphy T V, Majewski H
Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia.
J Auton Nerv Syst. 1990 Jul;30(3):221-32. doi: 10.1016/0165-1838(90)90253-f.
We used the alkylating agent N-ethylmaleimide in order to investigate G-proteins linked to release-modulating prejunctional receptors of sympathetic nerves in mouse atria incubated with [3H]-noradrenaline. The receptors tested were facilitatory beta-adrenoceptors and angiotensin II receptors and inhibitory neuropeptide Y receptors. In order to evaluate the specificity of the N-ethylmaleimide treatment, we tested N-ethylmaleimide against the second messenger pathways that are linked to beta-adrenoceptors (adenylate cyclase) and angiotensin II (protein kinase C). The results show that a 60-min preincubation with N-ethylmaleimide (3 microM) abolished the facilitatory effect of isoprenaline (0.1 microM) and angiotensin II (0.1 microM) on the stimulation-induced release of noradrenaline and reduced the inhibitory action of neuropeptide Y (0.3 microM). N-ethylmaleimide had no effect on the stimulatory action of either phorbol dibutyrate (0.01, 0.1 microM), forskolin (10 microM), or a combination of 8-bromo adenosine-3'5'-monophosphate (90 microM) and 3-isobutyl-1-methylxanthine (100 microM). However, at a higher concentration (10 microM), N-ethylmaleimide reduced the facilitatory effect of phorbol dibutyrate (0.1 microM) and the combination of 8-bromo adenosine-3',5'-monophosphate (90 microM) and 3-isobutyl-1-methylxanthine (100 microM). This suggests that N-ethylmaleimide at 3 microM but not 10 microM was selective for receptor-mediated modulation of noradrenaline release without directly affecting the adenylate cyclase (forskolin, 8-bromo adenosine-3',5'-monophosphate + 3-isobutyl-1-methylxanthine) or protein kinase C (phorbol dibutyrate) transduction pathways. In atria from mice pretreated with pertussis toxin (1.5 micrograms/mouse), N-ethylmaleimide preincubation (1 and 3 microM) resulted in a more pronounced reduction of the inhibitory action of neuropeptide Y (0.3 microM). The nature of this interaction is unclear. Since N-ethylmaleimide has been shown in other studies to inactivate G-proteins, the inhibitory effect of N-ethylmaleimide on prejunctional beta-adrenoceptors, angiotensin II receptors and neuropeptide Y receptors of sympathetic nerves may suggest that G-proteins are involved with these receptors, although other effects of N-ethylmaleimide on the receptor coupling processes cannot be ruled out. Moreover, it appears that the concentration of N-ethylmaleimide used is critical since a higher concentration (10 microM) resulted in non-specific effects on signal transduction mechanisms in the present experimental conditions.
我们使用烷基化剂N - 乙基马来酰亚胺，以研究与用[3H] - 去甲肾上腺素孵育的小鼠心房交感神经释放调节性节前受体相连的G蛋白。所测试的受体为促进性β - 肾上腺素能受体、血管紧张素II受体和抑制性神经肽Y受体。为了评估N - 乙基马来酰亚胺处理的特异性，我们针对与β - 肾上腺素能受体（腺苷酸环化酶）和血管紧张素II（蛋白激酶C）相连的第二信使途径测试了N - 乙基马来酰亚胺。结果表明，用N - 乙基马来酰亚胺（3 microM）预孵育60分钟消除了异丙肾上腺素（0.1 microM）和血管紧张素II（0.1 microM）对刺激诱导的去甲肾上腺素释放的促进作用，并降低了神经肽Y（0.3 microM）的抑制作用。N - 乙基马来酰亚胺对佛波酯（0.01、0.1 microM）、福斯高林（10 microM）或8 - 溴腺苷 - 3',5' - 单磷酸（90 microM）与3 - 异丁基 - 1 - 甲基黄嘌呤（100 microM）组合的刺激作用没有影响。然而，在较高浓度（10 microM）时，N - 乙基马来酰亚胺降低了佛波酯（0.1 microM）以及8 - 溴腺苷 - 3',5' - 单磷酸（90 microM）与3 - 异丁基 - 1 - 甲基黄嘌呤（100 microM）组合的促进作用。这表明3 microM而非10 microM的N - 乙基马来酰亚胺对受体介导的去甲肾上腺素释放调节具有选择性，而不会直接影响腺苷酸环化酶（福斯高林、8 - 溴腺苷 - 3',5' - 单磷酸 + 3 - 异丁基 - 1 - 甲基黄嘌呤）或蛋白激酶C（佛波酯）转导途径。在用百日咳毒素（1.5微克/小鼠）预处理的小鼠心房中，N - 乙基马来酰亚胺预孵育（1和3 microM）导致神经肽Y（0.3 microM）的抑制作用更明显降低。这种相互作用的性质尚不清楚。由于在其他研究中已表明N - 乙基马来酰亚胺可使G蛋白失活，N - 乙基马来酰亚胺对交感神经节前β - 肾上腺素能受体、血管紧张素II受体和神经肽Y受体的抑制作用可能表明G蛋白与这些受体有关，尽管不能排除N - 乙基马来酰亚胺对受体偶联过程的其他影响。此外，似乎所用N - 乙基马来酰亚胺的浓度很关键，因为在本实验条件下较高浓度（10 microM）会对信号转导机制产生非特异性影响。
