OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms
OX1 orexin receptors couple to adenylyl cyclase regulation via multiple mechanisms
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DOI：
10.1074/jbc.m407397200
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发表时间：
2005-02-25
影响因子：
4.8
通讯作者：
Kukkonen, JP
中科院分区：
文献类型：
--
作者：
Holmqvist, T;Johansson, L;Kukkonen, JP
本研究探讨了OX受体在中国仓鼠卵巢细胞中重组表达后对腺苷酸环化酶活性的调节机制。在完整细胞中，用食欲素-A刺激导致两种反应，弱（21%）、高效力（EC 50约为1 nM）抑制和强（4倍）、低效力（EC 50 =约为300 nM）刺激。百日咳毒素可逆转该抑制作用，提示G（i/o）参与了该过程。proteins.令人惊讶的是，食欲素-B在升高cAMP方面几乎与食欲素-A一样有效（pEC（50）=约500 nM）。cAMP升高不是由Ca ~（2+）升高或γ射线引起的。相反，它部分依赖于一种新的蛋白激酶C（PKC）亚型，PKC δ，使用药理学抑制剂确定。然而，单独的PKC刺激只能非常微弱地刺激cAMP的产生（1.1倍）。在G（s）活性存在的情况下，食欲素仍然升高cAMP;然而，效力大大增加（食欲素-A的EC 50 =约10 nm，食欲素-B的EC 50 =约100 nm），并且反应完全依赖于PKC δ。在透化细胞中，仅观察到PKC非依赖性低效力组分。该组分对抗-Ga敏感。抗体的我们的结论是，OX 1受体刺激腺苷酸环化酶通过一个低效力的G（s）耦合和一个高效力的磷脂酶C -> PKC耦合。前者或某些外源性G（s）激活是PKC显著激活腺苷酸环化酶所必需的。结果还表明，食欲素B激活的OX 1受体与G（s）的偶联几乎与食欲素A激活的受体一样有效，与Ca 2+升高和磷脂酶C激活相反，食欲素A的效力高10倍。
In this study, the mechanism of OX, orexin receptors to regulate adenylyl cyclase activity when recombinantly expressed in Chinese hamster ovary cells was investigated. In intact cells, stimulation with orexin-A led to two responses, a weak (21%), high potency (EC50 approximate to 1 nM) inhibition and a strong (4-fold), low potency (EC50 = approximate to300 nM) stimulation. The inhibition was reversed by pertussis toxin, suggesting the involvement of G(i/o). proteins. Orexin-B was, surprisingly, almost equally as potent as orexin-A in elevating cAMP (pEC(50) = approximate to500 nM). cAMP elevation was not caused by Ca2+ elevation or by Gbetagamma. In contrast, it relied in part on a novel protein kinase C (PKC) isoform, PKCdelta, as determined using pharmacological inhibitors. Yet, PKC stimulation alone only very weakly stimulated cAMP production (1.1-fold). In the presence of G(s) activity, orexins still elevated cAMP; however, the potencies were greatly increased (EC50 of orexin-A = approximate to10 nm and EC50 of orexin-B = approximate to100 nm), and the response was fully dependent on PKCdelta. In permeabilized cells, only a PKC-independent low potency component was seen. This component was sensitive to anti-Galpha(s). antibodies. We conclude that OX1 receptors stimulate adenylyl cyclase via a low potency G(s) coupling and a high potency phospholipase C --> PKC coupling. The former or some exogenous G(s) activation is essentially required for the PKC to significantly activate adenylyl cyclase. The results also suggest that orexin-B-activated OX1 receptors couple to G(s) almost as efficiently as the orexin-A-activated receptors, in contrast to Ca2+ elevation and phospholipase C activation, for which orexin-A is 10-fold more potent.