Mandla S, Tenenhouse H S
Department of Pediatrics, McGill University, McGill University-Montreal Children's Hospital Research Institute, Quebec, Canada.
Endocrinology. 1992 Apr;130(4):2145-51. doi: 10.1210/endo.130.4.1312447.
Forskolin has long been used to demonstrate the involvement of cAMP in the regulation of cellular function, by virtue of its ability to stimulate adenylate cyclase directly. Recently, however, forskolin has been shown to affect plasma membrane transporter and channel function in a manner unrelated to cAMP. The present study examines whether forskolin-mediated inhibition of a mitochondrial membrane-associated enzyme, 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), also occurs by a cAMP-independent mechanism. Both forskolin and PTH stimulated cAMP accumulation and inhibited 24-hydroxylase activity in a dose-dependent manner in fresh mouse renal tubules. However, the level of inhibition of 24-hydroxylase achieved with forskolin was consistently greater than that obtained with PTH, at comparable levels of cAMP. 1',9'-Dideoxyforskolin, a cyclase-inactive analog of forskolin, also inhibited 24-hydroxylase activity, without stimulating cAMP production. Moreover, both forskolin and 1',9'-dideoxyforskolin directly inhibited 24-hydroxylase in isolated renal mitochondria. Kinetic analysis revealed a competitive mode of inhibition for both agents; however, 1',9'-dideoxyforskolin proved to be a more potent inhibitor of 24-hydroxylase than forskolin (inhibitory constant, 0.25 vs. 22 microM, respectively). Finally, both forskolin and 1',9'-dideoxyforskolin also inhibited inducible 24-hydroxylase in renal tubules prepared from 1,25-(OH)2D3-treated mice. However, inducible 24-hydroxylase activity was less susceptible to inhibition by the diterpenes than the basal enzyme activity. The present study provides evidence for cAMP-independent inhibition of 24-hydroxylase by forskolin and represents the first demonstration of a cAMP-independent effect of forskolin on a protein that is not a plasma membrane-associated transporter or channel. Our data advocate caution in the interpretation of studies using forskolin to assess the role of cAMP in cellular processes.
由于福斯高林能够直接刺激腺苷酸环化酶，长期以来它一直被用于证明环磷酸腺苷（cAMP）参与细胞功能的调节。然而，最近研究表明，福斯高林以一种与cAMP无关的方式影响质膜转运体和通道功能。本研究旨在探讨福斯高林介导的对线粒体膜相关酶25-羟基维生素D3-24-羟化酶（24-羟化酶）的抑制作用是否也通过一种不依赖cAMP的机制发生。在新鲜的小鼠肾小管中，福斯高林和甲状旁腺激素（PTH）均以剂量依赖的方式刺激cAMP积累并抑制24-羟化酶活性。然而，在可比的cAMP水平下，福斯高林对24-羟化酶的抑制水平始终高于PTH。1',9'-二脱氧福斯高林是福斯高林的一种无环化酶活性的类似物，它也能抑制24-羟化酶活性，且不刺激cAMP的产生。此外，福斯高林和1',9'-二脱氧福斯高林都能直接抑制分离的肾线粒体中的24-羟化酶。动力学分析显示这两种药物均为竞争性抑制模式；然而，1',9'-二脱氧福斯高林被证明是比福斯高林更有效的24-羟化酶抑制剂（抑制常数分别为0.25和22 microM）。最后，福斯高林和1',9'-二脱氧福斯高林也能抑制由1,25-(OH)2D3处理的小鼠制备的肾小管中的诱导型24-羟化酶。然而，诱导型24-羟化酶活性比基础酶活性更不易受到二萜类化合物的抑制。本研究为福斯高林对24-羟化酶的非cAMP依赖性抑制提供了证据，并且首次证明了福斯高林对一种不是质膜相关转运体或通道的蛋白质具有非cAMP依赖性作用。我们的数据提醒人们在解释使用福斯高林评估cAMP在细胞过程中作用的研究时要谨慎。
