Xin Wenkuan, Feinstein Wei P, Britain Andrea L, Ochoa Cristhiaan D, Zhu Bing, Richter Wito, Leavesley Silas J, Rich Thomas C
Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina;
High Performance Computing, Louisiana State University, Baton Rouge, Louisiana;
Am J Physiol Cell Physiol. 2015 Sep 15;309(6):C415-24. doi: 10.1152/ajpcell.00090.2015. Epub 2015 Jul 22.
Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.
最近的研究表明，功能上离散的磷酸二酯酶（PDE）活性池调节着不同的细胞功能。虽然酶活性局部池的重要性已变得显而易见，但很少有研究估计离散亚细胞区室内的酶活性。在此，我们提出一种估计近膜PDE活性的方法。首先，使用传统的PDE活性测定法测量总PDE活性。其次，将已知的cAMP浓度透析到单个细胞中，并使用环核苷酸门控通道监测cAMP的空间扩散。第三，使用数学模型估计细胞内PDE活性的空间分布。使用这种三层方法，我们观察到两个药理学上不同的PDE活性池，一个对咯利普兰敏感的池和一个对8-甲氧基甲基异丁基黄嘌呤（8MM-IBMX）敏感的池。我们观察到，对咯利普兰敏感的PDE（PDE4）主要负责质膜附近的cAMP水解。最后，我们观察到，即使通过膜片吸管将100μM cAMP引入细胞，PDE4也能够降低质膜附近的cAMP水平。两室模型预测，质膜附近、环核苷酸门控通道附近的PDE活性显著低于细胞总PDE活性，并且cAMP的缓慢空间扩散使PDE活性能够有效水解近膜cAMP。这些结果表明，质膜附近的cAMP水平与其他亚细胞区室中的不同；PDE活性在细胞内并不均匀；AC和PDE活性的局部池负责控制不同亚细胞区室内的cAMP水平。
