Bajraktari Gzona, Burhenne Jürgen, Bugert Peter, Haefeli Walter Emil, Weiss Johanna
Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany.
Institute of Transfusion Medicine and Immunology, Heidelberg University, Medical Faculty Mannheim, German Red Cross Blood Service Baden-Württemberg-Hessen, Friedrich-Ebert-Strasse, 107, 68167 Mannheim, Germany.
Biochem Pharmacol. 2017 Dec 1;145:54-63. doi: 10.1016/j.bcp.2017.08.026. Epub 2017 Sep 28.
Sildenafil and tadalafil are widely-used phosphodiesterase 5 (PDE5) inhibitors for which no clear dose-response relationship could be established. Using isolated and/or recombinant PDE5, it has been demonstrated that cGMP can increase the affinity of this enzyme for sildenafil and tadalafil. We thus hypothesized that in cells expressing the nitric oxide - soluble guanylyl cyclase - cyclic guanosine monophosphate - PDE5 (NO-sGC-cGMP-PDE5) pathway such as platelets, the presence of NO increases the intracellular cGMP content and thus promotes the intracellular accumulation of sildenafil or tadalafil. As a cell model, isolated and washed human platelets were used. Platelet suspensions were incubated with sildenafil or tadalafil at different concentrations and for various time intervals with or without an NO donor to increase intraplatelet cGMP concentrations. Intracellular sildenafil or tadalafil was quantified by ultra-performance liquid chromatography tandem mass spectrometry and intracellular cGMP by an enzyme-linked immunosorbent assay. Sildenafil accumulated in platelets with an up to 4-fold higher accumulation when platelets were pretreated with an NO donor (p < .0001). Accumulation of tadalafil in platelets was even higher, whereas the increase was 2-fold when an NO donor was present (p < .001). This accumulation was time-dependent and happened concomitantly with a rise in intracellular cGMP. Our data demonstrate that intracellular cGMP increases intracellular PDE5 inhibitor concentrations most likely by raising the affinity of these compounds for PDE5. These findings suggest that PDE5 inhibitor action in humans is critically influenced by modulators of the activity of the NO pathway.
西地那非和他达拉非是广泛使用的磷酸二酯酶5（PDE5）抑制剂，但尚未建立明确的剂量反应关系。使用分离的和/或重组的PDE5已证明，环磷酸鸟苷（cGMP）可增加该酶对西地那非和他达拉非的亲和力。因此，我们推测在表达一氧化氮-可溶性鸟苷酸环化酶-环磷酸鸟苷-PDE5（NO-sGC-cGMP-PDE5）途径的细胞（如血小板）中，NO的存在会增加细胞内cGMP含量，从而促进西地那非或他达拉非在细胞内的蓄积。作为细胞模型，使用了分离并洗涤过的人血小板。将血小板悬浮液与不同浓度的西地那非或他达拉非孵育不同时间间隔，同时添加或不添加NO供体以增加血小板内cGMP浓度。通过超高效液相色谱串联质谱法定量细胞内的西地那非或他达拉非，通过酶联免疫吸附测定法定量细胞内cGMP。当用NO供体预处理血小板时，西地那非在血小板中的蓄积量增加了高达4倍（p <0.0001）。他达拉非在血小板中的蓄积量更高，而当存在NO供体时增加了2倍（p <0.001）。这种蓄积是时间依赖性的，并且与细胞内cGMP的升高同时发生。我们的数据表明，细胞内cGMP最有可能通过提高这些化合物对PDE5的亲和力来增加细胞内PDE5抑制剂的浓度。这些发现表明，人类中PDE5抑制剂的作用受到NO途径活性调节剂的关键影响。
