Characterization of inhibitors of phosphodiesterase 1C on a human cellular system

Abstract

Different inhibitors of the Ca 2+ ⁄ calmodulin-stimulated phosphodiester-ase 1 family have been described and used for the examination of phospho-diesterase 1 in cellular, organ or animal models. However, the inhibitors described differ in potency and selectivity for the different phosphodiester-ase family enzymes, and in part exhibit additional pharmacodynamic actions. In this study, we demonstrate that phosphodiesterase 1C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesterase 2, phosphodiesterase 3, phosphodiesterase 4 and phosphodiesterase 5 are also present. The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phos-phodiesterase 1C specific small interfering RNA. In addition, the measured K m values (cAMP, 1.7 lm; cGMP, 1.3 lm) are characteristic of phospho-diesterase 1C. We demonstrate that treatment with the Ca 2+ ionophore ionomycin increases intracellular Ca 2+ in a concentration-dependent way without affecting cell viability. Under conditions of enhanced intracellular Ca 2+ concentration, a rapid increase in cAMP levels caused by the ade-nylyl cyclase activator forskolin was abolished, indicating the involvement of Ca 2+-activated phosphodiesterase 1C. The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase 1 inhibitors in a concentration dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesterase 1 inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase 1 inhibitors developed by Schering-Plough (named compounds 31 and 30). We demonstrate that up to 10 lm 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine had no effect on the reduction of forskolin-stimulated cAMP levels by ionomycin, whereas the more selective and up to 10 000 times more potent phosphodi-esterase 1 inhibitors SCH51866, compound 31 and compound 30 inhibited the ionomycin-induced decline of forskolin-induced cAMP at nanomolar concentrations. Thus, our data indicate that SCH51866 and compounds 31 and 30 are effective phosphodiesterase 1 inhibitors in a cellular context, in contrast to the weakly selective and low-potency phosphodiesterase inhibi-tors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine. A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase 1 inhibitors. 8-Methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesterase 1-mediated functions. Abbreviations IL, interleukin; 8MM-IBMX, 8-methoxymethyl-3-isobutyl-1-methylxanthine; PDE, phosphodiesterase; si, small interfering.

PDE3 and PDE4 Isozyme-Selective Inhibitors Are Both Required for Synergistic Activation of Brown Adipose Tissue

“…We therefore confirmed the presence of PDE1 activity by measuring cGMP hydrolysis in the presence and absence of calcium and calmodulin in the whole BAT extract. This hydrolytic activity was increased by 2.7-fold by calcium and was fully inhibited by 100 nM SCH51866, a relatively selective PDE1 inhibitor (IC 50 for PDE1A, 10 nM) (Dunkern and Hatzelmann, 2007) (Fig. 2A).…”

Vinpocetine inhibits NF-κB–dependent inflammation via an IKK-dependent but PDE-independent mechanism

“…S6B). Finally, it has been recently reported that vinpocetine may inhibit PDE-4 activity in vitro (26). Because PDE-4 is a well-known antiinflammatory target (27), we analyzed the ability of vinpocetine to inhibit PDE-4 by PDE assay.…”

Age and disease related changes in the translocator protein (TSPO) system in the human brain: Positron emission tomography measurements with [11C]vinpocetine

“…The agent is widely used as a neuroprotective drug in the prevention and treatment of cerebrovascular diseases (Bereczki and Fekete, 2000;Nagy and Simon, 2004;Onishchenko et al, 2008;Fehér et al, 2009). In addition to its widely studied neuroprotective effects (Bönöczk et al, 2000;Tárnok et al, 2008;Nyakas et al, 2009) it is a well known phospho-diesterase (PDE) inhibitor (Dunkern and Hatzelmann, 2007;Deshmuk et al, 2009;Wunder et al, 2009) and voltage dependent sodium channel inhibitor (Ádám-Vizi, 2000). Most recently Jeon et al (2010) reported that vinpocetine is a potent antiinflammatory agent by targeting an IκB-related Kinases (IKK) dependent mechanism (cfr.…”