Báo cáo khoa học: Characterization of inhibitors of phosphodiesterase 1C on a human cellular system potx

Characterization of inhibitors of phosphodiesterase 1C on a human cellular system Torsten R Dunkern and Armin Hatzelmann Biochemistry Inflammation, ALTANA Pharma AG, Member of the Nycomed Group, Konstanz, Germany Keywords glioblastoma cell line; 8-methoxymethyl3-isoabutyl-1-methylxanthine; phosphodiesterase 1C; SCH51866; vinpocetine Correspondence T R Dunkern, Department of Biochemistry Inflammation RPR ⁄ B2, ALTANA Pharma AG, Member of the Nycomed Group, Byk-Gulden-Str 2, 78467 Konstanz, Germany Fax: +49 7531 84 2712 Tel: +49 7531 84 3121 E-mail: torsten.dunkern@altanapharma.com (Received 25 April 2007, revised 17 July 2007, accepted 19 July 2007) doi:10.1111/j.1742-4658.2007.06001.x Different inhibitors of the Ca2+ ⁄ calmodulin-stimulated phosphodiesterase family have been described and used for the examination of phosphodiesterase in cellular, organ or animal models However, the inhibitors described differ in potency and selectivity for the different phosphodiesterase 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 and phosphodiesterase are also present The identity of the phosphodiesterase 1C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesterase 1C specific small interfering RNA In addition, the measured Km values (cAMP, 1.7 lm; cGMP, 1.3 lm) are characteristic of phosphodiesterase 1C We demonstrate that treatment with the Ca2+ ionophore ionomycin increases intracellular Ca2+ in a concentration-dependent way without affecting cell viability Under conditions of enhanced intracellular Ca2+ concentration, a rapid increase in cAMP levels caused by the adenylyl cyclase activator forskolin was abolished, indicating the involvement of Ca2+-activated phosphodiesterase 1C The reduction of forskolin-stimulated cAMP levels was reversed by phosphodiesterase inhibitors in a concentration-dependent way Using this cellular system, we compared the cellular potency of published phosphodiesterase inhibitors, including 8-methoxymethyl-3-isobutyl-1-methylxanthine, vinpocetine, SCH51866, and two established phosphodiesterase inhibitors developed by ScheringPlough (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 phosphodiesterase 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 inhibitors in a cellular context, in contrast to the weakly selective and low-potency phosphodiesterase inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetine A172 cells therefore represent a suitable system in which to study the cellular effect of phosphodiesterase inhibitors 8-Methoxymethyl-3-isobutyl1-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 4812 FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS T R Dunkern and A Hatzelmann The superfamily of cyclic nucleotide-hydrolyzing phosphodiesterase (PDE) enzymes comprises 11 family members (PDE1–PDE11) Within each family, different genes encode different PDE subtypes, and alternative splicing of the messenger RNAs generates multiple isoforms The entire PDE superfamily consists of about 50 proteins [1] The historical classification into different PDE families is based on their substrate specificity for cAMP, cGMP or both, and mechanisms of activation and inhibitor sensitivity The PDE family PDE1, which consists of the subtypes PDE1A, PDE1B and PDE1C, each encoded by one gene, becomes activated upon Ca2+ ⁄ calmodulin binding Thus, PDE1 enzymes enable signaling crosstalk between the two important cellular second messengers Ca2+ and cyclic nucleotides, translating changes in Ca2+ concentrations into changes in cAMP ⁄ cGMP concentrations PDE1A is highly expressed in the brain as well as in spermatozoa PDE1B1 mRNA is present in neuronal cells of the cerebellum, caudate, and hippocampus, and in Purkinje cells [2] In addition, PDE1B1 was found in the heart and skeletal muscles [3] PDE1C, for which the three splicing variants PDE1C1, PDE1C2 and PDE1C3 have been described [4], is expressed in human arterial smooth muscle cells [5], nociceptive sensory neurons [6], the myocardium [7], and beta TC3 insulinoma cells [8] PDE1 enzymes have been reported to be involved in heterogeneous functions such as smooth muscle cell proliferation (PDE1C [5]), Cl– secretion by human colonic epithelial cells [9], interleukin (IL)-13 production by CD3 ⁄ CD28-stimulated human T-lymphocytes [10], the acrosome reaction of human spermatozoa [11], lipopolysaccharide-induced IL-6 release from alveolar epithelial cells [12], glucose-induced insulin secretion from insulinoma cells [8], contraction of ureteral smooth muscle cells [13], hypoxic pulmonary remodeling [14], granulocyte–macrophage colony-stimulating factor-induced human monocyte-to-macrophage differentiation (PDE1B [15]), and gall bladder relaxation [16] Until now, knowledge on the function of PDE1 in different cell types, organs or even animal models has come from the experimental use of more or less weak inhibitors with relatively low PDE1 selectivity, such as vinpocetine and 8-methoxymethyl-3-isobutyl-1-methylxanthine (8MM-IBMX) [with the exception of some PDE1-targeting small interfering (si)RNA approaches] [17] Unfortunately, changes in cyclic nucleotide concentrations, which are indicative of PDE-inhibitory action, were often not investigated in such studies This gives rise to the question of the validity of interpretations based on results obtained with the use of Characterization of inhibitors of PDE1C these compounds, especially as other modes of action besides PDE inhibition have been documented for the nootropic drug vinpocetine as well as for 8MM-IBMX, such as modulation of ion currents [18–20] and adenosine receptor inhibition [21], respectively Because of the lack of a cellular model that allows separate monitoring of PDE1 activity, no comparison of the cellular potency of PDE1 inhibitors described in the literature has been performed up to now This is important, because the cellular efficiency of PDE inhibitors does not only depend on their potency It is also affected by their physicochemical properties, such as lipophilicity, which influences membrane permeability, and the subcellular localization Furthermore, cellular potency is influenced by the concentrations of the substrates in the subcellular local environment, due to competition with the PDE inhibitors Thus, we developed a cellular model using PDE1Cexpressing A172 glioblastoma cells that allows us to quantitatively monitor the effects of the PDE1-inhibiting compounds 8MM-IBMX, vinpocetine, SCH51866, compound 30, and compound 31 (Fig 1) on PDE1C activity under conditions of enhanced intracellular Ca2+ concentration Results Human glioblastoma A172 cells express Ca2+/calmodulin-activated PDE1C As shown in Fig 2, we identified high Ca2+ ⁄ calmodulin-induced cAMP ⁄ cGMP PDE activity in the cellular extract of A172 cells; this therefore must be considered to be PDE1 enzyme activity In comparison to the described PDE1 activity of other cell types, such as eosinophils, macrophages, CD4+ ⁄ CD8+ T-lymphocytes and A549 epithelial cells [22], the measured PDE1 activity in A172 cells was up to 10 000-fold higher The similar PDE activity with regard to the rate of cAMP and cGMP hydrolysis was a first indication that this might be PDE1C activity This enzyme is well known to hydrolyze both cyclic nucleotides with similar efficiency, in contrast to PDE1A and PDE1B Our assumption was confirmed by determining the corresponding Km values, which were very similar for both cAMP (1.7 lm) and cGMP (1.3 lm) hydrolysis (published data for the other PDE1 subtypes: PDE1A2, Km [cAMP ⁄ cGMP] ¼ 112.7 ⁄ 5.0 lm; PDE1B1, Km [cAMP ⁄ cGMP] ¼ 24.3 ⁄ 2.7 lm) and were also similar to the published data for PDE1C [4] In addition, lower activities of PDE5 and PDE4 and minor activities for PDE2 and PDE3 were measured In accordance with the data for PDE activity, we FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 4813 Characterization of inhibitors of PDE1C Vinpocetin T R Dunkern and A Hatzelmann SCH51866 8MM-IBMX O O H N N N O O H N N N H N N O N N N O 30 N N N F O O N F F 31 N N N N N N Fig Molecular structures of the PDE1 inhibitors vinpocetine, 8MM-IBMX, SCH51886, and compounds 30 and 31 PDE activity pmol x min-1 x 108 cells-1 40000 30000 20000 10000 M G [c E5 PD E4 PD P] P] AM P] [c AM P] PD E3 [c [c G G E2 PD E1 [c [c PD E1 PD M M AM P] P] Fig Expression of family-specific PDE activities in A172 cells Cells were analyzed for PDE1 (cAMP and cGMP), PDE2, PDE3, PDE4 and PDE5 hydrolysis activity The mean values and standard deviations of 10 independent experiments are shown The substrates (cAMP ⁄ cGMP) used to determine each PDE activity are shown in parentheses confirmed the expression of PDE1C protein in A172 cells by western blotting (Fig 3A), in which recombinant PDE1C protein served as a molecular mass control More precisely, we detected two protein bands that probably reflected the PDE1C splice variants ⁄ isoforms PDE1C1 and presumably PDE1C3 (based on the determined molecular masses of 72 and 75 kDa) The polyclonal antibody used has been reported to 4814 detect different isoforms of PDE1C We confirmed that this antibody shows only negligible crossreactivity with PDE1A and PDE1B (data not shown) To verify the identity of the two observed protein bands and thus the specificity of the antibody used, we performed transfections with PDE1C (isotype-unspecific) siRNA smart pools As shown in Fig 3B, 48 h after transfection of A172 cells with 10 nm PDE1C-targeted siRNA, PDE1C mRNA expression was strongly reduced by about a factor of Consequently, 72 h after siRNA transfection, expression of the two protein bands was also reduced by about 60% in comparison to negative control siRNA-transfected cells (Fig 3C), which ultimately proved them to be identical to PDE1C Using the same PDE1C siRNA smart pool, we were also able to reduce PDE1C mRNA and protein expression in A549 cells, which further demonstrates the specificity of the siRNA (data not shown) Ionomycin treatment increases intracellular Ca2+ concentrations and activates PDE1C in A172 cells To monitor Ca2+-mediated PDE1C activation, we treated A172 cells with different concentrations of ionomycin As shown in Fig 4A, ionomycin (2–10 lm) increased intracellular Ca2+ concentration over 30 in a dose-dependent manner It was important to assess the concentrations of ionomycin that cells could tolerate over a 30 period Cells were therefore preincubated for 30 with a range of ionomycin concentrations up to 10 lm, and then with 10 lm ionomycin plus mm Ca2+ to elicit a maximum Ca2+ response (indicated by a dashed arrow in Fig 4A) This response no longer occurred when the ionomycin concentration in the 30 preincubation FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS T R Dunkern and A Hatzelmann Characterization of inhibitors of PDE1C Sf21/PDE1C1 A172 cells A 82.2 kDa PDE1C 63.2 kDa 10 30 20 B 2.5 µg C PDE1C 1.2 ERK2 PDE1C siRNA 5000 PDE1C siRNA PDE1C siRNA Negative control siRNA Negative control siRNA 0.2 10000 Lipid 0.4 phase was higher than lm, which suggests a cytotoxic effect of ionomycin at these higher concentrations The addition of 50 mm EGTA (as a control) at the end of the experiment (indicated by a solid arrow in Fig 4A) decreased the Ca2+ signal On the basis of these results, lm ionomycin (in the presence of mm Ca2+ in the medium, incubation time) was used in further experiments to rapidly increase intracellular Ca2+ concentrations (Fig 4B) Under these conditions, the elicited Ca2+ signal measured at was about 40% of the maximal possible Ca2+ signal and thus not in the saturation phase To further verify that these treatment conditions did not affect cell viability, cytotoxicity assays were performed In contrast to various concentrations of Triton X-100, which served as positive controls, treatment with lm ionomycin for 15 did not affect the viability of A172 cells (Fig 4C) Control Lipid 0.6 PDE1C protein expression (arbitrary densitometric units) 0.8 Lipid Fig PDE1C protein and mRNA are expressed in A172 gliobastoma cells (A) 10, 20 and 30 lg of A172 cell extracts were analyzed for PDE1C protein expression by western blotting Cell lysate from Sf21 insect cells (1 and 2.5 lg) transfected with human PDE1C1 was used as a control Molecular mass markers are shown to the right of the blot (B, C) PDE1C siRNA (smart pool; 10 nM) ⁄ negative control siRNA was transfected into A172 cells Forty-eight hours later, the cells were harvested for mRNA generation and subsequent cDNA synthesis The expression of PDE1C mRNA was analyzed by real-time PCR in relation to lipid-treated cells (B) The mean values and standard deviations of three experiments are shown Otherwise, cells were harvested 72 h after transfection, and analyzed for PDE1C protein expression by western blotting and densitometric analysis (C) To confirm equal protein loading, the blot was additionally incubated with an antibody to ERK2 PDE1C mRNA (relative expression) As shown in Fig 5, treatment of A172 cells with the adenylyl cyclase activator forskolin (10 lm) for 1, and 20 time-dependently increased intracellular cAMP concentrations up to three-fold in comparison to mock-treated controls However, pretreatment of the cells with ionomycin for to increase intracellular Ca2+ concentrations abolished or attenuated the forskolin-induced increase in cAMP, which we considered to be due to the activation of PDE1C in these cells under these conditions Inhibitors of PDE1 block the ionomycin-induced decrease in forskolin-induced cAMP concentrations Various inhibitors of PDE1 are described in the literature, including vinpocetine, 8MM-IBMX, SCH51866, and the Schering-Plough compounds 31 and 30 [23] FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 4815 Characterization of inhibitors of PDE1C T R Dunkern and A Hatzelmann 50000 A Control µM µM µM µM 10 µM RFU 40000 30000 20000 10000 30 15 Time (min) B 36000 31000 C Control Ionomycin Death cells (%) 100 RFU 26000 21000 50 16000 10 Time (min) Triton X-100 15 We investigated these compounds with regard to potency against PDE1A3, PDE1B1 and PDE1C1 and overall PDE selectivity (Table 1) The rank order of potency of these compounds against PDE1C1 is compound 31 > compound 30 > SCH51866 > 8MMIBMX > vinpocetine Indeed, the frequently used PDE1 inhibitors vinpocetine and 8MM-IBMX have low potency (IC50 values 100 lm and 10 lm, respectively) and selectivity vs other PDEs The IC50 values that we have determined for inhibition of the PDE1 enzymes by these compounds agree with those in the literature [23–26] Next, we analyzed the effect of these inhibitors on the ionomycin-induced decrease in forskolin-stimulated cAMP concentrations in A172 cells Figure 6A shows that SCH51866 reversed the ionomycin-induced decrease in cAMP concentrations in a concentrationdependent manner Even 10 nm of this PDE1 inhibitor 4816 0.020% Ionomycin 0.015% 0.005% 6000 0.010% 11000 Fig Ionomycin treatment up to lM concentration-dependently induces an increase in intracellular Ca2+ concentrations in A172 cells without inducing cytotoxicity (A) Fluo 3-mix dye-loaded A172 cells were treated for 30 with increasing concentrations (2–10 lM) of ionomycin The increase in intracellular Ca2+ concentrations was analyzed by scanning fluorometry After that time period, mM Ca2+ and ionomycin (to a final concentration of 10 lM) were added (dotted arrow) to elicit a maximum Ca2+ response to (a) confirm that the cells are still able to respond to a Ca2+ signal and (b) determine the maximal possible Ca2+ signal intensity Thereafter, EGTA (solid arrow) was added; this decreased the signal As shown in (B), the addition of ionomycin (straight arrow) and mM Ca2+ (dotted arrow) induced a rapid increase in intracellular Ca2+ concentrations (the mean values and standard deviations of three experiments are shown) (C) As measured by lactate dehydrogenase assays, treatment of A172 cells with lM ionomycin plus mM Ca2+ for 15 did not induce any cytotoxicity, whereas Triton X-100, which served as a positive control, induced cytotoxicity in a concentration-dependent manner proved to be significantly effective At this concentration, no other PDE expressed in A172 cells other than PDE1C was inhibited by this compound (Table 1), and thus the observed effect must be attributed exclusively to the inhibition of PDE1C In contrast, without addition of ionomycin and thus with basal intracellular Ca2+ concentrations, SCH51866 (up to 100 nm) did not potentiate the increase in cAMP induced by forskolin (Fig 6B) Compounds 31 and 30 proved to be inhibitors with higher enzymatic potency (Table 1) Accordingly, as shown in Fig 7A, these compounds also proved to be cellularly strongly effective Thus, compound 30 became significantly effective at 10 nm, and compound 31 at nm, in modulating intracellular cAMP concentrations The effect of these two compounds developed by Schering-Plough proved also to be concentration dependent However, at higher concentrations, these compounds (as also observed for FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS T R Dunkern and A Hatzelmann Characterization of inhibitors of PDE1C expressed in A172 cells, became inhibited Thus, the inhibition of these PDE family members might account for this effect Figure 7B demonstrates that the concentrations of compounds 31 and 30, which are effective in modulating cAMP in the presence of ionomycin, not modulate forskolin-induced cAMP concentrations in the absence of ionomycin In contrast to SCH51866, and compounds 31 and 30, the older, well-described and widely used PDE1 inhibitors 8MM-IBMX and vinpocetine from 10 nm up to 10 lm (which are concentrations reported in many publications to be effective on organs or cells) did not influence the ionomycin-induced decrease in cAMP concentrations (Fig 8A) This is indicative of their weak cellular potency, which is of course related to a low enzymatic potency (Table 1) In the absence of ionomycin, they also did not influence forskolininduced cAMP concentrations (Fig 8B) Control Ionomycin 1.50 Forskolin Forskolin + Ionomycin cAMP (pmol/1x106 cells) 1.25 1.00 0.75 0.50 0.25 0.00 Time (min) 20 Fig Pretreatment of A172 cells with ionomycin dampens a forskolin-induced increase in intracellular cAMP concentrations A172 cells pretreated with ionomycin (6 lM) or untreated were stimulated for adenylyl cyclase activation with forskolin (10 lM) or vehicle (control) cAMP was measured min, and 20 after forskolin stimulation in the corresponding cellular extracts by cAMPELISA The result of a representative experiment is shown SCH51866) also enhanced the forskolin-induced increase in intracellular cAMP concentrations, which might not be due to the inhibition of PDE1C At these concentrations of compounds 31 and 30, the cAMPhydrolyzing PDE2, PDE3 and PDE4, which are also Discussion The heterogeneous expression of human PDEs throughout the human body and their diverse functions make this enzyme superfamily an attractive target for pharmaceutical inhibitor development Whereas inhibitors of PDE5, PDE4 and PDE3 have undergone clinical trials in the past, are even approved as drugs, and are still under development for various indications such as asthma, pulmonary hypertension and chronic obstructive pulmonary disease, inhibitors of other PDE subtypes are mostly in preclinical development phases This is due to the limited information on the function of these enzymes, a lack of target-specific cellular test systems for drug screening purposes, and their drugability The cellular Table Inhibition of human PDE family members by vinpocetine, 8MM-IBMX, SCH51866 and compounds 31 and 30 The IC50 (log M) values of the different compounds obtained from at least three independent experiments using recombinant human PDEs (PDE1A3, PDE1B1, PDE1C1, PDE2A3, PDE3A1, PDE4D3, PDE4B2, PDE5A1, PDE7A1, PDE9A3, PDE9A5, PDE10A, PDE11A4) or PDEs from human platelets (PDE3, PDE5) are shown Dashes indicate that the corresponding measurements have not been performed Vinpocetine PDE1A3 PDE1B1 PDE1C1 PDE2A3 PDE3 ⁄ 3A1 PDE4D3 ⁄ B2 PDE5 ⁄ 5A1 PDE7A1 PDE9A3 ⁄ A5 PDE10A PDE11A4 > –⁄> –⁄> –⁄> > –⁄> > > ) ) ) ) ) ) ) ) ) ) ) 4.8 4.7 4 4 4 8MM-IBMX SCH51866 Compound 31 Compound 30 ) ) ) ) –⁄> ) –⁄) –⁄) >) –⁄> ) ) ) ) ) ) ) ) ) ) >) ) ) ) ) 9.2 ) 8.4 ) 9.3 5.7 – ⁄ ) 5.8 – ⁄ ) 6.2 – ⁄ ) 6.4 >)4 >)4 ) 4.8 ) 5.5 ) ) ) ) –⁄) –⁄) –⁄) >) >) ) ) 5.6 4.7 4.6 4.4 4.5 4 4.6 4.6 8.0 7.0 7.1 6.3 4.9 ⁄ – 5.3 ⁄ – 6.4 ⁄ – 4.9 ⁄ ) 4.7 5.4 4.6 FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 8.8 8.0 8.6 5.3 5.8 5.6 6.2 4 4.6 5.9 4817 Characterization of inhibitors of PDE1C T R Dunkern and A Hatzelmann *** A 15 *** cAMP (fold of control) ** * Fig The PDE1 inhibitor SCH51866 concentration-dependently reverses the ionomycin effect on the forskolin-induced increase in intracellular cAMP concentrations (A) A172 cells pretreated with ionomycin (6 lM) and SCH51866 () to ) log M final concentration) or untreated were stimulated for adenylyl cyclase activation with forskolin (10 lM) or vehicle (control) cAMP was measured after forskolin stimulation in the corresponding cellular extracts by cAMP-ELISA The mean values and standard deviations of three independent experiments are shown A statistical analysis (Student’s paired t-test) was performed to compare the forskolin plus ionomycin-treated group with those treated in addition with SCH51866 (B) A172 cells were treated only with SCH51866 () to ) log M final concentration) or vehicle (control), and stimulated for adenylyl cyclase activation with forskolin (10 lM); cAMP was then measured A statistical analysis (Student’s paired t-test) was used to compare the forskolin-treated group with control cells *P < 0.05, **P < 0.01, ***P < 0.005 SCH 51866 -5 log M SCH 51866 -6 log M SCH 51866 -7 log M SCH 51866 -8 log M Forskolin + Ionomycin Forskolin Control Forskolin + Ionomycin B * cAMP (fold of control) 15 * * 10 * SCH 51866 -6 log M SCH 51866 -7 log M SCH 51866 -8 log M Forskolin Control Forskolin functions of the PDE1 gene family have often been analyzed by using the well-described PDE inhibitors vinpocetine and 8MM-IBMX Unfortunately, vinpoce4818 tine and 8MM-IBMX possess other pharmacologic activities in addition to the inhibition of PDEs [18–21], which might interfere with the different findings described in the literature, such as lipopolysaccharide-induced IL-6 release from alveolar epithelial cells [12], or gall bladder relaxation [16], and might thus give rise to false interpretations In this article, we show that, in comparison to newer compounds such as SCH51866 [24], compound 31 and compound 30 [23], the ‘classic’ inhibitors vinpocetine and 8MM-IBMX (Fig 1) have low enzymatic potency and low PDE subtype specificity We asked: (a) what the effective cellular concentration of these inhibitors is; and (b) whether the difference in enzymatic potency would also transfer to differing cellular potency This need not necessarily be true, as cellular potency also depends on the physicochemical properties of inhibitors, such as lipid ⁄ liquid distribution and membrane permeability Thus, we searched for a cellular testing system that would allow us to measure a functional outcome, which is explicitly due and directly linked to the inhibition of PDE1 We showed that the human glioblastoma cell line A172 expresses high levels of Ca2+ ⁄ calmodulin-stimulated PDE1C activity and protein The identity of PDE1C was confirmed by Km value determination, demonstrating that the Ca2+ ⁄ calmodulin-stimulated PDE1 activity can hydrolyze cAMP and cGMP with nearly equal efficacy, as previously described [4] Furthermore, transfection of A172 cells with PDE1C siRNA reduced the expression of the protein (as shown by western blotting) Interestingly, it took 72 h after siRNA transfection for the PDE1C protein level to decrease, whereas mRNA was reduced earlier, suggesting a long half-life of this protein in A172 cells In FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS T R Dunkern and A Hatzelmann *** cAMP (fold of control) * ** ** * *** * * 30 -5 log M 30 -6 log M 30 -7 log M 30 -8 log M 30 -9 log M 31 -5 log M 30 -10 log M 31 -6 log M 31 -7 log M 31 -8 log M 31 -9 log M Forskolin + Ionomycin 31 -10 log M Forskolin + Ionomycin Control B Forskolin *** *** *** * *** *** *** 31 -8 log M 31 -9 log M 31 -10 log M 30 -8 log M 30 -9 log M 30 -10 log M Forskolin Control addition to PDE1C activity in A172 cells, we identified PDE4 activity and a minor amount of PDE3 activity in A172 cells (both capable of hydrolyzing cAMP to 5¢-AMP), as well as cGMP-hydrolyzing PDE5 In early experiments performed in our laboratories, A172 cells were incubated with PDE1 inhibitors with or without forskolin (to stimulate the adenylyl cyclase), and intracellular cAMP was subsequently measured However, we did not observe any effects of the PDE1 inhibitors on intracellular cyclic nucleotide concentrations within the PDE1-selective concentration range (data not shown) Thus, we speculated that under the given basal cell culture conditions, PDE1C might not be active in these cells To circumvent this, we treated A172 cells with ionoymcin, which concentration-dependently increased intracellular Ca2+ concentrations by Ca2+ store depletion and extracellular Ca2+ influx within a few minutes (as measured by Ca2+ imaging scanning fluorometry), which in theory should lead to an activation of PDE1C We determined an optimal ionomycin concentration for our experiments, which induced no cytotoxicity and increased Ca2+ concentrations by about 40% of the maximal inducible Ca2+ response in the time frame used for subsequent investigations Cells pretreated with ionomycin in such a way responded to forskolin with an attenuated or even abolished increase in intracellular cAMP concentrations This demonstrates that the ionomycin-induced increase in intracellular Ca2+ concentration leads to activation of the PDE1C enzyme, which then hydrolyzes cAMP generated by the forskolin-stimulated adenylyl cyclase Activation of PDE1C was seen after ionomycin treatment of the cells This cellular system proved to be highly useful for the cellular potency screening of PDE1 inhibitors Pretreatment of A cAMP (fold of control) Fig The PDE1 inhibitors compound 31 and compound 30 concentration-dependently reverse the ionomycin effect on the forskolin-induced increase in intracellular cAMP concentrations, starting at nM (A) A172 cells pretreated with ionomycin (6 lM) and compounds 31 and 30 () 10 to ) log M final concentration) or untreated were stimulated for adenylyl cyclase activation with forskolin (10 lM) or vehicle (control) cAMP was measured after forskolin stimulation in the corresponding cellular extracts by cAMP-ELISA A statistical analysis (Student’s paired t-test) was used to compare the forskolin plus ionomycin-treated group with those treated in addition with compounds 31 and 30 (B) Cells were treated only with compounds 31 and 30 () 10 to ) log M final concentration) or vehicle (control), and stimulated for adenylyl cyclase activation with forskolin (10 lM); cAMP was then measured A statistical analysis (Student’s paired t-test) was used to compare the forskolin-treated group with control cells *P < 0.05, **P < 0.01, ***P < 0.005 Characterization of inhibitors of PDE1C Forskolin A172 cells with the three recently described PDE1 inhibitors SCH51866 [24], compound 31 and compound 30 [23] reversed the attenuating effect of ionomycin on forskolin-induced cAMP concentrations That means that, in comparison to forskolin plus ionomycin-treated cells, the cAMP concentrations increased Interestingly, these compounds became effective even at 1–10 nm, which is close to the IC50 (measured at a competitive substrate concentration of 0.5 lm cAMP ⁄ cGMP) of these compounds for the FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 4819 Characterization of inhibitors of PDE1C A T R Dunkern and A Hatzelmann Fig The low selective PDE1 inhibitors vinpocetine and 8MMIBMX not reverse the ionomycin effect on the forskolin-induced increase in intracellular cAMP up to concentrations of 10 lM (A) A172 cells pretreated with ionomycin (6 lM) and vinpocetine ⁄ 8MMIBMX () to ) log M final concentration) or untreated were stimulated for adenylyl cyclase activation with forskolin (10 lM) or vehicle (control) cAMP was measured after forskolin stimulation in the corresponding cellular extracts by cAMP-ELISA (B) For comparison, A172 cells pretreated with vinpocetine ⁄ 8MM-IBMX () to ) log M) were stimulated with forskolin (10 lM) only, and cAMP was measured after The mean values (as fold of control) and standard deviations of three independent experiments are shown cAMP (fold of control) Vinpocetin -6 log M Vinpocetin -5 log M Vinpocetin -6 log M Vinpocetin -5 log M Vinpocetin -7 log M Vinpocetin -8 log M 8MM-IBMX -5 log M 8MM-IBMX -6 log M 8MM-IBMX -7 log M 8MM-IBMX -8 log M Control Forskolin Forskolin + Ionomycin Forskolin + Ionomycin 10 cAMP (fold of control) B Vinpocetin -7 log M Vinpocetin -8 log M 8MM-IBMX -5 log M 8MM-IBMX -6 log M 8MM-IBMX -7 log M 8MM-IBMX -8 log M Forskolin Control or compound 30 (at 100 nm to 10 lm) caused a greater increase in cAMP concentrations (in the presence of ionomycin and forskolin) than that induced by forskolin This increase must be attributed to the inhibition of other PDEs in addition to PDE1C at these concentrations by these compounds As expected on the basis of their low inhibitory potency against PDE1C, vinpocetine and 8MM-IBMX exhibited lower cellular potency Even up to 10 lm, no effects of these compounds were observed This is quite surprising, because there are several publications demonstrating effects of these drugs in this concentration range For example, Haddad et al [12] demonstrated on alveolar epithelial cells that 8MM-IBMX inhibits lipopolysaccharide-mediated IL-6 biosynthesis with an IC50 of 7.08 lm Vinpocetine has been shown to strengthen the contraction of smooth muscle cells and shorten action potentials at lm [13], and inhibit platelet-derived growth factor-induced proliferation of pulmonary artery smooth muscle cells at lm [14] Even at 30 nm, vinpocetine was shown to be effective in relaxing carbachol-constricted gall bladders [16] When considering these contradictory data, we should keep in mind that the nootropic drug vinpocetine exhibits additional pharmacologic actions, which might have affected the findings in the publications cited above For example, vinpocetine has been shown to modulate different types of K+ current [19] and to block the NaV1.8 tetrodotoxin-resistant Na+ channels of rats [20] Forskolin Conclusions human PDE1C enzyme Within this concentration range, or even higher, up to 100 nm, these three compounds not inhibit any of the PDEs expressed in A172 cells except for PDE1C Thus we can be sure to observe effects of these inhibitors on cAMP concentrations controlled by PDE1C At higher, nonselective concentrations, SCH51866 (at 1–10 lm), compound 31 4820 Our data demonstrate the suitability of the A172 cell system for determination of the PDE1C-specific effect of inhibitors on cAMP levels They further suggest caution in the interpretation of data arising from the experimental use of 8MM-IBMX and vinpocetine, because of their low potency and selectivity Thus, newer compounds, such as SCH51866, compound 31 FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS T R Dunkern and A Hatzelmann and compound 30, might be more useful as PDE1 inhibitors, due to their enhanced potency and selectivity Experimental procedures Materials The PDE inhibitors vinpocetine and 8MM-IBMX were purchased from Sigma-Aldrich GmbH (Taufkirchen, Germany) The PDE1-selective inhibitor SCH51866, compound 31 and compound 30 (Schering-Plough, Kenilworth, NJ, USA) [23], the selective PDE4 inhibitor piclamilast (RP73401) [27] and the PDE5 inhibitor sildenafil were prepared at the chemical facilities of ALTANA Pharma AG (Konstanz, Germany) The selective PDE3 inhibitor motapizone [28] was a generous gift from Sanofi-Aventis (formerly Rhone-Poulenc Rorer, Koln, Germany) ă The polyclonal rabbit antibody to PDE1C is a product of Fabgennix (Taufkirchen, Germany), and has been reported to be not cross-reactive with PDE1A, PDE1B or other PDE family members The polyclonal rabbit antibody to ERK2 was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA) Peroxidase-coupled secondary antibodies used for western blotting are a product of Jackson ImmunoResearch Laboratories Inc (West Grove, PA, USA) Cell culture The human glioblastoma cell line A172 was cultured in DMEM plus 4500 mgỈL)1 glucose (Gibco, Invitrogen Life Technologies, Grand Island, NY, USA), mm l-glutamine, mm sodium pyruvate and 10% heat-inactivated fetal bovine serum at 37 °C and 5% CO2 Measurements of PDE isoenzyme activities and preparation of cellular extracts Washed cells were sonicated in homogenization buffer (137 mm NaCl, 2.7 mm KCl, 8.1 mm Na2HPO4, 1.5 mm KH2PO4, 10 mm Hepes, mm EGTA, mm MgCl2, mm mercaptoethanol, lm pepstatin A, 10 lm leupeptin, 50 lm phenylmethanesulfonyl fluoride, 10 lm soybean trypsin inhibitor, mm benzamidine, pH 8.2) The lysates were used for PDE activity measurements as described by Thompson & Appleman [29] The assay mixture contained: 30 mm Tris ⁄ HCl (pH 7.4), mm MgCl2, 0.5 lm cAMP ⁄ cGMP, including [3H]cAMP or [3H]cGMP, 100 lm EGTA, PDE isoenzyme-specific activators and inhibitors, and lysates Incubations were performed for 30 at 37 °C, and terminated by adding 0.2 m HCl Assays were incubated on ice for 10 min, and then 25 lg of 5¢-nucleotidase (Crotalus atrox) was added for 10 at 37 °C Thererafter, mixtures were loaded onto QAE-Sephadex Characterization of inhibitors of PDE1C columns and eluted with 30 mm ammonium formate (pH 6.0) The radioactivity in the eluate was counted Selective inhibitors and activators of PDE isoenzymes were used to determine activities of PDE families as described previously [30], with modifications Briefly, PDE4 was calculated as the difference in PDE activities at 0.5 lm cAMP in the presence and absence of lm piclamilast (RP73401), and PDE3 as the difference between RP73401inhibited cAMP hydrolysis in the presence and absence of 10 lm motapizone The fraction of cGMP (0.5 lm) hydrolysis in the presence of 10 lm motapizone that was inhibited by 100 nm sildenafil represented PDE5 The increase in cAMP (0.5 lm) hydrolysis (in the presence of lm RP73401 and 10 lm motapizone) induced by lm cGMP represented PDE2 PDE1 was defined as the increment of cAMP hydrolysis (in the presence of lm RP73401 and 10 lm motapizone) or cGMP hydrolysis induced by mm Ca2+ and 100 nm calmodulin The Km values of the PDE1 activity were determined by measuring the specific activity of Ca2+ ⁄ calmodulin-stimulated cAMP or cGMP hydrolysis under different substrate concentrations and by transferring the data into a linear Lineweaver–Burk diagram PDE assay ⁄ inhibitor testing Inhibition of PDE activity by the inhibitors was measured on human PDE enzymes in a modified scintillation proximity assay (GE Healthcare UK Ltd, Little Chalfont, Buckinghamshire, UK) The test volume was 100 lL, and contained 20 mm Tris buffer (pH 7.4), 0.1 mg BSmL)1, mm Mg2+, 0.5 lm cAMP ⁄ cGMP (including [3H]cAMP and [3H]cGMP), lL of the respective substance dilution in dimethylsulfoxide and sufficient PDE to ensure that 15–20% of the cAMP was converted The reaction was started by adding the substrate (cAMP ⁄ cGMP) After incubation for 15 min, assays were stopped by adding scintillation proximity assay beads (50 lL, containing mm 8MMIBMX) After the beads had sedimented, the samples were analyzed in commercially available measuring appliances, and the corresponding IC50 values of the compounds were determined from the concentration–effect curves siRNA tranfection and real-time PCR For siRNA tranfection with subsequent RNA preparation, · 105 A172 cells were seeded into 24-well plates For transfection with subsequent western blot analysis, · 106 cells were seeded into 10 cm dishes The day after seeding, cells were transfected with 10 nm pan-PDE1C siRNA smart pool (Ambion Inc., Austin, TX, USA) [pool number M-007643– 00, sequences CCAAGGAGATTGAAGAATT (1), GAT CATGCACTGAAATTTA (2), GATGAAACCTCTCAA ACTG (3), and CATCATCGCTGGACAATGT (4)], using FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS 4821 Characterization of inhibitors of PDE1C T R Dunkern and A Hatzelmann lgỈmL)1 argfectin-50 lipid as the tranfection agent (Atugen, Berlin-Buch, Germany) For western blotting experiments, 72 h after transfection, cells were washed with NaCl ⁄ Pi, trypsinized, shock-frozen as a cell pellet in liquid nitrogen, and stored at ) 80 °C For mRNA analysis, 48 h after transfection, the cells were lysed with b-mercaptoethanol-supplemented RLT buffer provided with the RNeasyQuiagen RNA-preparation kit Thereafter, RNA isolation, quantification and cDNA synthesis from lg of RNA was performed according to the manufacturer’s standard protocols Successful cDNA synthesis was controlled by gel electrophoresis of an aliquot of the cDNA samples cDNA was used for subsequent real-time Taq-Man PCR analysis for PDE1C (Applied Biosystems, Foster City, CA, USA) The sense and antisense primer sequences were 5¢-TGTGAGT CCATTAATCGATGAAACC-3¢ and 5¢-ACCTGATCG CTTGGCATCTG-3¢, respectively The probe sequence was as follows: (FAM)-AGCTGATGCTATTCAAACTCGA ACGCCTCT-(TAMRA) A172 cells (1.5 · 105 cells in mm Ca2+-containing medium) were preincubated for in tubes in the presence or absence of the various PDE1 inhibitors Then, lm ionomycin (or Ca2+-containing medium in controls) was added, and the samples were incubated for a further at 37 °C Then, the adenylyl cyclase activator forskolin (final concentration 10 lm) was added (or Ca2+-containing medium in controls) At this time, the final assay volume of 300 lL was reached One minute later, the incubations were stopped by adding 15 lL of 3.3 m HCl After a further 15 of incubation at room temperature, the samples were centrifuged for at 10 000 g to pellet the cell debris using an Eppendorf centrifuge 5417R and rotor F45-30-11 The supernatant was diluted : 10, and analyzed for cAMP content using a commercially available ELISA kit (Assay Designs, Ann Arbor, MI, USA) Preparation of cell extracts Ca2+ measurements Frozen cell pellets were resuspended in buffer [20 mm Tris ⁄ HCl, pH 8.5, mm EDTA, 5% (v ⁄ v) glycerine, mm dithiothreitol, 0.5 mm phenylmethanesulfonyl fluoride] and sonicated [Bandelin sonicator, Sonopuls HD2070 plus cup booster BR30 (Berlin, Germany), · 15 s] The resulting suspension was centrifuged at 20 000 g for 10 at °C to pellet cellular debris using a 5417R centrifuge and F45-30-11 rotor (Eppendorf AC, Hamburg, Germany) Supernatants were collected, and protein concentration was determined by the Bradford assay before western blotting Measurements were performed with a scanning fluorometer and an integrated fluid transfer workstation (Flexstation; Molecular Devices, Sunnyvale, CA, USA) by using an excitation wavelength of 485 nm and an emission wavelength of 525 nm The data were collected as relative fluorescence units every 12 or 20 s A172 cells, · 105 cells per well, were seeded 24 h before measurement on 96-well microplates (black, clear bottom) in 100 lL of culture medium One hour before measurements, cells were loaded with the dye fluo 3-mix (calcium assay kit; Molecular Devices) by adding 50 lL of the dye per well Cells were then incubated for 50 at 37 °C and 5% CO2, and for a subsequent 10 at room temperature in the dark After an initial 60 s of measurement, different concentrations of ionomycin were added (in some experiments, additional mm Ca2+ was added at time point 20 s), and measurement was continued for a further 30 If desired, at the end of some experiments the maximal possible Ca2+ signal was investigated by adding 10 lm ionomycin and mm Ca2+ To reach a subsequent decrease in Ca2+ therafter at 35 min, 50 mm EGTA was added All measurements were performed in triplicate Western blot analysis Proteins (10–30 lg) were separated on a 7.5–12% SDS polyacrylamide gel and than blotted onto a nitrocellulose transfer membrane (Protran nitrocellulose transfer membrane; Schleicher & Schuell Bioscience GmbH, Dassel, Germany) for 2–3 h Membranes were blocked for h in 5% (w ⁄ v) milk powder in NaCl ⁄ Pi containing 0.1% Tween-20 (PBT), incubated for h with antibody to PDE1C (1 : 2000), washed three times in PBT for 10 min, and incubated for h with horseradish peroxidase-coupled secondary anti-rabbit serum (1 : 20000) After a final washing with PBT (three times for 10 each), the blots were developed by using a chemiluminescence detection system (Lumi Light Plus; Roche GmbH, Mannheim, Germany) and a luminescent image analyzer (Fujifilm LAS1000 Pro, Tokyo, Japan) To control for equal protein loading, blots were additionally incubated with a primary antibody to extracellular signal-related protein kinase (ERK2) (1 : 3000) 4822 cAMP measurements Cytotoxicity assay (lactate dehydrogenase assay) A172 cells, · 104, were seeded into 96-well plates and cultured for 24 h Ionomycin (6 lm) or different concentrations of Triton X-100 (positive controls) were then added for 15 For the generation of a standard curve, different cell numbers (1 · 103 up to 1.3 · 104 cells per well in triplicate) were seeded and treated 24 h later for FEBS Journal 274 (2007) 4812–4824 ª 2007 The Authors Journal compilation ª 2007 FEBS T R Dunkern and A Hatzelmann h with a supracytotoxic concentration of 1% Triton X-100, known to lyse all cells within this time After the various treatments, 50 lL of the cell culture medium supernatant was transferred to a 96-well plate, followed by 50 lL of a substrate solution (Promega, Madison, WI, USA; CytoTox96 assay), and after 15 of incubation, an additional 50 lL of a stop solution Thereafter, extinction was measured at a wavelength of 490 nm The numbers of dead cells induced by ionomycin and the different concentrations of Triton X-100 were calculated on the basis of the standard curve measurement Characterization of inhibitors of PDE1C 10 11 Acknowledgements We wish to acknowledge D Heinze, D Kubanek and N Spath from the Biochemistry Department Inamă mation of ALTANA Pharma AG for their excellent technical assistance 12 References Lugnier C (2006) Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents Pharmacol Therapeutics 109, 366–398 Polli JW & Kincaid RL (1992) Molecular cloning of DNA encoding a calmodulin-dependent phosphodiesterase enriched in striatum Proc Natl Acad Sci USA 89, 11079–11083 Yu J, Wolda SL, Frazier AL, Florio VA, 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(Ambion Inc., Austin, TX, USA) [pool number M-007643– 00, sequences CCAAGGAGATTGAAGAATT (1), GAT CATGCACTGAAATTTA (2), GATGAAACCTCTCAA ACTG (3), and CATCATCGCTGGACAATGT (4)], using FEBS Journal... lL of a substrate solution (Promega, Madison, WI, USA; CytoTox96 assay), and after 15 of incubation, an additional 50 lL of a stop solution Thereafter, extinction was measured at a wavelength of. .. with an attenuated or even abolished increase in intracellular cAMP concentrations This demonstrates that the ionomycin-induced increase in intracellular Ca2+ concentration leads to activation of