Armin Hatzelmann

Potent Inhibition of Ca2+ Release-activated Ca2+ Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2

Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels is essential for T-cell activation and proliferation. Recently, it has been shown that 3,5-bistrifluoromethyl pyrazole (BTP) derivatives are specific inhibitors of Ca2+-dependent transcriptional activity in T-cells (Trevillyan, J. M., Chiou, X. G., Chen, Y. W., Ballaron, S. J., Sheets, M. P., Smith, M. L., Wiedeman, P. E., Warrior, U., Wilkins, J., Gubbins, E. J., Gagne, G. D., Fagerland, J., Carter, G. W., Luly, J. R., Mollison, K. W., and Djuric, S. W. (2001) J. Biol. Chem. 276, 48118-48126). Whereas inhibition of Ca2+ signals was reported for BTP2 (Ishikawa, J., Ohga, K., Yoshino, T., Takezawa, R., Ichikawa, A., Kubota, H., and Yamada, T. (2003) J. Immunol. 170, 4441-4449), it was not found for BTP3 (Chen, Y., Smith, M. L., Chiou, G. X., Ballaron, S., Sheets, M. P., Gubbins, E., Warrior, U., Wilkins, J., Surowy, C., Nakane, M., Carter, G. W., Trevillyan, J. M., Mollison, K., and Djuric, S. W. (2002) Cell. Immunol. 220, 134-142). We show that BTP2 specifically inhibits CRAC channels in T-cells with an IC50 of ∼10 nm. It does not interfere with other mechanisms important for Ca2+ signals in T-cells, including Ca2+ pumps, mitochondrial Ca2+ signaling, endoplasmic reticulum Ca2+ release, and K+ channels. BTP2 inhibits Ca2+ signals in peripheral blood T-lymphocytes (in particular in CD4+ T-cells) and in human Jurkat T-cells. Inhibition of Ca2+ signals is independent of the stimulation method as Ca2+ entry was blocked following stimulation with anti-CD3, which activates the T-cell receptor, and also following stimulation with thapsigargin or inositol 1,4,5-trisphosphate. BTP2 also inhibited Ca2+-dependent gene expression (interleukins 2 and 5 and interferon γ) and proliferation of T-lymphocytes with similar IC50 values. BTP2 is the first potent and specific inhibitor of CRAC channels in primary T-lymphocytes. The inhibition of CRAC channels as well as Ca2+-dependent signal transduction with similar IC50 values in T-lymphocytes emphasizes the importance of CRAC channel activity during T-cell activation. Furthermore, BTP2 could prove to be a tool to finally unmask the molecular identity of CRAC channels.

Effects of the soluble guanylyl cyclase activator, YC-1, on vascular tone, cyclic GMP levels and phosphodiesterase activity.

The vasomotor and cyclic GMP‐elevating activity of YC‐1, a novel NO‐independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18. Similarly to SNP and NOC 18, YC‐1 (0.3–300u2003μM) caused a concentration‐dependent, endothelium‐independent relaxation that was greatly reduced by the sGC inhibitor 1‐H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ 10u2003μM; 59% inhibition of dilation induced by 100u2003μM YC‐1) suggesting the activation of sGC as one mechanism of action. Preincubation with YC‐1 (3 and 30u2003μM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO‐donors, SNP and NOC 18, by almost two orders of magnitude. Vasoactivity induced by SNP and YC‐1 displayed different kinetics as evidenced by a long‐lasting inhibition by YC‐1 (300u2003μM) on the phenylephrine (PE)‐induced contractile response, which was not fully reversible even after extensive washout (150u2003min) of YC‐1, and was accompanied by a long‐lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30u2003μM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound. Surprisingly, YC‐1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1–5 in vitro. In conclusion, YC‐1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC‐1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator.

Differential effects of non-selective and selective phosphodiesterase inhibitors on human eosinophil functions.

1 The effect of non‐selective (3‐isobutyl‐1‐methylxanthine, IBMX; theophylline) and type IV‐ or type III/IV‐selective (rolipram, RP 73401; zardaverine, tolafentrine) phosphodiesterase (PDE) inhibitors on human eosinophil functions was investigated. 2 For this purpose human eosinophils were purified from blood of healthy donors by a magnetic cell separation (MACS) technique to a purity ≥ 99%. From the stimuli investigated (complement C5a; N‐formyl‐methionyl‐leucyl‐phenylalanine, fMLP; platelet activating factor, PAF; opsonized zymosan) C5a was selected to test the influence of the above mentioned compounds on secretion of granule constituents (eosinophil cationic protein, ECP; eosinophil‐derived neurotoxin, EDN) as well as on formation of reactive oxygen species measured by luminol‐enhanced chemiluminescence in intact cells. For comparison, inhibition of PDE IV activity in the cytosol of disrupted cells, which contains about 75% of total PDE IV activity, was determined. 3 Both theophylline and IBMX inhibited the two cell responses with IC50 values which were in the range of their IC50 values obtained for inhibition of PDE IV activity in the cell‐free system. The β2‐adrenoceptor agonist, salbutamol (1 μmol 1−1), which by itself did not substantially influence the two cell responses, only marginally improved the potency of theophylline and IBMX in inhibiting ECP/EDN secretion. Only the IC50 value of IBMX for inhibition of chemiluminescence was lowered by about one order of magnitude in the presence of salbutamol. 4 In contrast, none of the selective PDE inhibitors tested substantially inhibited the two cell responses at concentrations up to 10 μmol 1−1. This was surprising because all of the compounds investigated inhibited PDE IV activity in the cell‐free system with IC50 values which were at least 30 fold lower than the highest concentration of the compounds used with intact cells. In combination with salbutamol, however, both ECP/EDN secretion and chemiluminescence was inhibited by rolipram and zardaverine with IC50 values similar to the IC50 values for inhibition of PDE IV activity. Although RP 73401 and tolafentrine also inhibited both cell responses in the presence of salbutamol, the potency of these two compounds in inhibiting eosinophil function in intact cells was at least two orders of magnitude lower than would have been expected from the inhibition of PDE IV activity in the cell‐free system. 5 These results indicate that (i) C5a‐stimulated human eosinophils are sensitive to inhibition by the non‐selective PDE inhibitors theophylline and IBMX, (ii) the inhibitory effect of these non‐selective PDE inhibitors cannot be mimicked by selective PDE IV or PDE III/IV inhibitors although human eosinophils almost exclusively contain PDE IV; (iii) the selective PDE inhibitors need an additional cyclic AMP trigger like a β2‐adrenoceptor agonist to be effective; but (iv) under the latter conditions inhibition of cell responses in intact cells does not correspond to inhibition of PDE IV activity in the cell‐free system. 6 We conclude that the non‐selective PDE‐inhibiting xanthines may inhibit C5a‐stimulated human eosinophil responses by other action(s) in addition to PDE IV inhibition, and that inhibition of PDE IV activity in the cell‐free system by the selective inhibitors may not generally represent the potency of the compounds in intact cells.

Phosphodiesterase profiles of highly purified human peripheral blood leukocyte populations from normal and atopic individuals: A comparative study

BACKGROUNDnSeveral previous reports have suggested an increased activity of cAMP phosphodiesterases (PDEs) and a higher sensitivity of these enzymes toward PDE inhibitors in leukocytes of patients suffering from atopic dermatitis.nnnOBJECTIVEnThe purpose of the present study was to comprehensively analyze and compare the PDE expression and activity profile of highly purified populations of leukocytes from normal and atopic blood donors. In addition, the influence of PDE inhibitors on function of leukocytes from normal and atopic individuals was investigated.nnnMETHODSnDensity gradient centrifugation, elutriation, and magnetic cell sorting techniques were used to purify eosinophils, monocytes, and B and T lymphocytes from peripheral human blood. Complementary DNA-polymerase chain reaction was used to analyze PDE4 subtype messenger RNA (mRNA) expression levels in addition to PDE isoenzyme activities. PDE4 inhibitor sensitivity was determined in monocyte homogenates from both groups. Functionally, suppression of lipopolysaccharide-induced synthesis of tumor necrosis factor-alpha in monocytes as well as phytohemagglutinin-induced T cell proliferation in peripheral blood mononuclear cell fractions by PDE4 and PDE3/4 inhibitors was compared.nnnRESULTSnIdentical PDE activities and mRNA expression profiles were found in all cells from normal and atopic donors except that there was an increase in the mRNA levels of PDE4A and PDE4B2 in atopic T cells, which was, however, not reflected in overall PDE4A activity. In addition, no differences in sensitivity of the functional responses to PDE inhibitors were noted. The mixed PDE3/4 inhibitor zardaverine was a more potent inhibitor of T cell proliferation than rolipram, a selective PDE4 inhibitor.nnnCONCLUSIONnNo evidence for alterations of PDE activities in atopy is provided by our findings.

Differential Expression and Function of Phosphodiesterase 4 (PDE4) Subtypes in Human Primary CD4+ T Cells: Predominant Role of PDE4D

Type 4 phosphodiesterases (PDE4) are critical regulators in TCR signaling by attenuating the negative constraint of cAMP. In this study, we show that anti-CD3/CD28 stimulation of human primary CD4+ T cells increases the expression of the PDE4 subtypes PDE4A, PDE4B, and PDE4D in a specific and time-dependent manner. PDE4A and PDE4D mRNAs as well as enzyme activities were up-regulated within 5 days, PDE4B showed a transient up-regulation with highest levels after 24 h. The induction was shown to be independent of different stimulation conditions and was similar in naive and memory T cell subpopulations. To elucidate the functional impact of individual PDE4 subtypes on T cell function, we used PDE4 subtype-specific short-interfering RNAs (siRNAs). Knockdown of either PDE4B or PDE4D inhibited IL-2 release 24 h after stimulation (time point of maximal IL-2 concentrations) to an extent similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast). Substantial amounts of IFN-γ or IL-5 were measured only at later time points. siRNA targeting PDE4D showed a predominant inhibitory effect on these cytokines measured after 72 h. However, the inhibition of all cytokines was most effective when PDE4 siRNAs were applied in combination. Although the effect of PDE4 inhibition on T cell proliferation is small, the PDE4D-targeting siRNA alone was as effective as the panPDE4 inhibitor, whereas PDE4A or PDE4B siRNAs had hardly an effect. In summary, individual PDE4 subtypes have overall nonredundant, but complementary, time-dependent roles in propagating various T cell functions and PDE4D is the form likely playing a predominant role.

Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation

1 CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function. 2 The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7‐like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected. 3 By cDNA‐PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found. 4 No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects. 5 Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time‐ and concentration‐dependent manner, which was increased in the presence of interleukin‐4 (IL‐4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole‐3′,5′‐cyclic monophosphorothioate, Sp‐isomer (dcl‐cBIMPS), respectively. However, at concentrations exceeding 100u2003μM db‐cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1u2003μM) and forskolin (10u2003μM) did not affect B cell proliferation, even when given in combination with rolipram. 6 Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp‐8‐Br‐cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram. 7 Importantly, PDE4 activity in LPS/IL‐4‐activated B lymphocytes decreased by about 50% compared to unstimulated control values. 8 We conclude that an increase in cyclic AMP, mediated by down‐regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL‐4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.

Phosphodiesterase isoenzyme families in human osteoarthritis chondrocytes – functional importance of phosphodiesterase 4

We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL‐1β‐induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized. Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL‐1β‐induced chondrocyte products (nitric oxide and prostaglandin E2) were measured in culture supernatants after 48u2003h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A‐D proteins were detected by immunoblotting. The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL‐1β‐induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL‐1β‐induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E2 (PGE2) enabled PDE4 inhibitors to reduce IL‐1β‐induced NO in this experimental setting. Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL‐1β and cyclic AMP‐mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein. Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects.

Effects of Roflumilast, a Phosphodiesterase-4 Inhibitor, on Hypoxia- and Monocrotaline-Induced Pulmonary Hypertension in Rats

Phosphodiesterase type 4 (PDE4) is involved in the hydrolysis of cAMP in pulmonary vascular smooth muscle (PA-SMC) and immune inflammatory cells. Given that intracellular cAMP accumulation inhibits contraction and growth of PA-SMCs as well as inflammatory cell functions, we investigated the effects of the PDE4 inhibitor 3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast), on pulmonary hypertension (PH) in rats. Treatment with roflumilast (0.5 or 1.5 mg · kg-1 day-1) from day 1 to day 21 after monocrotaline (MCT) injection (60 mg · kg-1 s.c.) attenuated PH development: pulmonary artery pressure, right ventricular hypertrophy, and muscularization of distal vessels on day 21 were decreased compared to control MCT-treated rats. Roflumilast (1.5 mg · kg-1 day-1) also reduced the increases in interleukin-6 and monocyte chemotactic protein-1 mRNAs observed in lung tissue on day 21 without affecting the rise in interleukin-1β mRNA on days 1 and 21. Roflumilast (1.5 mg · kg-1 day-1) from day 21 to day 42 after MCT reversed established PH, almost normalizing pulmonary artery pressure and structure, and suppressing proliferating cell nuclear antigen-positive cells in pulmonary vascular walls. Treatment with roflumilast similarly attenuated PH development due to chronic hypoxia. Treatment of human PA-SMCs with roflumilast N-oxide, the active metabolite of roflumilast, at concentrations up to 10-6 M, potentiated PA-SMC growth inhibition induced by prostacyclin (10-6 M) or interleukin-1β (10 ng · ml-1) but was inactive on its own. In conclusion, the PDE4 inhibitor roflumilast significantly attenuates pulmonary vascular remodeling and hypertension induced by chronic hypoxia or MCT and reverses established PH after MCT administration.

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

Different inhibitors of the Ca2+/calmodulin‐stimulated phosphodiesteraseu20031 family have been described and used for the examination of phosphodiesteraseu20031 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 phosphodiesteraseu20031C is expressed in the human glioblastoma cell line A172 with regard to mRNA, protein and activity level, and that lower activities of phosphodiesteraseu20032, phosphodiesteraseu20033, phosphodiesteraseu20034 and phosphodiesteraseu20035 are also present. The identity of the phosphodiesteraseu20031C activity detected was verified by downregulation of the mRNA and protein through human phosphodiesteraseu20031C specific small interfering RNA. In addition, the measured Km values (cAMP, 1.7u2003µm; cGMP, 1.3u2003µm) are characteristic of phosphodiesteraseu20031C. 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 phosphodiesteraseu20031C. The reduction of forskolin‐stimulated cAMP levels was reversed by phosphodiesteraseu20031 inhibitors in a concentration‐dependent way. Using this cellular system, we compared the cellular potency of published phosphodiesteraseu20031 inhibitors, including 8‐methoxymethyl‐3‐isobutyl‐1‐methylxanthine, vinpocetine, SCH51866, and two established phosphodiesteraseu20031 inhibitors developed by Schering‐Plough (named compoundsu200331 and 30). We demonstrate that up to 10u2003µm 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 10u2003000 times more potent phosphodiesteraseu20031 inhibitors SCH51866, compoundu200331 and compoundu200330 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 phosphodiesteraseu20031 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 phosphodiesteraseu20031 inhibitors. 8‐Methoxymethyl‐3‐isobutyl‐1‐methylxanthine and vinpocetine seem not to be suitable for the study of phosphodiesteraseu20031‐mediated functions.

Phosphodiesterase 2 inhibition diminished acute lung injury in murine pneumococcal pneumonia

Objective:Severe pneumococcal pneumonia frequently causes respiratory failure. Both pathogen factors and an uncontrolled host response may contribute to acute lung injury by impairing microvascular barrier function. Phosphodiesterase 2 (PDE2) was examined as a potential target in pneumonia-induced lung microvascular hyperpermeability. Design:Controlled, in vitro, ex vivo, and in vivo laboratory study. Subjects:Female Balb/C and C57Bl/6 mice, 8–12 weeks old. Interventions:Human umbilical vein endothelial cells and isolated mouse lungs were challenged with the pneumococcal exotoxin pneumolysin in the presence or absence of the selective PDE2 inhibitors 9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6one (PDP) or hydroxy-PDP. Transcellular electrical resistance or human serum albumin leakage in bronchoalveolar lavage fluid was determined, respectively. In addition, we induced pneumococcal pneumonia in mice and treated with hydroxy-PDP via continuous subcutaneous application by osmotic pumps. Human serum albumin leakage in bronchoalveolar lavage fluid was measured 48 hours after transnasal infection, and lung specimens were analyzed by TaqMan real-time polymerase chain reaction and Western blot for PDE2 gene and protein expression. Measurements and Main Results:In isolated perfused mouse lungs and in human umbilical vein endothelial cell monolayers, selective inhibition of PDE2 markedly decreased pneumolysin-induced hyperpermeability. Furthermore, in murine pneumococcal pneumonia, pulmonary PDE2-mRNA and -protein expression was significantly increased, and pneumonia-induced vascular permeability was distinctively reduced by PDE2 inhibition. Conclusions:PDE2 inhibition diminished microvascular leakage in pneumococcal pneumonia, and pulmonary PDE2 upregulation may play a crucial role in this respect. Selective PDE2 inhibitors thus may offer a promising therapeutic approach in severe pneumococcal pneumonia.