Christian Schudt

Influence of selective phosphodiesterase inhibitors on human neutrophil functions and levels of cAMP and Cai

SummaryChromatographic analysis of 3′,5′-cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the cytosol of human neutrophils shows the predominant presence of PDE IV (cAMP specific) and PDE V (cGMP specific). PDE IV is characterized by (1) cAMP selectivity, (2) a KM for cAMP of 1.2 M and (3) a typical rank order of IC 50-values for PDE inhibitors: 0.13, 0.17, 47 and 9.5 μM for PDE IV selective rolipram, PDE III/IV selective zardaverine, PDE III selective motapizone and unselective 3-isobutyl-l-methylxanthine (IBMX), respectively. Functions of polymorphonuclear leukocytes (PMN) such as N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide release and fMLP/thimerosal elicited leukotriene (LT) biosynthesis are inhibited by these PDE inhibitors with the same rank order and even lower IC50-values. Measurements of changes in cytosolic Cai in Fura-2 loaded PMN demonstrate a transient Cai increase after stimulation with 0.1 μM fMLP and an additional sustained elevation of Cai levels in the presence of thimerosal. PDE inhibitors suppress this sustained phase of Cai release with the same rank order of IC50-values as LT biosynthesis. The correlation between fMLP/thimerosal-induced LT biosynthesis and Cai levels reveal a Cai threshold of 150 nM for arachidonic acid metabolism. cAMP levels in PMN were elevated by PDE inhibitors alone by less than 2-fold. In the presence of fMLP however, cAMP was increased up to 10-fold and the efficacy of PDE inhibitors to increase cAMP paralleled their potency to inhibit PDE IV. It is concluded that (1) suppression of PMN functions is achieved by PDE IV inhibition, (2) necessary cAMP elevations are within 50% increase, (3) superoxide release was affected by cAMP/protein kinase A (PKA) directly whereas (4) for inhibition of LT biosynthesis a cAMP related reduction of Ca-influx is involved.

Zardaverine as a selective inhibitor of phosphodiesterase isozymes

The pyridazinone derivative zardaverine has recently been introduced as a potent bronchodilator in vivo and in vitro. In addition, zardaverine exerts a positive inotropic action on heart muscle in vitro. The actions of zardaverine are thought to be mediated via inhibition of phosphodiesterase (PDE) activity. Recent data suggest that there are multiple forms of phosphodiesterases and at least five different isozyme families are now recognized. In the present study, the effects of zardaverine on the different PDE isozymes were investigated in several tissues. PDE isozymes were separated by chromatography on Q-sepharose. Zardaverine inhibited the cyclic GMP-inhibitable PDE III from human platelets and the rolipram-inhibitable PDE IV from canine trachea and human polymorphonuclear (PMN) cells with IC50-values of 0.58, 0.79 and 0.17 microM, respectively. The pyridazinone derivative affected the calmodulin-stimulated PDE I, the cyclic GMP-stimulated PDE II and the cyclic GMP-specific PDE V only marginally at concentrations up to 100 microM. Zardaverine inhibits the ADP-induced aggregation of human platelets with an IC50 of 1.6 microM. This inhibition was synergistically increased by activators of adenylate cyclase such as PGE1 and forskolin. In human PMN cells, zardaverine inhibited the zymosan-induced superoxide anion generation with an IC50 of 0.40 microM. Again, this effect was increased by activators of adenylate cyclase. These data clearly demonstrate that zardaverine is a selective inhibitor of PDE III and PDE IV isozymes.

Adenosine receptor-blocking xanthines as inhibitors of phosphodiesterase isozymes

The pharmacological actions of methylxanthines such as theophylline and caffeine may be due to blockade of adenosine receptors and/or inhibition of phosphodiesterase (PDE) activities. In the last years, potent xanthines have been developed that display some selectivity for A1 and A2 adenosine receptors. Little is known about the PDE inhibitory potency of these xanthines. The aim of the present study was to determine the potencies of A1 and A2 receptor selective xanthines as inhibitors of several PDE isozymes, the PDE I-V subtypes. The IC50 values of 8-phenyl- and 8-cycloalkyl-1,3-dialkylxanthines for inhibition of PDE isozymes from different sources are up to 10,000-fold higher than their antagonistic potencies at adenosine receptors. However, the A1 receptor selective antagonists 1,3-diethyl-8-phenylxanthine and 1,3-dipropyl-8-cyclopentylxanthine are comparatively potent inhibitors of PDE IV activity with IC50 values in the 10 microM range and are, therefore, nearly as potent as the PDE IV selective inhibitor, rolipram. The A2 receptor selective 1,3-dipropyl-7-methylxanthine is about 10-300-fold more potent as an adenosine receptor antagonist than as a PDE inhibitor. The results indicate that some of these novel xanthines can be used as selective adenosine receptor antagonists without interference due to inhibitory effects on PDEs.

Prostanoid pattern and iNOS expression during chondrogenic differentiation of human mesenchymal stem cells

Availability of human chondrocytes is a major limiting factor regarding drug discovery projects and tissue replacement therapies. As an alternative human mesenchymal stem cells (hMSCs) from bone marrow are taken into consideration as they can differentiate along the chondrogenic lineage. However, it remains to be shown whether they could form a valid model for primary chondrocytes with regards to inflammatory mediator production, like nitric oxide (NO) and prostanoids. We therefore investigated the production of NO and prostanoids in hMSCs over the course of chondrogenic differentiation and in response to IL‐1β using primary OA chondrocytes as reference. Chondrogenic differentiation was monitored over 28 days using collagen I, collagen II, and collagen X expression levels. Expression levels of inducible nitric oxide synthase (iNOS), levels of NO, and prostanoids were assessed using PCR, Griess assay, and GC/MS/MS, respectively. The hMSCs collagen expression profile during course of differentiation was consistent with a chondrocytic phenotype. Contrary to undifferentiated cells, differentiated hMSCs expressed iNOS and produced NO following stimulation with IL‐1β. Moreover, this induction of iNOS expression was corticosteroid insensitive. The spectrum of prostanoid production in differentiated hMSCs showed similarities to that of OA chondrocytes, with PGE2 as predominant product. We provide the first detailed characterization of NO and prostanoid production in hMSCs in the course of chondrogenic differentiation. Our results suggest that differentiated hMSCs form a valid model for chondrocytes concerning inflammatory mediator production. Furthermore, we propose that IL‐1β stimulation, leading to corticosteroid‐insensitive NO synthesis, can be used as a sensitive marker of chondrogenesis. J. Cell. Biochem. 98: 798–809, 2006.