Claire Lugnier

Endothelium‐dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors

1 The effects of selective inhibitors of adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) and guanosine 3′: 5′‐cyclic monophosphate (cyclic GMP) phosphodiesterases (PDEs) were investigated on PDEs isolated from the rat aorta and on relaxation of noradrenaline (1 μm) precontracted rat aortic rings, with and without functional endothelium. 2 Four PDE forms were isolated by DEAE‐sephacel chromatography from endothelium‐denuded rat aorta: a calmodulin‐activated PDE (PDE I) which hydrolyzed preferentially cyclic GMP, two cyclic AMP PDEs (PDE III and PDE IV) and one cyclic GMP‐specific PDE (PDE V). The latter was selectively and potently inhibited by zaprinast. The two cyclic AMP PDEs were discriminated by specific inhibitors: one was inhibited by cyclic GMP (PDE III) and by new cardiotonic agents (milrinone, CI 930, LY 195115 and SK&F 94120); the other was inhibited by denbufylline and rolipram (PDE IV). None of these drugs significantly inhibited PDE I. 3 The PDE III inhibitors caused endothelium‐independent relaxations of rat aortic rings with the following EC50 values (μm concentration producing 50% relaxation): LY 195115: 3.4, milrinone: 5.7, CI 930; 7.8, SK&F 94120: 14.7. Neither NG‐monomethyl‐l‐arginine (l‐NMMA, 300 μm), an inhibitor of the l‐arginine‐NO pathway, nor l‐arginine (1 mm) modified the effect of PDE III inhibitors. However, methylene blue (10 μm) an inhibitor of soluble guanylate cyclase abolished relaxation induced by PDE III inhibitors except in the case of compound CI 930. 4 The specific PDE IV and PDE V inhibitors both produced endothelium‐dependent relaxations which were inhibited by l‐NMMA and by methylene blue (10 μm). In the presence of l‐NMMA, relaxation was restored by subsequent addition of l‐arginine. 5 The relaxant effects of denbufylline and rolipram were studied in the presence of drugs stimulating either adenylate cyclase (forskolin and isoprenaline) or soluble guanylate cyclase (sodium nitroprusside, SNP), or inhibiting PDE III (milrinone). In endothelium‐denuded rings, a relaxing effect of both denbufylline and rolipram was found in the presence of milrinone (EC50 values 1.7 and 12 μm, respectively) or SNP (EC50 values 12.3 and 124 μm, respectively), but not in the presence of forskolin or isoprenaline. However in the presence of functional endothelium, relaxations produced by PDE IV inhibitors were significantly potentiated by forskolin, isoprenaline, milrinone and SNP (respective EC50 values for denbufylline: 2, 2, 0.4 and 0.7 μm and for rolipram: 7, 13, 7 and 1.2 μm). 6 These results indicate that the relaxant effects of inhibitors of the cyclic AMP‐specific PDE IV are markedly enhanced by cyclic GMP elevating agents and by the PDE III inhibitor milrinone. They support the hypothesis that cyclic GMP enhances cyclic AMP‐mediated relaxation, possibly through the inhibition of the cyclic GMP‐inhibited PDE III.

Synthesis and anti-inflammatory activity of phthalimide derivatives, designed as new thalidomide analogues.

This paper describes the synthesis and anti-inflammatory activity of new N-phenyl-phthalimide sulfonamides (3a-e) and the isosters N-phenyl-phthalimide amides (4a-e), designed as hybrids of thalidomide (1) and aryl sulfonamide phosphodiesterase inhibitor (2). In these series, compound 3e (LASSBio 468), having a sulfonyl-thiomorpholine moiety, showed potent inhibitory activity on LPS-induced neutrophil recruitment with ED(50)=2.5mg kg(-1), which was correlated with its inhibitory effect on TNF-alpha level.

Cyclic nucleotide phosphodiesterase (PDE) isozymes as targets of the intracellular signalling network: benefits of PDE inhibitors in various diseases and perspectives for future therapeutic developments.

Cyclic nucleotide phosphodiesterases (PDEs) that specifically inactivate the intracellular messengers cAMP and cGMP in a compartmentalized manner represent an important enzyme class constituted by 11 gene‐related families of isozymes (PDE1 to PDE11). Downstream receptors, PDEs play a major role in controlling the signalosome at various levels of phosphorylations and protein/protein interactions. Due to the multiplicity of isozymes, their various intracellular regulations and their different cellular and subcellular distributions, PDEs represent interesting targets in intracellular pathways. Therefore, the investigation of PDE isozyme alterations related to various pathologies and the design of specific PDE inhibitors might lead to the development of new specific therapeutic strategies in numerous pathologies.

Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells.

Experiments were carried out in order to isolate and characterize the cyclic nucleotide phosphodiesterase activities in primary and low passages of cultured bovine aortic endothelial cells. The subcellular characterization of the cyclic nucleotide hydrolytic activity showed that both cAMP and cGMP hydrolytic activities were predominant in the cytosolic rather than the particulate fraction of the endothelial cell homogenate. At a low substrate concentration (0.25 microM), the major hydrolytic activity was for cAMP while at a high concentration (20 microM) it was for both cAMP and cGMP. Both cAMP and cGMP hydrolytic activities were insensitive to calmodulin. Cytosolic cyclic nucleotide phosphodiesterase activity was resolved into two distinct phosphodiesterase forms using HPLC. The first eluted form was designated cGS-PDE: it hydrolysed both cAMP and cGMP and its cAMP hydrolytic activity was markedly enhanced by the presence of cGMP. The second form was designated cAMP-PDE: it selectively hydrolysed cAMP. The cytosolic cAMP-PDE was inhibited by micromolar concentrations of cAMP-PDE inhibitors such as trequinsin, rolipram, dipyridamole or papaverine. The cGS-PDE was inhibited by micromolar concentrations of trequinsin, dipyridamole and papaverine and was insensitive to rolipram, except for the hydrolysis of cAMP which was inhibited in the micromolar range. Both the cAMP-PDE and the cGS-PDE were relatively insensitive to the selective cGMP-PDE inhibitor, zaprinast which was about 750-fold less potent on endothelial PDEs than on smooth muscle cGMP-PDE. The identification of selective and specific PDE inhibitors of the different PDE forms may allow a better understanding of the regulation and the role of cyclic nucleotides in endothelial cells.

Delphinidin inhibits endothelial cell proliferation and cell cycle progression through a transient activation of ERK-1/-2

Epidemiological studies have shown that a diet rich in fruits and vegetables might reduce the risk of cardiovascular diseases and protect against cancer by mechanisms that have not been elucidated yet. This study was aimed to define the effect of delphinidin, a vasoactive polyphenol belonging to the class of anthocyanin, on bovine aortic endothelial cells (BAECs) proliferation. Delphinidin inhibited serum- and vascular endothelium growth factor-induced BAECs proliferation. This antiproliferative effect of delphinidin, is triggered by ERK-1/-2 activation, independent of nitric oxide pathway and is correlated with suppression of cell progression by blocking the cell cycle in G(0)/G(1) phase. Furthermore, suppression of cell cycle progression is associated with the modulation of the mitogenic signaling transduction cascade. This includes over-expression of caveolin-1 and p21(WAF1/Cip1) and down-expression of Ras and cyclin D1. In conclusion, the antiproliferative effect of delphinidin may be of importance in preventing both plaque development and stability in atherosclerosis and tumor dissemination in cancer.

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1

The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (DeltaPsim). TQ-induced apoptosis, confirmed by the presence of hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression, reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.

Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation

1 The involvement of cyclic AMP‐dependent protein kinase (PKA) and cyclic GMP‐dependent protein kinase (PKG) in the effects of cyclic AMP‐elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent‐protein kinase activities and ATP‐induced calcium signalling ([Ca2+]i) was studied in rat aorta. Cyclic AMP‐elevating agents used were a β‐adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram). 2 In rat intact aorta, the relaxant effect induced by isoprenaline (0.01–0.3 μM) was decreased by a specific inhibitor of PKA, H‐89, whereas a specific inhibitor of PKG, Rp‐8‐Br‐cyclic GMPS, was without effect. No significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 μM was used. At the same concentration, isoprenaline did not modify ATP‐induced changes in [Ca2+]i in smooth muscle cells. Neither H‐89 nor Rp‐8‐Br‐cyclic GMPS modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01–0.3 μM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved. 3 The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp‐8‐Br‐cyclic GMPS with no significant effect of H‐89. Neither SK&F 94120, nor rolipram at 30 μM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP‐induced transient increase in [Ca2+]i. This decrease was abolished by Rp‐8‐Br‐cyclic GMPS whereas H‐89 had no significant effect. These results suggest that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross‐activation of PKG by cyclic AMP in rat aorta. 4 The combination of 5 μM SK&F 94120 with rolipram markedly potentiated the relaxant effect of rolipram. This relaxation was decreased by H‐89 and not significantly modified by Rp‐8‐Br‐cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PKA rather than PKG is involved in this type of vasorelaxation. The differences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are differently localized in the cell. 5 These findings support a role for both PKA and PKG in cyclic AMP‐mediated relaxation in rat aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.

Study of the mechanisms involved in the vasorelaxation induced by (−)-epigallocatechin-3-gallate in rat aorta

This study investigated several mechanisms involved in the vasorelaxant effects of (−)‐epigallocatechin‐3‐gallate (EGCG). EGCG (1 μM–1 mM) concentration dependently relaxed, after a transient increase in tension, contractions induced by noradrenaline (NA, 1 μM), high extracellular KCl (60 mM), or phorbol 12‐myristate 13‐acetate (PMA, 1 μM) in intact rat aortic rings. In a Ca2+‐free solution, EGCG (1 μM–1 mM) relaxed 1 μM PMA‐induced contractions, without previous transient contraction. However, EGCG (1 μM–1 mM) did not affect the 1 μM okadaic acid‐induced contractions. Removal of endothelium and/or pretreatment with glibenclamide (10 μM), tetraethylammonium (2 mM) or charybdotoxin (100 nM) plus apamin (500 nM) did not modify the vasorelaxant effects of EGCG. In addition, EGCG noncompetitively antagonized the contractions induced by NA (in 1.5 mM Ca2+‐containing solution) and Ca2+ (in depolarizing Ca2+‐free high KCl 60 mM solution). In rat aortic smooth muscle cells (RASMC), EGCG (100 μM) reduced increases in cytosolic free Ca2+ concentration ([Ca2+]i) induced by angiotensin II (ANG II, 100 nM) and KCl (60 mM) in 1.5 mM CaCl2‐containing solution and by ANG II (100 nM) in the absence of extracellular Ca2+. In RASMC, EGCG (100 μM) did not modify basal generation of cAMP or cGMP, but significantly reversed the inhibitory effects of NA (1 μM) and high KCl (60 mM) on cAMP and cGMP production. EGCG inhibited the enzymatic activity of all the cyclic nucleotide PDE isoenzymes present in vascular tissue, being more effective on PDE2 (IC50∼17) and on PDE1 (IC50∼25). Our results suggest that the vasorelaxant effects of EGCG in rat aorta are mediated, at least in part, by an inhibition of PDE activity, and the subsequent increase in cyclic nucleotide levels in RASMC, which, in turn, can reduce agonist‐ or high KCl concentration‐induced increases in [Ca2+]i.

Decreased Expression and Activity of cAMP Phosphodiesterases in Cardiac Hypertrophy and Its Impact on β-Adrenergic cAMP Signals

Rationale: Multiple cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP in cardiomyocytes but the role of PDEs in controlling cAMP signaling during pathological cardiac hypertrophy is poorly defined. Objective: Evaluate the &bgr;-adrenergic regulation of cardiac contractility and characterize the changes in cardiomyocyte cAMP signals and cAMP-PDE expression and activity following cardiac hypertrophy. Methods and Results: Cardiac hypertrophy was induced in rats by thoracic aortic banding over a time period of 5 weeks and was confirmed by anatomic measurements and echocardiography. Ex vivo myocardial function was evaluated in Langendorff-perfused hearts. Engineered cyclic nucleotide-gated (CNG) channels were expressed in single cardiomyocytes to monitor subsarcolemmal cAMP using whole-cell patch-clamp recordings of the associated CNG current (ICNG). PDE variant activity and protein level were determined in purified cardiomyocytes. Aortic stenosis rats exhibited a 67% increase in heart weight compared to sham-operated animals. The inotropic response to maximal &bgr;-adrenergic stimulation was reduced by ≈54% in isolated hypertrophied hearts, along with a ≈32% decrease in subsarcolemmal cAMP levels in hypertrophied myocytes. Total cAMP hydrolytic activity as well as PDE3 and PDE4 activities were reduced in hypertrophied myocytes, because of a reduction of PDE3A, PDE4A, and PDE4B, whereas PDE4D was unchanged. Regulation of &bgr;-adrenergic cAMP signals by PDEs was blunted in hypertrophied myocytes, as demonstrated by the diminished effects of IBMX (100 &mgr;mol/L) and of both the PDE3 inhibitor cilostamide (1 &mgr;mol/L) and the PDE4 inhibitor Ro 201724 (10 &mgr;mol/L). Conclusions: &bgr;-Adrenergic desensitization is accompanied by a reduction in cAMP-PDE and an altered modulation of &bgr;-adrenergic cAMP signals in cardiac hypertrophy.

Section Review: Cardiovascular & Renal: Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiovascular diseases

Cyclic nucleotide phosphodiesterases (PDEs) comprise at least seven families of isozymes coded by related but distinct genes, grouped on the basis of their structural and enzymatic characteristics. Five of these families are known to be present in the cardiovascular system. A number of potent inhibitors have been synthesised with relative selectivity for some PDEs. However, there is no selective inhibitor of PDE1 (calmodulin-activated), and only one compound has been reported which selectively inhibits PDE2 (stimulated by cGMP). Available information is limited to pharmacological and therapeutic properties of drugs selectively inhibiting two PDEs specific for cAMP (PDE3, inhibited by milrinone-like cardiotonics, and PDE4, inhibited by rolipram) and a cGMP-PDE (PDE5, inhibited by zaprinast). Differential expression of PDEs and differential subcellular localisation provide the possibility of selectively targeting cardiovascular and platelet functions with selective PDE inhibitors. The resulting effects incl...