Antiplatelet Activity,P2Y1 and P2Y12 Inhibition,and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background

Diadenosine tetraphosphate (Ap₄A), a constituent of platelet dense granules, and its P¹,P⁴-dithio and/or P²,P³-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y₁ and P2Y₁₂. The most active of those analogs, diadenosine 5′,5″″-P¹,P⁴-dithio-P²,P³-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

Objective

To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y₁ and P2Y₁₂ receptor antagonism, and their metabolism in human plasma.

Methods

We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y₁-mediated changes in cytosolic Ca²⁺, P2Y₁₂-mediated changes in VASP phosphorylation, and metabolism in human plasma.

Results

The inhibition of ADP-induced human platelet aggregation and human platelet P2Y₁₂ receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P¹- and P⁴-phosphorothioate groups, the S_PS_P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R_PR_P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S_PR_P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P²,P³-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S_PS_P and the R_PR_P diastereomers.

Conclusions

The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y₁ and P2Y₁₂ receptors.     

Integrin αvβ5 in endothelial cells of rat splenic sinus: an immunohistochemical and ultrastructural study

Localization of integrins β1-8, α1, α2, α3, α5, α6 and αv in sinus endothelial cells of the rat spleen was examined by immunofluorescence microscopy. Labeling for anti-integrin β5 and integrin αv was detected and colocalized in the entire circumference of endothelial cells. Labeling for integrin β5, vinculin and actin filaments demonstrated that they lay close to each other in the basal part of the endothelial cells. Although the other integrin βs, including integrin β1 and integrins α1, α2, α3, α5 and α6 in combination with integrin β1, were localized in leukocytes, slightly large cells, megakaryocytes and/or platelets in the sinus lumen and splenic cords, they were not detected in endothelial cells. Labeling for vitronectin, a component of the extracellular-matrix-binding integrin αvβ5, was strongly stained in the periphery of the wall of sinuses, as was collagen IV and, in addition, was localized in the cytoplasm of endothelial cells. Ultrastructural localization of integrin β5, vitronectin and clathrin was examined by immunogold electron microscopy to elucidate the involvement of integrin αvβ5 in the endocytosis of vitronectin in sinus endothelial cells. Electron microscopy with detergent extraction revealed abundant coated pits and coated vesicles in endothelial cells. Immunogold labeling for vitronectin was present in pits, vesicles and the stacked endoplasmic reticulum. Double-labeling for integrin β5 or integrin αv and clathrin revealed that they were colocalized in some vesicles in close proximity to the apical and lateral plasma membrane of the endothelial cells. The possible functional roles of integrin αvβ5 in endothelial cells of the splenic sinus are discussed.     

Synthesis and P2Y receptor activity of nucleoside 5′-phosphonate derivatives

Ribose-based nucleoside 5′-diphosphates and triphosphates and related nucleotides were compared in their potency at the P2Y receptors with the corresponding nucleoside 5′-phosphonate derivatives. Phosphonate derivatives of UTP and ATP activated the P2Y₂ receptor but were inactive or weakly active at P2Y₄ receptor. Uridine 5′-(diphospho)phosphonate was approximately as potent at the P2Y₂ receptor as at the UDP-activated P2Y₆ receptor. These results suggest that removal of the 5′-oxygen atom from nucleotide agonist derivatives reduces but does not prevent interaction with the P2Y₂ receptor. Uridine 5′-(phospho)phosphonate as well as the 5′-methylenephosphonate equivalent of UMP were inactive at the P2Y₄ receptor and exhibited maximal effects at the P2Y₂ receptor that were ?50% of that of UTP suggesting novel action of these analogues.     

5′-Deoxypyridoxal inhibition of glucocorticoid receptor binding in HeLa S3 cells and rat thymocytes

Recent evidence suggests a possible role for pyridoxal 5′-phosphate, the active physiological form of vitamin B₆, in steroid hormone action (1–3). We now report that 5′-deoxypyridoxal, a synthetic vitamin B₆ antagonist, causes a rapid and complete loss of dexamethasone receptor binding in cytosol preparations in whole HeLa S₃ cells or in rat thymocytes. This effect is concentration and time dependent, and is specific for 5′-deoxypyridoxal. In whole cell incubations of either HeLa S₃ cells or rat thymocytes at 37°C, a 50% reduction in [³H]dexamethasone binding was observed in the presence of 0.25 mM 5′-deoxypyridoxal. Cytotoxicity was not evident in rat thymocytes or HeLa S₃ cells incubated with 3.0mM or 1.0mM 5′-deoxypyridoxal respectively. Pyridoxal 5′-phosphate, 5′-deoxypyridoxine and 5′-deoxypyridoxamine were ineffective in causing a loss of steroid receptor binding. These studies suggest that 5′-deoxypyridoxal may be an effective non-steroidal compound which can effect the binding of glucocorticoids to specific receptor proteins in whole cells.     

Synthesis and P2Y₂ receptor agonist activities of uridine 5'-phosphonate analogues

We explored the influence of modifications of uridine 5'-methylenephosphonate on biological activity at the human P2Y(2) receptor. Key steps in the synthesis of a series of 5-substituted uridine 5'-methylenephosphonates were the reaction of a suitably protected uridine 5'-aldehyde with [(diethoxyphosphinyl)methylidene]triphenylphosphorane, C-5 bromination and a Suzuki-Miyaura coupling. These analogues behaved as selective agonists at the P2Y(2) receptor, with three analogues exhibiting potencies in the submicromolar range. Although maximal activities observed with the phosphonate analogues were much less than observed with UTP, high concentrations of the phosphonates had no effect on the stimulatory effect of UTP. These results suggest that these phosphonates bind to an allosteric site of the P2Y(2) receptor.     

Inhibition of 3β- and 17β-hydroxysteroid dehydrogenase activities in rat Leydig cells by perfluorooctane acid

Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in rat testis microsomes and Leydig cells. The IC₅₀s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3β-HSD with an IC₅₀ of 53.2 ± 25.9 μM and 17β-HSD3 with an IC₅₀ 17.7 ± 6.8 μM. PFOA inhibited intact Leydig cell 3β-HSD with an IC₅₀ of 146.1 ± 0.9 μM and 17β-HSD3 with an IC₅₀ of 194.8 ± 1.0 μM. The inhibitions of 3β-HSD and 17β-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3β-HSD and 17β-HSD3 in rat Leydig cells.     

Synthesis and Properties of P1, P2-, P1, P3- and P1, P4-Dinucleoside Di-, Tri- and Tetraphosphate mRNA 5′-Cap Analogues

Abstract

Chemically synthesized dinucleoside P¹, P²-di-, P¹, P³-tri- and P¹, P⁴-tetraphosphates, derivatives of 5′-linked 7-methylguanosine and guanosine were characterized with respect to their structural properties and functional effect on eukaryotic translation inhibition.     

Different effects of thymidine and 5-fluorouracil 2′-deoxyriboside on biosynthetic events in cultured P815Y mast cells

1. Addition of 2mm-thymidine, although resulting in the cessation of cell division, allows the continuation of phospholipid and protein synthesis and results in an increase in mean cell volume for at least 15h. 2. 5-Fluorouracil 2'-deoxyriboside inhibits cell division but differs from thymidine by inhibiting the synthesis of phospholipid and protein in a more marked manner. 3. The relation between these results and the P815Y cell cycle is discussed.     

Effects of ischaemia,reperfusion and α1 receptor stimulation on the inositoltrisphosphate receptor population in rat heart atria and ventricles

Binding sites specific for inositol 1,4,5-trisphosphate (InsP₃) have been demonstrated in sarcoplasmic reticulum vesicles isolated from heart muscle. Scatchard analysis of a binding isotherm indicated a high as well as a low affinity binding site [1]. In this study a comparison was made between InsP₃ binding to crude microsomal membranes prepared from rat heart atria and ventricles respectively. Results obtained showed a four-fold higher incidence of binding to atrial membranes. Furthermore, the receptor populations of the atria and ventricles behaved differently during conditions causing fluctuations in tissue InsP₃ levels, viz. ischaemia, reperfusion and ₁-adrenergic stimulation. Reperfusion, as well as phenylephrine stimulation, caused an increase in InsP₃ levels associated with down-regulation of the ventricular InsP₃ receptor population while binding to atrial binding sites was elevated. In the ventricular population this down-regulation was the result of a reduction in B_max alone with no changes in the Kd values of the high- or the low-affinity binding sites. The reason(s) for the differential response of the atrial and ventricular InsP₃ receptor populations to changes in InsP₃ levels, remains to be established.     

The role of purinergic P2Y12 and P2Y13 receptors in ADPβS-induced inhibition of the cardioaccelerator sympathetic drive in pithed rats

Purinergic Signalling - ATP is a cotransmitter released with other neurotransmitters from sympathetic nerves, where it stimulates purinergic receptors. Purinergic adenosine P1 receptors (coupled to...     

Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGFβ-1 in cultured rat nucleus pulposus cells

Introduction  

Although transforming growth factor β1 (TGFβ1) is known to be a potent inhibitor of proliferation in most cell types, it accelerates proliferation in certain mesenchymal cells, such as articular chondrocytes and nucleus pulposus cells. The low ability for self-renewal of nucleus pulposus cells is one obstacle in developing new therapeutic options for intervertebral disc diseases, and utilizing cytokines is one of the strategies to regulate nucleus pulposus cell proliferation. However, the precise cell cycle progression and molecular mechanisms by which TGFβ1 stimulates cell growth remain unclear. The aim of this study was to elucidate a mechanism that enables cell proliferation with TGFβ1 stimulation.     

Role of 11β-OH-C(19) and C(21) steroids in the coupling of 11β-HSD1 and 17β-HSD3 in regulation of testosterone biosynthesis in rat Leydig cells

Here we describe further experiments to support our hypothesis that bidirectional 11β-HSD1-dehydrogenase in Leydig cells is a NADP(H) regenerating system. In the absence of androstenedione (AD), substrate for 17β-HSD3, incubation of Leydig cells with corticosterone (B) or several C₁₉- and C₂₁-11β-OH-steroids, in the presence of [³H]-11-dehydro-corticosterone (A), stimulated 11β-HSD1-reductase activity. However, in presence of 30 μM AD, testosterone (Teso) synthesis is stimulated from 4 to 197 picomole/25,000 cells/30 min and concomitantly inhibited 11β-HSD1-reductase activity, due to competition for the common cofactor NADPH needed for both reactions. Testo production was further significantly increased (p < 0.05) to 224-267 picomole/25,000 cells/30 min when 10 μM 11β-OH-steroids (in addition to 30 μM AD) were also included. Similar results were obtained in experiments conducted with lower concentrations of AD (5 μM), and B or A (500 nM).Incubations of 0.3-6.0 μM of corticosterone (plus or minus 30 μM AD) were then performed to test the effectiveness of 17β-HSD3 as a possible NADP⁺ regenerating system. In the absence of AD, increasing amounts (3-44 pmol/25,000 cells/30 min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30 μM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3-5-fold producing 4-210 pmol/25,000 cells/30 min of 11-dehydro-corticosterone. We conclude that 11β-HSD1 is enzymatically coupled to 17β-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems.     

Roxithromycin treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of Caveolin-1 in rat airway smooth muscle cells

Background

Roxithromycin (RXM) has been widely used in asthma treatment; however, the mechanism has not been fully understood. The aim of our study was to investigate the underlying mechanism of RXM treatment in mediating the effect of transforming growth factor (TGF)-β1 on airway smooth muscle cells (ASMCs) proliferation and caveolinn-1 expression.

Methods

Firstly, the rat ovalbumin (OVA) model was built according to the previous papers. Rat ASMCs were prepared and cultured, and then TGF-β1 production in ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the proliferation of ASMCs was determined using cell counting kit (CCK-8) assay. Additionally, the expressions of caveolin-1, phosphorylated-ERK1/2 (p-ERK1/2) and phosphorylated–AKT (p-AKT) in ASMCs treated with or without PD98059 (an ERK1/2 inhibitor), wortannin (a PI3K inhibitor), β-cyclodextrin (β-CD) and RXM were measured by Western blot. Finally, data were evaluated using t–test or one-way ANOVA, and then a P value < 0.05 was set as a threshold.

Results

Compared with normal control, TGF-β1 secretion was significantly increased in asthmatic ASMCs; meanwhile, TGF-β1 promoted ASMCs proliferation (P < 0.05). However, ASMCs proliferation was remarkably inhibited by RXM, β-CD, PD98059 and wortmannin (P < 0.05). Moreover, the expressions of p-ERK1/2 and p-AKT were increased and peaked at 20 min after TGF-β1 stimulation, and then suppressed by RXM. Further, caveolin-1 level was down-regulated by TGF-β1 and up-regulated by inhibitors and RXM.

Conclusion

Our findings demonstrate that RXM treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of caveolin-1, which may be the potential mechanism of RXM protection from chronic inflammatory diseases, including bronchial asthma.     

Regulation of Gβγi-Dependent PLC-β3 Activity in Smooth Muscle: Inhibitory Phosphorylation of PLC-β3 by PKA and PKG and Stimulatory Phosphorylation of Gαi-GTPase-Activating Protein RGS2 by PKG

In gastrointestinal smooth muscle, agonists that bind to G_i-coupled receptors activate preferentially PLC-β3 via Gβγ to stimulate phosphoinositide (PI) hydrolysis and generate inositol 1,4,5-trisphosphate (IP₃) leading to IP₃-dependent Ca²⁺ release and muscle contraction. In the present study, we identified the mechanism of inhibition of PLC-β3-dependent PI hydrolysis by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). Cyclopentyl adenosine (CPA), an adenosine A₁ receptor agonist, caused an increase in PI hydrolysis in a concentration-dependent fashion; stimulation was blocked by expression of the carboxyl-terminal sequence of GRK2(495–689), a Gβγ-scavenging peptide, or Gα_i minigene but not Gα_q minigene. Isoproterenol and S-nitrosoglutathione (GSNO) induced phosphorylation of PLC-β3 and inhibited CPA-induced PI hydrolysis, Ca²⁺ release, and muscle contraction. The effect of isoproterenol on all three responses was inhibited by PKA inhibitor, myristoylated PKI, or AKAP inhibitor, Ht-31, whereas the effect of GSNO was selectively inhibited by PKG inhibitor, Rp-cGMPS. GSNO, but not isoproterenol, also phosphorylated Gα_i-GTPase-activating protein, RGS2, and enhanced association of Gα_i3-GTP and RGS2. The effect of GSNO on PI hydrolysis was partly reversed in cells (i) expressing constitutively active GTPase-resistant Gα_i mutant (Q204L), (ii) phosphorylation-site-deficient RGS2 mutant (S46A/S64A), or (iii) siRNA for RGS2. We conclude that PKA and PKG inhibit Gβγ_i-dependent PLC-β3 activity by direct phosphorylation of PLC-β3. PKG, but not PKA, also inhibits PI hydrolysis indirectly by a mechanism involving phosphorylation of RGS2 and its association with Gα_i-GTP. This allows RGS2 to accelerate Gα_i-GTPase activity, enhance Gαβγ_i trimer formation, and inhibit Gβγ_i-dependent PLC-β3 activity.