Evaluation studies : review annual/ Edit.: Aikin

728 hal.: tab.ill.; 23 cm

Evaluation studies : review annual/ Edit.: Aikin

728 hal.: tab.ill.; 23 cm

Evaluation studies : review annual/ Edit.: Aikin

728 hal.: tab.ill.; 23 cm

Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis

Background Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets. Methods To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASPS239) by flow cytometry and Western blot. ANOVA followed by Bonferroni’s test and Student’s t-test were used as appropriate. Results We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite. Conclusions All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC

Peroxisome-proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARα and PPARδ (also known as PPARβ) (but not PPARγ). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARα (but not PPARδ or PPARγ) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4β-PMA and PGF2α, and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4β-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4β-PMA in the absence of a PPAR ligand was decreased by the NF-κB (nuclear factor κB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-κB in addition to PPAR phosphorylation. Use of NF-κB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARα to increase PTGS2 levels in bovine endometrial stromal cells