cyclic-gmp and sulotroban

The importance of endothelial contraction in the genesis of inflammatory edema has been reported. ROS are metabolites synthesized in pathological conditions in that a significant intravascular fluid leak occurs, such as ischemia-reperfusion. Present experiments were designed to test the hypothesis that ROS, particularly H2O2, may elicit the contraction of endothelial cells, and to explore the mechanisms involved. Bovine aortic endothelial cells incubated with H2O2 showed a significant reduction in planar cell surface area (PCSA), and a significant increase in myosin light chain phosphorylation (MLCP), with a time- and dose-dependent pattern, without any significant toxicity. This effect of H2O2 was not blocked by sulotroban (TxA2 antagonist) or BN 52021 (PAF antagonist). Lanthanum chloride (calcium channel blocker) and EGTA partially inhibited the increase in MLCP induced by H2O2. H7 and staurosporine, PKC inhibitors, and PKC down-regulation (phorbol myristate acetate treatment, 24 h) also blocked H2O2-dependent endothelial contraction, measured as PCSA or MLCP. H2O2 increased the intracellular calcium concentration, an effect blunted by EGTA and lanthanum chloride. H2O2 also increased the phosphorylation of an 80 kD polypeptide, probably MARCKS, a PKC substrate. In summary, the present results demonstrate the ROS-dependent contraction of endothelial cells, an effect that could explain the intravascular fluid leak observed in some pathophysiological situations. Calcium and PKC may be involved in the development of this contraction.

Topics: Animals; Aorta; Cattle; Cell Size; Cells, Cultured; Cyclic GMP; Diterpenes; Endothelium, Vascular; Free Radical Scavengers; Ginkgolides; Hydrogen Peroxide; Kinetics; Lactones; Myosin Light Chains; Phosphorylation; Reactive Oxygen Species; Sulfonamides

Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Calcium; Cyclic AMP; Cyclic GMP; Cytosol; Epoprostenol; Humans; Intracellular Fluid; Nitroprusside; Platelet Activation; Prostaglandin Endoperoxides, Synthetic; Receptors, Thromboxane; Signal Transduction; Sulfonamides; Terpenes; Thapsigargin; Thromboxane A2

The relationship between agonist-sensitive calcium compartments and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in human platelets. In this context, calcium mobilization from intracellular pools and manganese influx was investigated in relation to the effect of altered cyclic-nucleotide levels. For maximal calcium release from intracellular stores, thapsigargin, compared to a receptor agonist like thrombin, requires the platelet's self-amplification mechanism, known to generate thromboxane A2. With this lipid mediator formed, thapsigargin released calcium and stimulated manganese influx in a manner similar to thrombin. Blocking the thromboxane receptor by addition of sulotroban (BM13.177) or, alternatively, increasing platelet cAMP or cGMP using prostacyclin or sodium nitroprusside, dramatically reduced the ability of thapsigargin to release calcium from intracellular compartments. The same experimental conditions significantly reduced the rate of manganese influx initiated by thapsigargin compared to thrombin. The experiments indicate that thapsigargin-sensitive compartments play only a minor role in inducing manganese influx compared to the receptor-sensitive compartment. Cyclic nucleotides accelerate the redistribution of an agonist-elevated platelet calcium into the thapsigargin-sensitive compartment, from which calcium can be released by inhibition of the Ca(2+)-ATPase. In human platelets, thapsigargin-induced calcium increase and influx were responsible for only part the calcium release resulting from inhibition of the corresponding ATPase; another part results from the indirect effect of thapsigargin acting via thromboxane-A2-receptor activation. Cyclic nucleotides are therefore an interesting regulatory device which can modify the thapsigargin response by not allowing the self-amplification mechanism of platelets to operate.

Topics: Blood Platelets; Calcium; Cell Compartmentation; Cyclic AMP; Cyclic GMP; Cytosol; Epoprostenol; Homeostasis; Humans; In Vitro Techniques; Manganese; Nitroprusside; Sulfonamides; Terpenes; Thapsigargin; Thrombin