The ability of the beta-adrenergic agonist, isoproterenol, to attenuate the thrombin-induced increase in endothelial permeability was examined by measuring 125I-labeled albumin clearance across endothelial cell monolayers. Bovine pulmonary artery endothelial cells (CCL-209) were grown to confluence on gelatinized, polycarbonate micropore filters and mounted on modified Boyden chambers with Dulbecco's modified Eagle's medium (DMEM) and 0.5% bovine serum albumin. alpha-Thrombin at 0.2 nM to 2 microM produced a dose-related increase (P less than 0.01) in 125I-labeled albumin clearance from the DMEM control value. Light and electron microscopy revealed that the thrombin-induced increase in permeability correlated with changes in cell shape and rearrangement of filamentous actin. Coincubation of 2 microM isoproterenol with 2 microM alpha-thrombin reduced (P less than 0.01) the thrombin-induced increase in albumin clearance and the observed morphological changes. This attenuation was not caused by inhibition of thrombin's enzymatically active site, since isoproterenol did not impair thrombin's fibrinogen clotting activity nor its amidolytic cleavage of an artificial substrate (Spectrozyme-TH). Coincubation of 20 microM propranolol, a beta-adrenergic antagonist, with 2 microM isoproterenol and thrombin blocked the permeability-decreasing effect of isoproterenol. Both 2 microM isoproterenol and 2 pM alpha-thrombin alone decreased (P less than 0.01) albumin clearance below the DMEM control value. These results suggest that isoproterenol can reduce the thrombin-induced increase in endothelial permeability in vitro by directly maintaining actin filaments and the shape of endothelial cells.
Interaction of thrombin with vascular endothelial cells was investigated as a mechanism promoting platelet activation and adherence to endothelial monolayers. We found that pretreatment of endothelium with alpha-thrombin in the absence of platelets results in the attachment of platelets to endothelial cells after the removal of fluid-phase alpha-thrombin. This activity was eliminated by exposure of alpha-thrombin-pretreated endothelial cells to active site inhibitors of alpha-thrombin or by adding alpha-thrombin in the presence of excess diisopropyl fluorophosphate-inhibited thrombin, suggesting retention of active alpha-thrombin by a receptor-mediated mechanism. Morphological data and the results of [14C]serotonin release studies indicate that platelets are activated by alpha-thrombin-pretreated endothelium and that adherence represents aggregates of activated platelets as well as individual platelets. Adherence on alpha-thrombin-pretreated endothelium is dependent on divalent cations. Platelets also adhered to aortic segments pretreated with thrombin. The data of the current studies support the contention that alpha-thrombin can promote adherence of activated platelets to endothelial cells because of the binding and retention of alpha-thrombin to endothelial cells in a manner in which it remains active and available for platelet activation.
The endothelial cells lining the vessel wall can modulate vasomotor tone by releasing vasoactive factors, such as endothelial-derived constricting factors. We observed that a-thrombin, but not catalytically inactivated a-thrombin, mediated the release of two pulmonary vasoconstrictor peptides into the venous effluent of guinea pig lungs. These peptides elicited a slow-onset, long-lasting pulmonary vasoconstriction similar to the effect of endothelin, an endothelial-derived 21-amino acid vasoconstrictor peptide previously isolated from cells in culture. One of the isolated peptides coelutes with endothelin upon reverse-phase HPLC with an acetonitrile gradient and has a molecular weight comparable to endothelin as determined by gel-permeation HPLC. The other vasoconstrictor peptide elutes earlier than endothelin on reverse-phase HPLC and exhibits a lower molecular weight. The studies show the release of endothelin-like pulmonary vasoconstrictor peptides in the intact lung by a-thrombin, a central regulatory enzyme in hemostasis.The vascular endothelium produces vasodilators, prostacyclin and the endothelial-derived relaxing factor (1, 2), and vasoconstrictors collectively termed endothelial-derived constrictor factors (EDCF) (3). One EDCF has been identified as endothelin, a 21-amino acid peptide initially isolated from cultured porcine aortic endothelial cells (4). Originally the human and porcine endothelins were found to have identical sequences (5), and the DNA sequence of the rat gene indicated 6 amino acid replacements in the aminoterminal half of the molecule (6). The two intrachain disulfide bonds and the hydrophobic carboxyl terminus [which appear necessary for the vasoconstrictor activity (7)] were conserved. More recently, three genes have been identified for endothelin in a human DNA library by Inoue et al. (8). These authors have suggested the nomenclature of ET-1 to designate the original porcine/human endothelin, ET-2, which has a Trp6,Leu7 substitution, and ET-3, the peptide initially designated as rat endothelin (8).Endothelin is released constitutively by cultured aortic endothelial cells (4). The endothelin-induced vasoconstriction is not inhibitied by a-adrenergic, cholinergic, histaminergic, and serotonergic antagonists (3,4), nor by cyclooxygenase or lipoxygenase inhibitors (4). The vasoconstrictor response requires extracellular Ca2", is associated with increased intracellular free Ca2", and may be inhibited by dihydropyridine Ca2l channel antagonists (4,9). A number of stimuli and vasoactive mediators such as thrombin, norepinephrine, or the Ca2l ionophore A23187 stimulate endothelin mRNA transcription in vitro (4). Endothelin may participate in hypertensive responses (10-13) because of its vasoconstrictor properties. However, whether endothelin or endothelin-like peptides are released in vivo is not known. In this study, we examined whether endothelin or endothelin-like peptides are released in the intact circulation. Studies were made in perfused guinea pig lungs challenged with a...
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