Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells.

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.

A potent tryptase inhibitor nafamostat mesilate dramatically suppressed pulmonary dysfunction induced in rats by a radiographic contrast medium.

(1) Intravenous injection of ioxaglate (4 g iodine kg(-1)), an iodinated radiographic contrast medium, caused a marked protein extravasation, pulmonary oedema and a decrease in the arterial partial oxygen pressure in rats. (2) All of these reactions to ioxaglate were reversed by the pretreatment with gabexate mesilate (10 and 50 mg kg(-1), 5 min prior to injection) or nafamostat mesilate (3 and 10 mg kg(-1)), in which the inhibition was complete after injection of nafamostat mesilate (10 mg kg(-1)). (3) Both gabexate mesilate and nafamostat mesilate inhibited the activity of purified human lung tryptase, although the latter compound was far more potent than the former. (4) Ioxaglate enhanced the nafamostat-sensitive protease activity in the extracellular fluid of rat peritoneal mast cell suspensions. (5) Tryptase enhanced the permeability of protein through the monolayer of cultured human pulmonary arterial endothelial cells. Ioxaglate, when applied in combination with rat peritoneal mast cells, also produced the endothelial barrier dysfunction. These effects of tryptase and ioxaglate were reversed by nafamostat mesilate. (6) Consistent with these findings, immunofluorescence morphological analysis revealed that tryptase or ioxaglate in combination with mast cells increased actin stress fibre formation while decreasing VE-cadherin immunoreactivity. Both of these actions of tryptase and ioxaglate were reversed by nafamostat mesilate. (7) These findings suggest that tryptase liberated from mast cells plays a crucial role in the ioxaglate-induced pulmonary dysfunction. In this respect, nafamostat mesilate may become a useful agent for the cure or prevention of severe adverse reactions to radiographic contrast media.

Calcium-dependent injury of human microvascular endothelial cells induced by a variety of iodinated radiographic contrast media.

RATIONALE AND OBJECTIVES: The aim of the present study was to determine the possible mechanisms underlying the endothelial cell damage induced by iodinated radiographic contrast materials (RCM). METHODS: The cultured human skin microvascular endothelial cells (HMVECs) were exposed to various contrast media, and the cell viability was measured by mitochondrial enzyme activity. Nuclear damage was assessed by Hoechst 33342 staining and a fluorescent single-cell gel electrophoresis. The effects of contrast materials on the cellular ATP content and intracellular free Ca2+ concentration were subsequently examined. RESULTS: Although the iodinated RCM tested all caused the cell injury in HMVECs, ionic RCM including amidotrizoate and ioxaglate were more potent in producing the cell damage than nonionic RCM. It is unlikely that the contrast material-induced cell damage is associated with hyperosmolality, since hyperosmolar solution of mannitol or NaCl had no marked influence on the endothelial cell viability. Nuclear damage was noted in cells exposed to amidotrizoate. Amidotrizoate lowered cellular ATP content while elevating the intracellular free Ca2+ concentration. It was notable that the RCM-induced endothelial cell damage was reversed by the chelation of intracellular Ca2+ with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the removal of extracellular Ca2+. CONCLUSIONS: Both ionic and nonionic contrast materials caused nuclear damage of endothelial cells. The decrease in tissue ATP content and elevation of intracellular Ca2+ are likely to contribute to the contrast materials-induced endothelial cell damage.