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Monocrotaline pyrrole interacts with actin and increases thrombin-mediated permeability in pulmonary artery
D W Wilson1, M W Lamé, S K Dunston
1Departments of Veterinary Pathology, Microbiology, and Immunology, University of California at Davis, Davis, California 95616, USA.
Abstract:
One of the earliest morphologic changes evident in the monocrotaline (MCT) model of pulmonary hypertension in rats is microvascular leak. Whether this represents a direct effect of MCT metabolites or is secondary to inflammatory and thrombotic changes remains uncertain. To determine whether MCT directly affects endothelial cell permeability barrier function, we characterized the interaction of the reactive pyrrole intermediate of MCT (MCTP) with endothelial cell actin and characterized its effects on thrombin-mediated signal transduction and monolayer permeability. Bovine pulmonary endothelial cells (BPAEC) treated with MCTP had altered distribution of filamentous actin evident by fluorescence microscopy. Correlative Western blots and autoradiography of actin isolated from BPAEC treated with 14C-MCTP showed comigration of actin and MCTP-derived 14C. MCTP treatment did not alter cellular free Ca2+ concentrations nor did it interfere with thrombin-mediated intracellular Ca2+ signal. Pretreatment with MCTP significantly augmented the thrombin-mediated transudation of Evan's blue albumin in BPAEC monolayers apparently by increasing the size of intercellular gaps. We conclude that MCTP directly interacts with actin to alter its polymerization state but does not significantly affect endothelial cell response to contractile stimulus. Our results suggest that MCTP may affect endothelial cell barrier function through alterations in intracellular junctions.
Insights
Monocrotaline metabolite (MCTP) directly disrupts endothelial cell actin, increasing microvascular leak in pulmonary hypertension models. This occurs independently of calcium signaling, suggesting MCTP alters cell junctions.
Area of Science:
- Pulmonary Hypertension Research
- Endothelial Cell Biology
- Toxicology
Background:
- Pulmonary hypertension (PH) involves early microvascular leak in the monocrotaline (MCT) model.
- The precise mechanism of MCT-induced leak, whether direct or indirect, remains unclear.
Purpose of the Study:
- To investigate if MCT metabolites directly impact endothelial cell barrier function.
- To characterize the interaction of MCT pyrrole (MCTP) with endothelial actin and its effect on permeability.
Main Methods:
- Bovine pulmonary artery endothelial cells (BPAEC) were treated with MCTP.
- Actin distribution was assessed via fluorescence microscopy.
- MCTP-actin interaction was confirmed using 14C-MCTP and Western blotting.
- Effects on calcium signaling and thrombin-induced permeability were evaluated.
Main Results:
- MCTP altered filamentous actin distribution in BPAEC.
- 14C-MCTP co-migrated with actin, indicating direct interaction.
- MCTP did not affect intracellular calcium levels or thrombin signaling.
- MCTP pretreatment enhanced thrombin-induced albumin leakage by widening intercellular gaps.
Conclusions:
- MCTP directly interacts with endothelial actin, affecting its polymerization state.
- MCTP impairs endothelial barrier function independent of contractile signaling.
- MCTP likely disrupts endothelial barrier integrity via alterations in intercellular junctions.