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Thrombin inhibits myosin light chain dephosphorylation in endothelial cells
D M Shasby1, T Stevens, D Ries
1Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242, USA.
Abstract:
Histamine and thrombin increase myosin light-chain kinase-mediated phosphorylation of myosin light chain (MLC) in human umbilical vein endothelial cells (HUVEC). The increase in MLC phosphorylation caused by thrombin persists longer (330 min) than the increase caused by histamine (<5 min), although both increase cell calcium similarly. We hypothesized that some of the longer duration of the increase in MLC phosphorylation caused by thrombin was because of inhibition of myosin dephosphorylation by thrombin. Calyculin A, an inhibitor of type 1 and 2A protein phosphatases, caused a time-dependent increase in MLC phosphorylation in unstimulated HUVEC. As thrombin-stimulated phosphorylation approached its peak at 15 min, calyculin A caused progressively less of an increase in MLC phosphorylation in thrombin-stimulated HUVEC, and no increase at the peak of thrombin stimulation. In HUVEC in which cell calcium was maintained at 600 nM, thrombin increased MLC phosphorylation above the level caused by increased calcium alone at a time coinciding with the peak of thrombin stimulation. However, when phosphatase activity was already inhibited with calyculin A, thrombin did not further increase MLC phosphorylation in cells in which calcium was maintained at 600 nM calcium. Thrombin increases MLC phosphorylation in HUVEC not only by increasing cell calcium but also by inhibiting calyculin A-sensitive dephosphorylation of MLC.
Insights
Thrombin prolongs myosin light-chain phosphorylation in endothelial cells by inhibiting dephosphorylation, unlike histamine. This suggests thrombin actively suppresses myosin dephosphorylation pathways.
Area of Science:
- Endothelial cell biology
- Molecular signaling
- Vascular physiology
Background:
- Histamine and thrombin elevate myosin light-chain phosphorylation (MLC) in human umbilical vein endothelial cells (HUVEC).
- Thrombin's effect on MLC phosphorylation is significantly longer-lasting than histamine's, despite similar calcium increases.
- The prolonged effect of thrombin suggests a mechanism beyond calcium signaling, potentially involving altered dephosphorylation rates.
Purpose of the Study:
- To investigate the role of myosin dephosphorylation in the differential duration of MLC phosphorylation induced by thrombin and histamine.
- To determine if thrombin inhibits protein phosphatases responsible for MLC dephosphorylation.
Main Methods:
- Utilized Calyculin A, a protein phosphatase inhibitor, to assess its impact on MLC phosphorylation in HUVEC.
- Compared MLC phosphorylation levels in response to thrombin and histamine under varying conditions, including phosphatase inhibition and controlled calcium levels.
- Measured MLC phosphorylation over time in response to stimuli and phosphatase inhibition.
Main Results:
- Calyculin A increased MLC phosphorylation in unstimulated HUVEC, confirming its inhibitory effect on phosphatases.
- Thrombin's ability to further increase MLC phosphorylation was diminished when phosphatases were inhibited by Calyculin A.
- Thrombin increased MLC phosphorylation independently of calcium levels by inhibiting calyculin A-sensitive dephosphorylation.
Conclusions:
- Thrombin enhances MLC phosphorylation not only by increasing intracellular calcium but also by actively inhibiting the dephosphorylation of MLC.
- This inhibition of myosin dephosphorylation by thrombin contributes to the prolonged elevation of MLC phosphorylation observed in HUVEC.
- The findings reveal a dual mechanism for thrombin-induced MLC phosphorylation, involving both activation and deactivation pathway modulation.