Related Experiment Videos
ATP induces dephosphorylation of myosin light chain in endothelial cells
T Noll1, M Schäfer, U Schavier-Schmitz
1Physiologisches Institut, Justus-Liebig-Universität, D-35392 Giessen, Germany. thomas.noll@physiologie.med.uni-giessen.de
Abstract:
In cultured porcine aortic endothelial monolayers, the effect of ATP on myosin light chain (MLC) phosphorylation, which controls the endothelial contractile machinery, was studied. ATP (10 microM) reduced MLC phosphorylation but increased cytosolic Ca(2+) concentration ([Ca(2+)](i)). Inhibition of the ATP-evoked [Ca(2+)](i) rise by xestospongin C (10 microM), an inhibitor of the inositol trisphosphate-dependent Ca(2+) release from endoplasmic reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC dephosphorylation was prevented in the presence of calyculin A (10 nM), an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activates MLC dephosphorylation in a Ca(2+)-independent manner. In the presence of calyculin A, MLC phosphorylation was incremented after addition of ATP, an effect that could be abolished when cells were loaded with the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid acetoxymethyl ester (10 microM). Thus ATP also activates a Ca(2+)-dependent kinase acting on MLC. In summary, ATP simultaneously stimulates a functional antagonism toward both phosphorylation and dephosphorylation of MLC in which the dephosphorylation prevails. In endothelial cells, ATP is the first physiological mediator identified to activate MLC dephosphorylation by a Ca(2+)-independent mechanism.
Insights
Adenosine triphosphate (ATP) influences endothelial cell contraction by regulating myosin light chain (MLC) phosphorylation. ATP primarily promotes MLC dephosphorylation independently of calcium, but also activates a calcium-dependent kinase.
Area of Science:
- Endothelial cell biology
- Cellular signaling
- Biochemistry
Background:
- Myosin light chain (MLC) phosphorylation regulates endothelial cell contraction.
- Adenosine triphosphate (ATP) is a key signaling molecule in vascular tissues.
- Understanding ATP's role in endothelial function is crucial for cardiovascular research.
Purpose of the Study:
- To investigate the effect of ATP on MLC phosphorylation in porcine aortic endothelial cells.
- To elucidate the role of calcium ions ([Ca(2+)](i)) and protein phosphatases in ATP-mediated MLC phosphorylation.
- To identify the primary mechanism by which ATP modulates endothelial contractile machinery.
Main Methods:
- Primary cell culture of porcine aortic endothelial cells.
- Measurement of MLC phosphorylation and cytosolic calcium concentration ([Ca(2+)](i)).
- Pharmacological inhibition of calcium release (xestospongin C) and protein phosphatases (calyculin A).
Main Results:
- ATP (10 microM) decreased MLC phosphorylation and increased [Ca(2+)](i).
- ATP-induced MLC dephosphorylation occurred independently of the calcium rise.
- Inhibition of protein phosphatases PP-1 and PP-2A by calyculin A revealed a Ca(2+)-dependent kinase activation by ATP.
- ATP exerts a dual effect, promoting dephosphorylation (prevailing) and phosphorylation of MLC.
Conclusions:
- ATP activates MLC dephosphorylation in a Ca(2+)-independent manner in endothelial cells.
- ATP also stimulates a Ca(2+)-dependent kinase that phosphorylates MLC.
- ATP's net effect is to promote MLC dephosphorylation, influencing endothelial contractility through a novel Ca(2+)-independent pathway.