Differential effects of myosin light chain kinase inhibition on contractility, force development and myosin light chain 20 phosphorylation of rat cervical and thoracic duct lymphatics
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The intrinsic contractile activity of lymphatics varies in different regions of the body. We have previously shown that cervical lymphatics possess an inherently higher frequency but lower tone at a given pressure when compared to thoracic duct lymphatics. However, the molecular mechanisms modulating the contractile characteristics of these lymphatics are not well understood. Since myosin light chain 20 (MLC(20)) phosphorylation appears to underlie the tonic component of lymphatic contraction, we hypothesized that the thoracic duct would be more sensitive to the modulation of MLC(20) phosphorylation when compared to cervical lymphatics. To test our hypothesis, the contractile activities and MLC(20) phosphorylation of thoracic duct and cervical lymphatics were determined in the absence or presence of the specific myosin light chain kinase (MLCK) inhibitor ML-7 under both isobaric and isometric conditions. Addition of ML-7 at each concentration tested led to a decrease in tone in both vessel types. While ML-7 (10(-6) m) significantly reduced the phasic contraction frequency of cervical lymphatics, it completely stopped phasic contractions of thoracic duct at that concentration. Under isometric conditions the active peak and plateau components of tension were both significantly higher in thoracic duct compared to cervical lymphatics. ML-7 (10(-5) m) significantly decreased both the active peak and plateau tensions of thoracic duct, whereas only the active peak tension of cervical lymphatics was decreased. In thoracic duct MLC(20) di-phosphorylation, but not mono-phosphorylation, was significantly decreased with increasing transmural pressure, whereas in cervical vessels only at the higher pressures tested did MLC(20) di-phosphorylation decrease. ML-7 treatment of the thoracic duct caused a significant decrease in both the mono- and di-phosphorylated forms of MLC(20). However, in cervical vessels ML-7 treatment produced an increase in the mono-phosphorylated MLC(20) form while di-phosphorylated MLC(20) was significantly decreased. These data indicate that thoracic duct has an enhanced sensitivity to MLCK inhibition when compared to cervical lymphatics and while the status of the mono- and di-phosphorylation forms of MLC(20) affects both tonic and phasic components of lymphatic contractions, the pressure-dependent changes in tonic contractions are modulated by the status of the di-phosphorylation of MLC(20) in the lymphatics.
author list (cited authors)
Nepiyushchikh, Z. V., Chakraborty, S., Wang, W., Davis, M. J., Zawieja, D. C., & Muthuchamy, M.