Abstract
Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle. The rate of phosphorylation of MLCK is slower than the rates of increase in cytosolic Ca2+ concentrations and phosphorylation of the regulatory light chain of myosin in intact tracheal smooth muscle cells in culture. In permeable cells, increasing the Ca2+ concentration increased the extent of myosin light chain and MLCK phosphorylation. The Ca2+ concentration required for half-maximal phosphorylation was 500 nM for MLCK and 250 nM for myosin light chain. Addition of KN-62 or a synthetic peptide CK II, inhibitors of multifunctional Ca2+/calmodulin-dependent protein kinase II activity, abolished MLCK phosphorylation. Under these conditions, the Ca2+ concentration required for half-maximal light chain phosphorylation decreased to 170 nM. Thus, the Ca2+ concentrations required for MLCK phosphorylation are greater than those required for light chain phosphorylation in smooth muscle cells. Furthermore, phosphorylation of MLCK decreases the Ca2+ sensitivity of light chain phosphorylation. These results can be explained by a regulatory scheme in which calmodulin available for myosin light chain kinase activation is limiting. This is supported by the retention of calmodulin when tracheal smooth muscle cells and tissues are permeabilized in relaxing solution and by the low mobility of rhodamine-calmodulin in intact tracheal smooth muscle cells.
Highlights
Myosin light chain kinase (MLCK) is phosphorylated be a primary determinantof light chainphosphorylation
Preincubation of permeable cells with the CK I1 peptide (10p ~10, min) inhibited the Caz+-dependentdecrease in the myosin light chain kinase activity ratio (Fig. 8). This response was associated with an increase in the extentof myosin light chain phosphorylation (Fig. 8).These results aresimilar to the results obtained with KN-62
The findings obtained in these studiesprovide evidencethat the decrease in the myosin light chain kinase activity ratio associated with an increase in KCaMis dependent on increases in [Ca2+Jiin smooth muscle cells and is probably mediated via phosphorylation by CaM kinase 11
Summary
Cells-The recells to stimuwith Labview software (National Instruments, AusTtXin),on a MacIn- lation with endothelin(10 m)is illustrated inFig. 1. Ca2+-Values of myosin light chain kinase activity ratio and light chainphosphorylation at different times after increasing [Ca"], in smoothmuscle cells made permeable withp-escin for 15 minareillustratedin Fig. 2. The value for calmodulin content in muscle cells with KN-62 (1.5 p ~ 3,0 min) abolished the CaZ+intact tracheal tissue (39 PM)compares favorably with values dependent decrease in the myosin light chain kinase activity reported for other smooth muscles (Ruegg et al, 1984). Preincubation of permeable cells with the CK I1 peptide (10p ~10, min) inhibited the Caz+-dependentdecrease in the myosin light chain kinase activity ratio (Fig. 8). This response was associated with an increase in the extentof myosin light chain phosphorylation (Fig. 8).These results aresimilar to the results obtained with KN-62
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
