AJP - Renal AJP: Lung Cellular and Molecular Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 QUICK SEARCH:   [advanced]


     


Am J Physiol Renal Physiol 294: F1487-F1492, 2008. First published April 9, 2008; doi:10.1152/ajprenal.00060.2008
0363-6127/08 $8.00
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/6/F1487    most recent
00060.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takeya, K.
Right arrow Articles by Walsh, M. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takeya, K.
Right arrow Articles by Walsh, M. P.

INNOVATIVE METHODOLOGY

A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles

Kosuke Takeya,1,2 Kathy Loutzenhiser,1 Mitsuya Shiraishi,2 Rodger Loutzenhiser,1 and Michael P. Walsh2

Departments of 1Pharmacology and Therapeutics and 2Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Smooth Muscle Research Group, Calgary, Alberta, Canada

Submitted 4 February 2008 ; accepted in final form 2 April 2008

Phosphorylation of the 20-kDa myosin regulatory light chains (LC20) plays a key role in the regulation of smooth muscle contraction. The level of LC20 phosphorylation is governed by the relative activities of myosin light chain kinase and phosphatase pathways. The regulation of these two pathways differs in different smooth muscle types and in the actions of different vasoactive stimuli. Little is known concerning the regulation of LC20 phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure LC20 phosphorylation are not sensitive enough for quantification in small arterioles. We describe here a novel approach to address this important issue. Using SDS-PAGE with polyacrylamide-bound Mn2+-phosphate-binding tag and enhanced Western blot analysis, we were able to detect LC20 phosphorylation using as little as 5 pg (250 amol) of isolated LC20. Phosphorylated and unphosphorylated LC20 were detected in single isolated afferent arterioles, and LC20 phosphorylation levels could be accurately quantified in pooled samples of three arterioles (<300 cells). The phosphorylation level of LC20 in the afferent arteriole was 6.8 ± 1.7% under basal conditions and increased to 34.7 ± 5.1% and 44.6 ± 6.6% in response to 30 mM KCl and 10–8 M angiotensin II, respectively. The application of this technique will enable investigations of the different determinants of LC20 phosphorylation in afferent and efferent arterioles and provide insights into the signaling pathways that regulate LC20 phosphorylation in the renal microvasculature under physiological and pathophysiological conditions.

afferent arteriole; Phos-tag SDS-PAGE



Address for reprint requests and other correspondence: K. Takeya, Univ. of Calgary Faculty of Medicine, Smooth Muscle Research Group, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada (e-mail: ktakeya{at}ucalgary.ca)




This article has been cited by other articles:


Home page
J. Physiol.Home page
R. P. Johnson, A. F. El-Yazbi, K. Takeya, E. J. Walsh, M. P. Walsh, and W. C. Cole
Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine-855 by Rho kinase contributes to the arterial myogenic response
J. Physiol., June 1, 2009; 587(11): 2537 - 2553.
[Abstract] [Full Text] [PDF]


Home page
Mol. Pharmacol.Home page
E. Ihara, P. L. Beck, M. Chappellaz, J. Wong, S. A. Medlicott, and J. A. MacDonald
Mitogen-Activated Protein Kinase Pathways Contribute to Hypercontractility and Increased Ca2+ Sensitization in Murine Experimental Colitis
Mol. Pharmacol., May 1, 2009; 75(5): 1031 - 1041.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Gui, X.-L. Zheng, J. Zheng, and M. P. Walsh
Inhibition of rat aortic smooth muscle contraction by 2-methoxyestradiol
Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H1935 - H1942.
[Abstract] [Full Text] [PDF]




HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.