A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles.
Phosphorylation of the 20-kDa myosin regulatory light chains (LC(20)) plays a key role in the regulation of smooth muscle contraction. The level of LC(20) 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 LC(20) phosphorylation in the renal microcirculation. The available pharmacological probes are often nonspecific, and current techniques to directly measure LC(20) 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 Mn(2+)-phosphate-binding tag and enhanced Western blot analysis, we were able to detect LC(20) phosphorylation using as little as 5 pg (250 amol) of isolated LC(20). Phosphorylated and unphosphorylated LC(20) were detected in single isolated afferent arterioles, and LC(20) phosphorylation levels could be accurately quantified in pooled samples of three arterioles (<300 cells). The phosphorylation level of LC(20) 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 LC(20) phosphorylation in afferent and efferent arterioles and provide insights into the signaling pathways that regulate LC(20) phosphorylation in the renal microvasculature under physiological and pathophysiological conditions.
Takeya
Kosuke
K
Department of Pharmacology, Univ. of Calgary Faculty of Medicine, Smooth Muscle Research Group, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada. ktakeya@ucalgary.ca
Loutzenhiser
Kathy
K
Shiraishi
Mitsuya
M
Loutzenhiser
Rodger
R
Walsh
Michael P
MP
D000818:Animals; D001160:Arterioles / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D007668:Kidney / Q000098:blood supply* / Q000378:metabolism; D007834:Lasers; D049449:Luminescence; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar
D018994:Myosin Light Chains