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

Abstract

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.

Myosin Regulatory Light Chain Diphosphorylation Slows Relaxation of Arterial Smooth Muscle

“…Samples (40 l) were subjected to phosphate affinity SDS-PAGE using an acrylamide-pendant phosphate-binding tag (Phos-tag SDS-PAGE with 12.5% acrylamide) at 30 mA/gel for 70 min in mini-gels in which 0.05 mM Phos-tag acrylamide (NARD Institute, Japan) and 0.1 mM MnCl 2 were incorporated into the running gel (49). Separated proteins were transferred to PVDF membranes (Roche Applied Science) overnight at 27 volts and 4°C in 25 mM Tris-HCl, pH 7.5, 192 mM glycine, 10% (v/v) methanol.…”

Rho-associated kinase plays a role in rabbit urethral smooth muscle contraction, but not via enhanced myosin light chain phosphorylation

“…Nonphosphorylated and phosphorylated forms of LC20 were separated by Phos-tag SDS-PAGE (23,35). Muscle protein samples stored in SDS-PAGE sample buffer at Ϫ20°C were electrophoresed at 30 mA/gel for 60 min in minigels in which 0.05 mM Phos-tag acrylamide (NARD Institute, Amagasaki, Japan) and 0.1 mM MnCl2 were incorporated into the running gel.…”

Impaired Feedback Regulation of the Receptor Activity and the Myofilament Ca2+ Sensitivity Contributes to Increased Vascular Reactiveness after Subarachnoid Hemorrhage

“…Phos-tag is a compound that specifically binds to a phosphate group. Therefore, SDS-PAGE containing polyacrylamide-bound Mn 2 + Phos-tag (Phos-tag SDS-PAGE) causes a mobility shift in protein, depending on the degree of phosphorylation (Takeya et al, 2008). The samples for analysis were obtained during the measurement of tension in the intact preparations.…”