New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation

Sandra Puetz,Lena Reimann,Bettina Warscheid,Gabriele Pfitzer,Mechthild M Schroeter,Mona S Hunger,Lubomir T Lubomirov,Heike Piechura,Doris Metzler

doi:10.1007/s10974-012-9306-9

Abstract

Nitrovasodilators and agonists, via an increase in intracellular cyclic nucleotide levels, can induce smooth muscle relaxation without a concomitant decrease in phosphorylation of the regulatory light chains (RLC) of myosin. However, since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca2+], and hence, a decreased activity of MLCK, we tested the hypothesis that the site responsible for the elevated RLC phosphorylation is not Ser19. Smooth muscle strips from gastric fundus were isometrically contracted with ET-1 which induced an increase in monophosphorylation from 9 ± 1 % under resting conditions (PSS) to 36 ± 1 % determined with 2D-PAGE. Electric field stimulation induced a rapid, largely NO-mediated relaxation with a half time of 8 s, which was associated with an initial decline in RLC phosphorylation to 18 % within 2 s and a rebound to 34 % after 30 s whereas relaxation was sustained. In contrast, phosphorylation of RLC at Ser19 probed with phosphospecific antibodies declined in parallel with force. LC/MS and western blot analysis with phosphospecific antibodies against monophosphorylated Thr18 indicate that Thr18 is significantly monophosphorylated during sustained relaxation. We therefore suggest that (i) monophosphorylation of Thr18 rather than Ser19 is responsible for the phosphorylation rebound during sustained EFS-induced relaxation of mouse gastric fundus, and (ii) that relaxation can be ascribed to dephosphorylation of Ser19, the site considered to be responsible for regulation of smooth muscle tone.

Highlights

There is general consent that phosphorylation of the 20 kDa regulatory light chain (RLC) of myosin at Ser19 is a prerequisite for the actin activation of MgATPase activityJ Muscle Res Cell Motil (2012) 33:471–483 of smooth muscle myosin
Since cyclic nucleotide-induced relaxation is associated with a decrease in intracellular [Ca2?], and a decreased activity of MLCK, we tested the hypothesis that the site responsible for the elevated regulatory light chains (RLC) phosphorylation is not Ser19
Not unequivocally found several authors reported that relaxation mediated by NO-donors and isoprenaline of different types of smooth muscle tissues from different species occurs without dephosphorylation of RLC or that dephosphorylation was only transient leading to the statement of Kate and Michael Barany: 0it can be concluded that RLC dephosphorylation is not a prerequisite of smooth muscle relaxation0 (Barany and Barany 1993)

Summary

Introduction

There is general consent that phosphorylation of the 20 kDa regulatory light chain (RLC) of myosin at Ser is a prerequisite for the actin activation of MgATPase activityJ Muscle Res Cell Motil (2012) 33:471–483 of smooth muscle myosin (reviewed in Kamm and Stull 1985; Pfitzer 2001; Somlyo and Somlyo 2003). In 32P labelled intact carotid arteries were associated with a concomitant increase in RLC phosphorylation (Barron et al 1979) which is reversed upon relaxation (Barron et al 1980) These proof-of-concept yet correlative studies were complemented by skinned fibre experiments in which a contraction could be elicited in the absence of Ca2? Many laboratories showed that contractions elicited by a variety of agonists in different types of smooth muscle from different species are associated with an increase in RLC phosphorylation (reviewed in Arner and Pfitzer 1999; Kamm and Stull 1985; Kim et al 2008; Somlyo and Somlyo 2003)

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Conclusion