The key event which regulates the contractile activity of smooth muscle is the reversible phosphorylation of the regulatory light chains of myosin (RLC) on Ser19. However, several groups reported independently that cAMP as well as cGMP induced relaxation was uncoupled from RLC phosphorylation seemingly violating the phosphorylation theory of regulation of smooth muscle contraction. It was postulated that smooth muscle relaxation involves RLC phosphorylation independent mechanisms. However, none of these previous investigators determined which amino acid residue of RLC is phosphorylated during relaxation and it should be remembered that RLC can be phosphorylated on several residues. Therefore, we determined which site is phosphorylated during cGMP/cAMP induced relaxation using gastric fundus smooth muscle as model. Consistent with the previous reports we found that during sustained relaxation RLC phosphorylation determined with 2D-PAGE did not decline. However, western blot analysis with phosphospecific antibodies in combination with LC/MS analysis revealed, that this was due to phosphorylation of Thr18 whereas phosphorylation of Ser19 declined in parallel with relaxation as predicted by the phosphorylation theory. To conclude, the apparent uncoupling between cyclic nucleotide induced relaxation and RLC phosphorylation is due to an increase in phosphorylation of Thr18, and in keeping with the phosphorylation theory, Ser19, the site responsible for regulating smooth muscle actomyosin interaction, is dephosphorylated. The functional relevance of Thr18 phosphorylation remains elusive at present.

Supported by the DFG