Highly sensitive myosin phosphorylation analysis in the renal afferent arteriole

The regulation of smooth muscle contraction and relaxation involves phosphorylation and dephosphorylation of regulatory proteins, particularly myosin. To elucidate the regulatory mechanisms, analyzing the phosphorylation signal transduction is crucial. Although a pharmacological approach with selective inhibitors is sensitive and a useful technique, it leads to speculation regarding a signaling pathway but does not provide direct evidence of changes at a molecular level. We developed a highly sensitive biochemical technique to analyze phosphorylation by adapting Phos-tag SDS-PAGE. With this technique, we successfully analyzed myosin light chain (LC(20)) phosphorylation in tiny renal afferent arterioles. In the rat afferent arterioles, endothelin-1 (ET-1) induced diphosphorylation of LC(20) at Ser19 and Thr18 as well as monophosphorylation at Ser19 via ET(B) receptor activation. Considering that LC(20) diphosphorylation can decrease the rate of dephosphorylation and thus relaxation, we concluded that LC(20) diphosphorylation contributes, at least in part, to the prolonged contraction induced by ET-1 in the renal afferent arteriole.