Abstract
O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.
Highlights
Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate blood flow to all tissues and organs through contraction and relaxation
We determined whether proteins that play an important role in the regulation of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activity are directly affected by O-GlcNAcylation
O-GlcNAcylation is an innovative way to think about signaling events both in physiological conditions and in disease states
Summary
Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate blood flow to all tissues and organs through contraction and relaxation. A variety of contractile proteins, ion channels and signaling molecules regulate contraction of VSMCs, but the phosphorylation/ dephosphorylation of myosin light chain (MLC) is a key event in the regulation of smooth muscle contraction. The binding of calcium (Ca2+) by calmodulin activates smooth muscle myosin light chain kinase (MLCK) by inducing a conformational change that allows MLCK to phosphorylate the 20-kDa regulatory MLC at serine [1,2]. Ca2+-dependent phosphorylation of MLC is followed by a Rho-kinase-mediated Ca2+ sensitization process, which maintains the contracted state of VSMCs by inhibiting MLC phosphatase (MLCP) activity via phosphorylation of the MLCP target subunit (MYPT-1) [2,3,4]. The phosphorylation state of MLC depends on the balance between MLCK, which phosphorylates MLC leading to smooth muscle cell contraction, and MLCP, which dephosphorylates MLC, resulting in smooth muscle.
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