Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. Wehypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase(MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading toincreased vascular contractile responsiveness. The vascular responses were measuredby isometric force displacement. Thoracic aorta and vascular smooth muscle cells(VSMCs) from rats were incubated with vehicle or with PugNAc, which increasesO-GlcNAcylation. In addition, we determined whether proteins that play an importantrole in the regulation of MLCK and MLCP activity are directly affected byO-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximaleffect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCPinhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolishedthe differences in PE-response between the groups. The effect of PugNAc was notobserved when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed thatdifferences in the PE-induced contractile response between the groups were abolishedby the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatasetarget subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosinphosphatase activity. PugNAc incubation produced a time-dependent increase invascular phosphorylation of myosin light chain and decreased phosphorylation levelsof AMP-activated protein kinase, which decreased the affinity of MLCK forCa²⁺/calmodulin. Our data suggest that proteins that play an importantrole in the regulation of MLCK and MLCP activity are directly affected byO-GlcNAcylation, favoring vascular contraction.