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

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.

O‑GlcNAcylation contributes to intermittent hypoxia‑associated vascular dysfunction via modulation of MAPKs but not CaMKII pathways

“…The alterations in OGA activity may interfere with the contractile apparatus required for PE-induced contraction via MAPK signaling, since MAPK blockade had no effects on vasoconstrictor sensitivity to PE in the vessels that had not received PugNAc treatment. This could be proven by preliminary data showing that PugNAc increases contractions to PE through the activation of the Ras homolog family member A (RhoA)/Rho-kinase pathway ( 47 ). In addition, an interaction between ERK1/2 and Rho-kinase has been reported, where ERK1/2 activation depends on Rho-kinase signaling ( 48 ).…”

“…With respect to arterial hypertension and the associated vascular abnormality, several specific pathways, such as PKC, MAPK, PI3K/Akt and RhoA/Rho-kinase, are targets for O-GlcNAcylation ( 16 , 17 , 21 ). It has been proposed that O-GlcNAc modification of contractile regulators downstream of those specific cascades, such as MLC kinase, MYPT1 and MLC, can interfere with their phosphorylated activities, thus directly influencing vascular smooth muscle function ( 16 , 17 , 47 , 49 ).…”

O-Linked β-N-Acetylglucosamine Modification: Linking Hypertension and the Immune System

“…In this sense, there is direct evidence that elevated levels of protein O-GlcNAcylation in endothelial cells impairs endothelium-dependent relaxation ( 44 , 47 – 49 ), demonstrating that O-GlcNAcylation leads to endothelial dysfunction. Furthermore, O-GlcNAcylation of vascular smooth muscle cells augments vascular response to contractile agonists ( 50 , 51 ) and favors vascular calcification ( 52 – 54 ), resulting in high blood pressure ( 25 ).…”

Protein O‐GlcNAcylation in the heart

“…Increased protein O-GlcNAcylation by PUGNAc in rat thoracic aorta and vascular smooth muscle cells is also associated with augmented vascular constriction induced by phenylephrine. 109,110 Reduced O-GlcNAc level in aortic smooth muscle cells prevents glucosamine-induced vessel constriction 111 and downregulates matricellular protein thrombospondin-1 (TSP-1) expression. 105 TSP-1 is a potent pro-atherogenic protein that is upregulated in diabetic patients and animal models, and involved in vascular wall injury and atherosclerotic lesions.…”