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Braz. j. med. biol. res ; 47(10): 826-833, 10/2014. graf
Article in English | LILACS | ID: lil-722174


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.

Animals , Male , Muscle, Smooth, Vascular/physiology , Myosin Light Chains/metabolism , Protein Processing, Post-Translational/physiology , Vasoconstriction/physiology , Aorta, Thoracic , Acetylglucosamine/analogs & derivatives , Acetylglucosamine/pharmacology , Acylation/drug effects , Acylation/physiology , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Azepines/pharmacology , Blotting, Western , Enzyme Inhibitors/pharmacology , Hypoglycemic Agents/pharmacology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myosin-Light-Chain Kinase/metabolism , Myosin-Light-Chain Phosphatase/metabolism , Oxazoles/pharmacology , Oximes/pharmacology , Phenylcarbamates/pharmacology , Phenylephrine/agonists , Phosphorylation/drug effects , Phosphorylation/physiology , Rats, Wistar , Ribonucleotides/pharmacology , Vasoconstriction/drug effects , Vasoconstrictor Agents/pharmacology , beta-N-Acetylhexosaminidases/antagonists & inhibitors
Braz. j. med. biol. res ; 44(11): 1080-1087, Nov. 2011. ilus
Article in English | LILACS | ID: lil-604269


Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

Humans , Calcium Channels/metabolism , Calcium Signaling/physiology , Calcium/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Cardiovascular Diseases/metabolism , Lung Diseases/metabolism
Braz. j. med. biol. res ; 42(11): 1058-1067, Nov. 2009. ilus
Article in English | LILACS | ID: lil-529110


Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 µM) or indomethacin (10 µM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 µM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 µM, a nonselective K+ channel blocker), Tram-34 (10 µM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 µM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20°C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of α-1 and α-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.

Animals , Male , Mice , Endothelium, Vascular/enzymology , Hypertension/physiopathology , Mesenteric Arteries/enzymology , Sodium-Potassium-Exchanging ATPase/physiology , Vascular Resistance/physiology , Endothelium, Vascular/physiology , Enzyme Inhibitors/pharmacology , Hypertension/chemically induced , Mesenteric Arteries/physiology , Ouabain/pharmacology