scholarly journals Muscle-Specific F-Box Only Proteins Facilitate BK Channel β 1 Subunit Downregulation in Vascular Smooth Muscle Cells of Diabetes Mellitus

2010 ◽  
Vol 107 (12) ◽  
pp. 1454-1459 ◽  
Author(s):  
Dai-min Zhang ◽  
Tongrong He ◽  
Zvonimir S. Katusic ◽  
Hon-Chi Lee ◽  
Tong Lu
Keyword(s):  
Diabetes Mellitus ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Bk Channel

Abstract TP434: Regulation of BK Channel ß 1 Subunit Expression by Muscle Ring Finger-1 Protein in Diabetic Vessels

Stroke ◽  
2013 ◽  
Vol 44 (suppl_1) ◽  
Author(s):  
Tong Lu ◽  
Yi Fu ◽  
Monte S Willis ◽  
Hon-Chi Lee
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Protein Level ◽  
Ring Finger ◽  
Fold Increase ◽  
Muscle Cells ◽  
Diabetic Mouse ◽  
Bk Channel

BK channels, abundantly expressed in vascular smooth muscle cells (VSMCs), are the key determinant of vascular physiology. BK channel activity is tightly regulated by its accessory β 1 (BK-β 1 ) subunits. BK-β 1 protein expression is down-regulated in diabetic vessels, contributing to diabetic vascular complications including hypertension, stroke and coronary heart disease. However, BK-β 1 mRNA is not reduced in diabetic vessels suggesting increased BK-β 1 turnover. Since ubiquitin-proteasome system (UPS) accounts for 80~90% of intracellular protein degradations in mammalian cells, we hypothesize that muscle ring finger-1 (MuRF-1) protein, the muscle specific E3 ligase, accelerates vascular BK-β 1 degradation in diabetes mellitus. In this study, we found that BK-β 1 protein level was decreased by 1.95±0.14 fold, accompanied by a 2.83±0.16 fold increase of MuRF-1 expression in streptozotocin-induced diabetic mouse vessels (n=3, p<0.05 vs. control). Moreover, response of vascular BK channel activation by DHS-1 (a specific BK-β 1 activator) was absent in diabetic mouse vascular smooth muscle cells (VSMCs). Similar results were observed in human VSMCs cultured with 22 mM glucose (HG) compared to those cultured with 5 mM glucose. Knockdown of MuRF-1 by siRNA (100 nM) produced a 4.2-fold decrease of MuRF-1 protein level and a 1.8-fold increase in BK-β 1 expression in HG-cultured VSMCs. In addition, the total BK-β 1 expression was 2.58±0.04 fold lower but the ubiquitinated BK-β 1 protein was 5.18±0.04 fold higher in HEK293 cells 48-h after transfection with MuRF-1, compared to those with MuRF-1ΔR (an inactive mutant with the Ring structure deletion) transfection (n=3, p<0.05). Immunoprecipitation experiments confirmed that the N-terminus of BK-β 1 was physically associated with MuRF-1. Inhibition of UPS activity by 10 μM MG-132 (a proteasome inhibitor) enhanced BK-β 1 expression by 1.70±0.01 fold (n=3, p<0.05 vs. control). Our results indicate that MuRF-1 participated in ubiquitin-mediated vascular BK-β 1 protein proteasomal degradation and delineated a fundamental mechanism underlying vasculopathy in diabetes.

Isolation of Multiple Types of Plasminogen Activator Inhibitors from Vascular Smooth Muscle Cells

1989 ◽  
Vol 61 (03) ◽  
pp. 517-521 ◽  
Author(s):  
Walter E Laug ◽  
Ruedi Aebersold ◽  
Ambrose Jong ◽  
Willian Rideout ◽  
Barbara L Bergman ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Affinity Purification ◽  
Muscle Cells ◽  
Exchange Chromatography ◽  
Natural Resistance ◽  
Protease Nexin ◽  
Pai 1

SummaryLarge arteries have a natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors. Vascular smooth muscle cells (VSMC) representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture. These cells were found to produce large amounts of inhibitors of plasminogen activators (PA). Fractionation of VSMC-conditioned medium by heparin-affigel chromatography separated three immunologically and functionally distinct PA inhibitors (PAI), namely PAI-1, PAI-2 and protease-nexin I. The three inhibitors were characterized by functional assays and immunoblotting. PA inhibitor 2 (PAI-2) had little affinity for heparin, whereas PA inhibitor 1 (PAI-l) bound to heparin and was eluted from the column at NaCl concentrations of 0. 1 to 0.35 M. Protease-nexin I, eluted at NaCl concentrations of 0.5 M and higher. Most of the PAI-1 was present in the latent, inactive form. PAI-1 was further purified by ion exchange chromatography on a Mono-Q column. Partial sequencing of the purified PAI-1 confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI-1. Thus, human VSMC produce all three presently known PAI and these can be separated in a single heparin affinity purification step.

Thrombin Prevents the Expression of Inducible Nitric Oxide Synthase in Vascular Smooth Muscle Cells by a Proteolytically-Activated Thrombin Receptor

1995 ◽  
Vol 74 (03) ◽  
pp. 980-986 ◽  
Author(s):  
Valérie B Schini-Kerth ◽  
Beate Fißithaler ◽  
Thomas T Andersen ◽  
John W Fenton ◽  
Paul M Vanhoutte ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Inducible Nitric Oxide Synthase ◽  
Muscle Cells ◽  
Thrombin Receptor ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

SummaryProteolytically active forms of thrombin (α- and γ-thrombin) and thrombin receptor peptides inhibited the release of nitrite, a stable endproduct of nitric oxide, evoked by interleukin-1 β(IL-1 β) in cultured vascular smooth muscle cells while proteolytically inactive forms [D-Phe-Pro-Arg chloromethyl ketone-α-thrombin (PPACK-α- thrombin) and diisopropylphosphoryl-α-thrombin (DIP-α-thrombin)] had either no or only minimal inhibitory effects. Under bioassay conditions, perfusates from columns containing IL-1 β-activated vascular smooth muscle cells or cells treated with IL-1βplus PPACK-α-thrombin relaxed detector blood vessels. These relaxations were abolished by the inhibitor of nitric oxide synthesis, NG-nitro-L arginine. No relaxations were obtained with untreated cells or IL-1 β-treated cells in the presence of α-thrombin. The expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells by IL-1 β was impaired by α-thrombin. These results demonstrate that thrombin regulates the expression of the inducible nitric oxide synthase at a transcriptional level via the proteolytic activation of the thrombin receptor in vascular smooth muscle cells

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