Mg(2+),ATP-dependent plasma membrane calcium pump of smooth muscle cells. I. Structural organization and properties

Objective: Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses PPIase activity and scaffold function. CyPA regulates Angiotensin II (Ang II) induced reactive oxygen species (ROS) production in vascular smooth muscle cells. However, the mechanism of this CyPA regulation remains unclear. We hypothesized that CyPA regulates plasma membrane translocation of NADPH oxidase cytosolic subunit, p47phox, which is required for NADPH oxidase structural organization and activity. Methods and results: Immunofluorescence studies in rat aortic smooth muscle cells revealed that CyPA translocated from the cytosol to the plasma membrane in response to Ang II in a time dependent manner with a peak at 10min (46.4±5.4 fold increase). Mouse Aortic Smooth Muscle Cells (MASM) were isolated from mice lacking CyPA (CyPA-/-) and wild type controls (WT), treated with Ang II (100nM) and immunofluorescence analysis was performed. Ang II induced p47phox plasma membrane translocation at 10min in WT mice. However, p47 phox translocation was significantly inhibited in CyPA -/- MASM. CyPA and p47phox colocalized at the plasma membrane in response to Ang II. Further analysis using subcellular fractionation studies confirmed that Ang II induced p47phox plasma membrane translocation was inhibited in CyPA -/- MASM compared to WT (1.2±2.7 vs 4.3±3.4 fold increase). Coimmunoprecipitation analyses confirmed that Ang II increased CyPA association with p47phox in a time dependent manner (2.5±3.4 fold increase at 10min). Finally, pretreatment with the PPIase activity inhibitor, cyclosporine A (1uM), could not inhibit CyPA association with p47phox and CyPA mediated p47phox translocation to the plasma membrane. Conclusion: These data suggest that Ang II promotes an association between CyPA and p47phox that enhances plasma membrane translocation of p47phox. This is proposed to increase the NADPH oxidase activity thereby increasing cellular ROS production. This process is independent of the PPIase activity of CyPA. Therefore, inhibition of the CyPA and p47phox association could be a future therapeutic target for Ang II induced ROS regulated cardiovascular diseases such as atherosclerosis and abdominal aortic aneurysm formation.

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Involvement of Organic Cation Transporter-3 and Plasma Membrane Monoamine Transporter in Serotonin Uptake in Human Brain Vascular Smooth Muscle Cells

Frontiers in Pharmacology ◽

10.3389/fphar.2013.00014 ◽

2013 ◽

Vol 4 ◽

Cited By ~ 6

Author(s):

Rachel W. S. Li ◽

Cui Yang ◽

Y. W. Kwan ◽

S. W. Chan ◽

Simon M. Y. Lee ◽

...

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Vascular Smooth Muscle ◽

Human Brain ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Organic Cation ◽

Organic Cation Transporter ◽

Muscle Cells ◽

Organic Cation Transporter 3

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Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

AJP Cell Physiology ◽

10.1152/ajpcell.00221.2012 ◽

2013 ◽

Vol 305 (8) ◽

pp. C829-C845 ◽

Cited By ~ 22

Author(s):

Hanaa K. B. Motawea ◽

Selvi C. Jeyaraj ◽

Ali H. Eid ◽

Srabani Mitra ◽

Nicholas T. Unger ◽

...

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Cyclic Amp ◽

Cell Surface ◽

Smooth Muscle Cells ◽

Blood Vessels ◽

Muscle Cells ◽

Small Gtpase ◽

Actin Binding ◽

Surface Translocation

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.

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Simultaneous measurement of Ca2+ transients and changes in the cell volume and microviscosity of the plasma membrane in smooth muscle cells

Biochemical Pharmacology ◽

10.1016/0006-2952(96)00138-4 ◽

1996 ◽

Vol 52 (1) ◽

pp. 49-63 ◽

Cited By ~ 3

Author(s):

Martin Ochsner

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Smooth Muscle Cells ◽

Cell Volume ◽

Simultaneous Measurement ◽

Muscle Cells

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A natural protective mechanism against hyperglycaemia in vascular endothelial and smooth-muscle cells: role of glucose and 12-hydroxyeicosatetraenoic acid

Biochemical Journal ◽

10.1042/bj3620413 ◽

2002 ◽

Vol 362 (2) ◽

pp. 413-422 ◽

Cited By ~ 28

Author(s):

Evgenia ALPERT ◽

Arie GRUZMAN ◽

Hanan TOTARY ◽

Nurit KAISER ◽

Reuven REICH ◽

...

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Smooth Muscle Cells ◽

Transport System ◽

Muscle Cells ◽

Vascular Endothelial ◽

Vascular Cells ◽

Hexose Transport ◽

Glut 1 ◽

Glucose Levels

Bovine aortic endothelial and smooth-muscle cells down-regulate the rate of glucose transport in the face of hyperglycaemia, thus providing protection against deleterious effects of increased intracellular glucose levels. When exposed to high glucose concentrations these cells reduced the mRNA and protein content of their typical glucose transporter, GLUT-1, as well as its plasma-membrane abundance. Inhibition of the lipoxygenase (LO) pathway, and particularly 12-LO, reversed this glucose-induced down-regulatory process and restored the rate of hexose transport to the level seen in vascular cells exposed to normal glucose levels. This reversal was accompanied by increased levels of GLUT-1 mRNA and protein, as well as of its plasma-membrane content. Exposure of the vascular cells to elevated glucose concentrations increased by 2–3-fold the levels of cell-associated and secreted 12-hydroxyeicosatetraenoic acid (12-HETE), the product of 12-LO. Inhibition of 15- and 5-LO, cyclo-oxygenases 1 and 2, and eicosanoid-producing cytochrome P450 did not modify the hexose-transport system in vascular cells. These results suggest a role for HETEs in the autoregulation of hexose transport in vascular cells. 8-Iso prostaglandin F2α, a non-enzymic oxidation product of arachidonic acid, had no effect on the hexose-transport system in vascular cells exposed to hyperglycaemic conditions. Taken together, these findings show that hyperglycaemia increases the production rate of 12-HETE, which in turn mediates the down-regulation of GLUT-1 expression and the glucose-transport system in vascular endothelial and smooth-muscle cells.

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Movement of plasma-membrane sterols to the endoplasmic reticulum in cultured cells

Biochemical Journal ◽

10.1042/bj2480237 ◽

1987 ◽

Vol 248 (1) ◽

pp. 237-242 ◽

Cited By ~ 16

Author(s):

J P Slotte ◽

E L Bierman

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Muscle ◽

Plasma Membrane ◽

Smooth Muscle Cells ◽

Human Skin ◽

Cultured Cells ◽

Muscle Cells ◽

Skin Fibroblasts ◽

Human Skin Fibroblasts ◽

Arterial Smooth Muscle Cells

The spontaneous turnover of plasma-membrane sterols, as measured by their transfer to the endoplasmic reticulum, was measured in quiescent cultured human skin fibroblasts and monkey arterial smooth-muscle cells. The plasma-membrane sterol pool was pulse-labelled with trace amounts of either [3H]desmosterol or [3H]cholesterol. We then measured the enzymic conversion of [3H]desmosterol into [3H]cholesterol and of [3H]cholesterol into [3H]cholesteryl esters in intact cells. Depending on the probe used, markedly different transfer or conversion rates were found in these cells. In quiescent human skin fibroblasts, incubated in a serum-free medium, about 1.1% of the plasma-membrane [3H]desmosterol was converted into [3H]cholesterol/h, whereas in monkey arterial smooth-muscle cells the corresponding rate was 0.4%. Under similar experimental conditions, these cells esterified less than 0.02% (fibroblasts) and 0.12% (smooth-muscle cells) of the plasma-membrane [3H]cholesterol/h. The movement of sterols from the plasma membrane to the endoplasmic reticulum, as measured by the conversion of [3H]desmosterol into [3H]cholesterol was not blocked by colchicine, but was markedly enhanced by 3% (w/v) dimethyl sulphoxide. In all, these results indicate that plasma-membrane sterols of cultured cells are continuously transferred to the interior of the cell at a rate substantially higher than previously appreciated. This turnover of plasma-membrane sterol molecules took place even when there was no mass transfer of sterols into the cells.

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