Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

2006 ◽  
Vol 290 (5) ◽  
pp. C1341-C1349 ◽  
Author(s):  
Jyoti Pande ◽  
Kanwaldeep K. Mallhi ◽  
Ahilya Sawh ◽  
Magdalena M. Szewczyk ◽  
Fiona Simpson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cultured Cells ◽  
Extracellular Domain ◽  
Force Of Contraction ◽  
Human Embryonic Kidney ◽  
Aortic Smooth Muscle ◽  
293 Cells ◽  
Arterial Smooth Muscle Cells ◽  
Endothelium Dependent Relaxation

Plasma membrane Ca2+ pumps (PMCA) that expel Ca2+ from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca2+-Mg2+-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a Ki value of 46 ± 5 μM, making it 10× more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca2+-Mg2+-ATPase in human embryonic kidney-293 cells with a higher Ki value (105 ± 11 μM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 μM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca2+] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.

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

1987 ◽  
Vol 248 (1) ◽  
pp. 237-242 ◽  
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.

Phosphatidylinositol 3,5-bisphosphate increases intracellular free Ca2+ in arterial smooth muscle cells and elicits vasocontraction

2011 ◽  
Vol 300 (6) ◽  
pp. H2016-H2026 ◽  
Author(s):  
Neerupma Silswal ◽  
Nikhil K. Parelkar ◽  
Michael J. Wacker ◽  
Marco Brotto ◽  
Jon Andresen
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ryanodine Receptors ◽  
Muscle Cells ◽  
Arterial Smooth Muscle ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
Arterial Smooth Muscle Cells

Phosphoinositide (3,5)-bisphosphate [PI(3,5)P2] is a newly identified phosphoinositide that modulates intracellular Ca2+ by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca2+, we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca2+ stores PI(3,5)P2 would increase intracellular Ca2+ in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P2 was present in the mouse aorta and that exogenously applied PI(3,5)P2 readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P2 elevated intracellular Ca2+, and it also contracted aortic rings. Both the rise in intracellular Ca2+ and the contraction caused by PI(3,5)P2 were prevented by antagonizing RyRs, while the majority of the PI(3,5)P2 response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca2+ stores with thapsigargin or caffeine and/or ryanodine blunted the Ca2+ response and greatly attenuated the contraction elicited by PI(3,5)P2. The removal of extracellular Ca2+ or addition of verapamil to inhibit voltage-dependent Ca2+ channels reduced but did not eliminate the Ca2+ or contractile responses to PI(3,5)P2. We also found that PI(3,5)P2 depolarized aortic smooth muscle cells and that LaCl3 inhibited those aspects of the PI(3,5)P2 response attributable to extracellular Ca2+. Thus, full and sustained aortic contractions to PI(3,5)P2 required the release of SR Ca2+, probably via the activation of RyR, and also extracellular Ca2+ entry via voltage-dependent Ca2+ channels.

scholarly journals THE EFFECT OF PROPARGYLGLYCINE ON THE ENDOTHELIUM-DEPENDENT RELAXATION AND ACTIVITY OF MAIN SOURCES OF SUPEROXIDE ANION GENERATION DURING AGING

2020 ◽  
Vol 66 (4) ◽  
pp. 7-11
Author(s):  
K.O. Drachuk ◽  
◽  
N.A. Dorofeyeva ◽  
V. F. Sagach ◽  
◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Superoxide Anion ◽  
Healthy Adult ◽  
Adult Rats ◽  
Aortic Smooth Muscle ◽  
Superoxide Anion Generation ◽  
Average Value ◽  
Old Rats ◽  
Endothelium Dependent Relaxation ◽  
Dependent Pathway

The objective of the present study was to investigate the effect of propargylglycine (PAG), an inhibitor of the CSE-dependent pathway of hydrogensulfide synthesis, on the endotheliumdependent relaxation of aortic smooth muscle (SM) in old rats. It was found a violation of SM relaxation in this group of animals. The average value of its amplitude was 7.5 ± 1.4% compared with 64.9 ± 3.5% in healthy adult rats. PAG significantly improved endothelium-dependent relaxation in old rats, the average value of its amplitude increased from 7.5 ± 1.4% to 44,5 ± 3,2%. This effect was due to suppression of superoxide anion generation by inhibition of xanthineoxidase activity.

scholarly journals Parallel Isolation and Characterization of Porcine Smooth Muscle, Endothelial and Mesenchymal Stromal Cells for Bioengineering Applications

2021 ◽  
Author(s):  
Sara Morini ◽  
Iris Pla-Palacín ◽  
Pilar Sainz-Arnal ◽  
Natalia Sánchez-Romero ◽  
Maria Falceto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cultured Cells ◽  
Umbilical Vein ◽  
Building Blocks ◽  
Cell Types ◽  
Aortic Smooth Muscle ◽  
Isolation And Characterization ◽  
Umbilical Vein Endothelial Cells

Abstract There is significant interest in the pig as the animal model of choice for organ transplantation and the study of tissue engineering (TE) products and applications. Currently, efforts are being taken to bioengineer solid organs to reduce donor shortages for transplantation. For complex organs such as the lung, heart, and liver, the vasculature represents a fundamental feature. Thus, to generate organs with a functional vascular network, the different cells constituting the building blocks of the blood vessels should be procured. However, due to species' specificities, porcine cell isolation, expansion, and characterization are not entirely straightforward compared to human cell procurement. Here, we report the establishment of simple and suitable methods for the isolation and characterization of distinct porcine cells for bioengineering purposes.We successfully isolated, expanded and characterized porcine bone marrow-derived mesenchymal stromal (pBM-MSC), aortic smooth muscle (pASMC), and umbilical vein endothelial cells (pUVEC). We demonstrated that the three cell types showed specific immunophenotypical features. Moreover, we demonstrated that pBM-MSC could preserve their multipotency in vitro, and pUVEC were capable of maintaining their functionality in vitro.These cultured cells could be further expanded and represent a useful cellular tool for TE purposes (i.e., for recellularization approaches of vascularized organs or in vitro angiogenesis studies).

PDGF/MEK/ERK axis represses Ca2+ clearance via decreasing the abundance of plasma membrane Ca2+ pump PMCA4 in pulmonary arterial smooth muscle cells

2021 ◽  
Vol 320 (1) ◽  
pp. C66-C79
Author(s):  
Liyu Deng ◽  
Jidong Chen ◽  
Ting Wang ◽  
Bin Chen ◽  
Lei Yang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Pulmonary Artery ◽  
Smooth Muscle Cells ◽  
Vascular Remodeling ◽  
Muscle Cells ◽  
Arterial Smooth Muscle ◽  
Pulmonary Arterial ◽  
Arterial Smooth Muscle Cells ◽  
Pulmonary Arterial Smooth Muscle

Pulmonary arterial hypertension (PAH) is a rare and lethal disease characterized by vascular remodeling and vasoconstriction, which is associated with increased intracellular calcium ion concentration ([Ca2+]i). Platelet-derived growth factor-BB (PDGF-BB) is the most potent mitogen for pulmonary arterial smooth muscle cells (PASMCs) and is involved in vascular remodeling during PAH development. PDGF signaling has been proved to participate in maintaining Ca2+ homeostasis of PASMCs; however, the mechanism needs to be further elucidated. Here, we illuminate that the expression of plasma membrane calcium-transporting ATPase 4 (PMCA4) was downregulated in PASMCs after PDGF-BB stimulation, which could be abolished by restraining the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK). Functionally, suppression of PMCA4 attenuated the [Ca2+]i clearance in PASMCs after Ca2+ entry, promoting cell proliferation and elevating cell locomotion through mediating formation of focal adhesion. Additionally, the expression of PMCA4 was decreased in the pulmonary artery of monocrotaline (MCT)- or hypoxia-induced PAH rats. Moreover, knockdown of PMCA4 could increase the right ventricular systolic pressure (RVSP) and wall thickness (WT) of pulmonary artery in rats raised under normal conditions. Taken together, our findings demonstrate the importance of the PDGF/MEK/ERK/PMCA4 axis in intracellular Ca2+ homeostasis in PASMCs, indicating a functional role of PMCA4 in pulmonary arterial remodeling and PAH development.

scholarly journals A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment.

1987 ◽  
Vol 105 (2) ◽  
pp. 819-831 ◽  
Author(s):  
A A Rogalski
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Cardiac Muscle ◽  
Cultured Cells ◽  
Molecular Forms ◽  
Cell Coupling ◽  
Membrane Structures ◽  
Adult Chicken ◽  
Attachment Sites ◽  
Membrane Attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.

Sign in / Sign up

Export Citation Format

Share Document