scholarly journals Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis.

1984 ◽  
Vol 32 (4) ◽  
pp. 347-357 ◽  
Author(s):  
K Chen ◽  
T N Wight
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Acridine Orange ◽  
Alcian Blue ◽  
Ruthenium Red ◽  
Cationic Dyes ◽  
Monolayer Films ◽  
Arterial Smooth Muscle ◽  
Chondroitinase Abc ◽  
Safranin O

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.

scholarly journals Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1333-1343 ◽  
Author(s):  
TN Wight ◽  
MG Kinsella ◽  
A Keating ◽  
JW Singer
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Layer ◽  
Ruthenium Red ◽  
Adherent Cells ◽  
Sulfate Proteoglycan ◽  
Chondroitinase Abc ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H- activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1333-1343 ◽  
Author(s):  
TN Wight ◽  
MG Kinsella ◽  
A Keating ◽  
JW Singer
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Layer ◽  
Ruthenium Red ◽  
Adherent Cells ◽  
Sulfate Proteoglycan ◽  
Chondroitinase Abc ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

Abstract Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H- activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle

2001 ◽  
Vol 280 (1) ◽  
pp. H208-H215 ◽  
Author(s):  
Pin-Lan Li ◽  
Wang-Xian Tang ◽  
Hector H. Valdivia ◽  
Ai-Ping Zou ◽  
William B. Campbell
Keyword(s):  
Smooth Muscle ◽  
Lipid Bilayers ◽  
Ryanodine Receptors ◽  
Fold Increase ◽  
Ruthenium Red ◽  
Dependent Manner ◽  
High Concentration ◽  
Open Probability ◽  
Arterial Smooth Muscle ◽  
Concentration Dependent Manner

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca2+release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca2+ release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs+ current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NPo) of the channels in the presence of ryanodine (0.01–1 μM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 μM) and ruthenium red (40–80 μM) substantially inhibited the activity of RYR/Ca2+ release channels. Caffeine (0.5–5 mM) markedly increased the NPo of these Ca2+release channels of the SR, but d- myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NPo of these Ca2+release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 μM) into the cis bath solution produced a 2.9-fold increase in the NPo of these RYR/Ca2+release channels. An eightfold increase in the NPo of the RYR/Ca2+ release channels (0.0056 ± 0.001 vs. 0.048 ± 0.017) was observed at a concentration of cADPR of 1 μM. The effect of cADPR was completely abolished by ryanodine (50 μM). In the presence of cADPR, Ca2+-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca2+ release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca2+ release through the activation of RYRs on the SR of these smooth mucle cells.

scholarly journals Arterial chondroitin sulfate proteoglycan: localization with a monoclonal antibody.

1988 ◽  
Vol 36 (10) ◽  
pp. 1211-1221 ◽  
Author(s):  
M W Lark ◽  
T K Yeo ◽  
H Mar ◽  
S Lara ◽  
I Hellström ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Chondroitin Sulfate ◽  
Chondroitin Sulfate Proteoglycan ◽  
Elastic Fibers ◽  
Immunogold Electron Microscopy ◽  
Sulfate Proteoglycan ◽  
Arterial Smooth Muscle ◽  
Matrix Components

We generated a monoclonal antibody (Mab) against a large chondroitin sulfate proteoglycan (CSPG) isolated from bovine aorta. This Mab (941) immunoprecipitates a CSPG synthesized by cultured monkey arterial smooth muscle cells. The immunoprecipitated CSPG is totally susceptible to chondroitinase ABC digestion and possesses a core glycoprotein of Mr approximately 400-500 KD. By use of immunofluorescence light microscopy and immunogold electron microscopy, the PG recognized by this Mab was shown to be deposited in the extracellular matrix of monkey arterial smooth muscle cell cultures in clusters which were not part of other fibrous matrix components and not associated with the cell's plasma membrane. With similar immunolocalization techniques, the CSPG antigen was found enriched in the intima and present in the medial portions of normal blood vessels, as well as in the interstitial matrix of thickened intimal lesions of atherosclerotic vessels. Immunoelectron microscopy revealed that this CSPG was confined principally to the space within the extracellular matrix not occupied by other matrix components, such as collagen and elastic fibers. These results indicate that this particular proteoglycan has a specific but restricted distribution in the extracellular matrix of arterial tissue.

ANP signaling inhibits TGF-β-induced Smad2 and Smad3 nuclear translocation and extracellular matrix expression in rat pulmonary arterial smooth muscle cells

2007 ◽  
Vol 102 (1) ◽  
pp. 390-398 ◽  
Author(s):  
Peng Li ◽  
Suzanne Oparil ◽  
Lea Novak ◽  
Xu Cao ◽  
Weibin Shi ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Nuclear Translocation ◽  
Muscle Cells ◽  
Arterial Smooth Muscle ◽  
Pulmonary Arterial ◽  
Arterial Smooth Muscle Cells ◽  
Pulmonary Arterial Smooth Muscle ◽  
Tgf Β1

Atrial natriuretic peptide (ANP) and transforming growth factor (TGF)-β play important counterregulatory roles in pulmonary vascular adaptation to chronic hypoxia. To define the molecular mechanism of this important interaction, we tested whether ANP-cGMP-protein kinase G (PKG) signaling inhibits TGF-β1-induced extracellular matrix (ECM) expression and defined the specific site(s) at which this molecular merging of signaling pathways occurs. Rat pulmonary arterial smooth muscle cells (PASMCs) were treated with ANP (1 μM) or cGMP (1 mM) with or without pretreatment with PKG inhibitors KT-5823 (1 μM) or Rp-8-bromo-cGMP (Rp-8-Br-cGMP 50 μM), then exposed to TGF-β1 (1 ng/ml) for 5–360 min (for pSmad nuclear translocation and protein analysis) or 24 h (for ECM mRNA expression). Nuclear translocation of pSmad2 and pSmad3 was assessed by fluorescent confocal microscopy. ANP and cGMP inhibited TGF-β1-induced pSmad2 and pSmad3 nuclear translocation and expression of periostin, osteopontin, and plasminogen activator inhibitor-1 mRNA and protein, but not TGF-β1-induced phosphorylation of Smad2 and Smad3. KT-5823 and Rp-8-Br-cGMP blocked ANP/cGMP-induced activation of PKG and inhibition of TGF-β1-stimulated nuclear translocation of pSmad2 and pSmad3 in PASMCs. These results reveal for the first time a precise site at which ANP-cGMP-PKG signaling exerts its antifibrogenic effect on the profibrogenic TGF-β1 signaling pathway: by blocking TGF-β1-induced pSmad2 and pSmad3 nuclear translocation and ECM expression in PASMCs. Blocking nuclear translocation and subsequent binding of pSmad2 and pSmad3 to TGF-β-Smad response elements in ECM genes may be responsible for the inhibitory effects of ANP on TGF-β-induced expression of ECM molecules.

Sign in / Sign up

Export Citation Format

Share Document