scholarly journals Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

2008 ◽  
Vol 295 (5) ◽  
pp. H2178-H2187 ◽  
Author(s):  
Irina A. Ionova ◽  
Jeannette Vásquez-Vivar ◽  
Jennifer Whitsett ◽  
Anja Herrnreiter ◽  
Meetha Medhora ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
De Novo ◽  
Regulatory Protein ◽  
Cell Types ◽  
No Production ◽  
Inos Protein ◽  
Protein Dimers ◽  
Adult Cardiac Myocytes ◽  
Salvage Pathways

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.

Physiological and hypoxic O2 tensions rapidly regulate NO production by stimulated macrophages

2008 ◽  
Vol 294 (4) ◽  
pp. C1079-C1087 ◽  
Author(s):  
Mary A. Robinson ◽  
James E. Baumgardner ◽  
Virginia P. Good ◽  
Cynthia M. Otto
Keyword(s):  
Protein Concentration ◽  
Cultured Cells ◽  
Raw 264.7 Cells ◽  
Hypoxic Exposure ◽  
Convection Cell ◽  
No Production ◽  
Inos Protein ◽  
Substrate Limitation ◽  
No Consumption

Nitric oxide (NO) production by inducible NO synthase (iNOS) is dependent on O2 availability. The duration and degree of hypoxia that limit NO production are poorly defined in cultured cells. To investigate short-term O2-mediated regulation of NO production, we used a novel forced convection cell culture system to rapidly (response time of 1.6 s) and accurately (±1 Torr) deliver specific O2 tensions (from <1 to 157 Torr) directly to a monolayer of LPS- and IFNγ-stimulated RAW 264.7 cells while simultaneously measuring NO production via an electrochemical probe. Decreased O2 availability rapidly (≤30 s) and reversibly decreased NO production with an apparent KmO2 of 22 (SD 6) Torr (31 μM) and a Vmax of 4.9 (SD 0.4) nmol·min−1·10−6 cells. To explore potential mechanisms of decreased NO production during hypoxia, we investigated O2-dependent changes in iNOS protein concentration, iNOS dimerization, and cellular NO consumption. iNOS protein concentration was not affected ( P = 0.895). iNOS dimerization appeared to be biphasic [6 Torr ( P = 0.008) and 157 Torr ( P = 0.258) >36 Torr], but it did not predict NO production. NO consumption was minimal at high O2 and NO tensions and negligible at low O2 and NO tensions. These results are consistent with O2 substrate limitation as a regulatory mechanism during brief hypoxic exposure. The rapid and reversible effects of physiological and pathophysiological O2 tensions suggest that O2 tension has the potential to regulate NO production in vivo.

scholarly journals Epithelial argininosuccinate synthetase is dispensable for intestinal regeneration and tumorigenesis

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Jonathan H. M. van der Meer ◽  
Ruben J. de Boer ◽  
Bartolomeus J. Meijer ◽  
Wouter L. Smit ◽  
Jacqueline L. M. Vermeulen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Intestinal Epithelium ◽  
De Novo ◽  
Cell Types ◽  
Intestinal Epithelial ◽  
Argininosuccinate Synthetase ◽  
Important Amino Acid ◽  
Tissue Specimens ◽  
Colorectal Cancer Patients

AbstractThe epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.

Abstract 897: Alliance of Blood Derived Endothelial Cells and Adipose Stromal Cells in Human Vasculogenesis

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Dmitry O Traktuev ◽  
Daniel N Prater ◽  
Aravind R Sanjeevaiah ◽  
Stephanie Merfeld-Clauss ◽  
Brian H Johnstone ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Stromal Cells ◽  
De Novo ◽  
Functional Integration ◽  
Cell Types ◽  
Specific Marker ◽  
Pronounced Difference ◽  
Adipose Stromal Cells ◽  
Small Vessels

Introduction Both Endothelial progenitor cells (EPC) and adipose stromal cells (ASC) are under investigation as therapies for cardiovascular diseases. Both cell types are capable of modulating vascular assembly and are, thereby, capable of directly promoting revascularization of ischemic tissues. We have shown that EPC differentiate into endothelial cells to form small vessels, whereas ASC have pericytic properties and naturally stabilize vessels. In this study we tested the possibility that ASC would interact with EPC to assemble de novo vessels in collagen in an in vivo chimeric implant. Methods and Results Collagen implants embedded with either umbilical cord blood EPC or adult ASC or a 4:1 mixture of both (2x10 6 cells/ml) were implanted subcutaneously into NOD/SCID mice. After 14 d implants were harvested and evaluated by immunohistochemistry. There was a pronounced difference among the groups in vascular network assembly. The majority of vessels formed in the EPC and ASC monocultures were small capillaries bounded by a single endothelial layer. Conversely, 100% of the plugs embedded with both cell types were highly invaded with multilayered arteriolar vessels. The density of the CD31 + vessels in the EPC and co-culture plugs was 26.6 ± 5.8 and 122.4 ± 9.8 per mm 2 , respectively. No CD31 + cells of human origin were detected in the ASC monocultures, indicating that ASC, which do not express this EC-specific marker, engage murine EC or form pseudovessels in this system. The density of α-SMA + vessels with lumens per mm 2 was 13.1 ± 3.6 (EPC), 10.2 ± 3.5 (ASC) and 124.7 ± 19.7 (co-culture). The total overlap of CD31 + and SMA + vessels demonstrates that mature, multilayered conduits were formed with the co-culture. Moreover, the majority of these vessels were filled with erythrocytes (92.5 ± 16.2 per mm 2 ), indicating inosculation with the native vasculature, which was confirmed by ultrasound with echogenic microbubbles and persisted to at least 4 months. Conclusion This study is the first to demonstrate that non-transformed human EPC and ASC cooperatively form mature and stable vasculature with subsequent functional integration into a host vasculature system.

Adult cardiac myocytes in primary culture: cell characteristics and insulin-receptor interaction

1985 ◽  
Vol 249 (2) ◽  
pp. H212-H221 ◽  
Author(s):  
J. Eckel ◽  
G. van Echten ◽  
H. Reinauer
Keyword(s):  
Cardiac Myocytes ◽  
Structural Integrity ◽  
Cultured Cells ◽  
Insulin Binding ◽  
Receptor Interaction ◽  
Scatchard Analysis ◽  
Apparent Dissociation Constant ◽  
Adult Cardiac Myocytes ◽  
Receptor Number

Calcium-tolerant adult cardiac myocytes were kept in culture under serum-free conditions in the presence of physiological concentrations of insulin. Up to 4 days, 70% of cells retained their in vivo rodshaped morphology without gross structural alterations. During that period a constant ATP-to-ADP ratio was observed with a mean value of 10.6 +/- 0.5 (n = 4). The rate of [14C]phenylalanine incorporation remained unaltered up to 63 h in culture. Insulin binding to cultured cells was found to be time-and temperature-dependent, reversible, and highly specific. Scatchard analysis of equilibrium binding data showed a curvilinear plot with a high-affinity segment yielding an apparent dissociation constant of 4.5 X 10(-10) mol/l and a receptor number of 125,000 sites/cell. Both affinity and receptor number remained unaltered between 18 and 66 h in culture. [14C]phenylalanine incorporation was stimulated by 108% in cardiocytes cultured in the presence of high concentrations of insulin (1.7 X 10(-7) mol/l) for 63 h, when compared with control cells cultured in the absence of insulin. These data demonstrate the retention of structural integrity, insulin receptors, and insulin responsiveness in primary cultured adult cardiac myocytes and provide a useful model for long-term studies on the regulation of insulin action on the heart.

scholarly journals Constitutive nitric oxide production by rat alveolar macrophages

1998 ◽  
Vol 274 (3) ◽  
pp. L360-L368 ◽  
Author(s):  
P. R. Miles ◽  
L. Bowman ◽  
A. Rengasamy ◽  
L. Huffman
Keyword(s):  
Nitric Oxide ◽  
Alveolar Macrophage ◽  
Alveolar Macrophages ◽  
Lung Surfactant ◽  
Calcium Ionophore ◽  
Cell Types ◽  
Alveolar Type ◽  
No Production ◽  
No Formation

Results from previous studies suggest that alveolar macrophages must be exposed to inflammatory stimuli to produce nitric oxide (⋅ NO). In this study, we report that naive unstimulated rat alveolar macrophages do produce ⋅ NO and attempt to characterize this process. Western blot analysis demonstrates that the enzyme responsible is an endothelial nitric oxide synthase (eNOS). No brain or inducible NOS can be detected. The rate of ⋅ NO production is ∼0.07 nmol ⋅ 106cells−1 ⋅ h−1, an amount that is less than that produced by the eNOS found in alveolar type II or endothelial cells. Alveolar macrophage ⋅ NO formation is increased in the presence of extracellularl-arginine, incubation medium containing magnesium and no calcium, a calcium ionophore (A-23187), or methacholine. ⋅ NO production is inhibited by N G-nitro-l-arginine methyl ester (l-NAME) but not by N G-nitro-l-arginine,l- N 5-(1-iminomethyl)ornithine hydrochloride, or aminoguanidine. Incubation with ATP, ADP, or histamine also inhibits ⋅ NO formation. Some of these properties are similar to and some are different from properties of eNOS in other cell types. Cellular ⋅ NO levels do not appear to be related to ATP or lactate content. Alveolar macrophage production of ⋅ NO can be increased approximately threefold in the presence of lung surfactant or its major component, dipalmitoyl phosphatidylcholine (DPPC). The DPPC-induced increase in ⋅ NO formation is time and concentration dependent, can be completely inhibited by l-NAME, and does not appear to be related to the degradation of DPPC by alveolar macrophages. These results demonstrate that unstimulated alveolar macrophages produce ⋅ NO via an eNOS and that lung surfactant increases ⋅ NO formation. This latter effect may be important in maintaining an anti-inflammatory state in vivo.

scholarly journals The Steroidogenic Acute Regulatory Protein Is Expressed in Steroidogenic Cells of the Day-Old Brain

Endocrinology ◽  
2004 ◽  
Vol 145 (10) ◽  
pp. 4775-4780 ◽  
Author(s):  
Steven R. King ◽  
Stephen D. Ginsberg ◽  
Tomohiro Ishii ◽  
Roy G. Smith ◽  
Keith L. Parker ◽  
...  
Keyword(s):  
De Novo ◽  
Metastatic Lymph Node ◽  
Regulatory Protein ◽  
Cell Types ◽  
Adult Brain ◽  
Peripheral Tissues ◽  
Cytochrome P450scc ◽  
Star Protein ◽  
Steroidogenic Acute Regulatory ◽  
The Brain

Abstract Although recent research has focused on the fundamental role(s) of steroids synthesized de novo in the brain on development, the mechanism by which production of these neurosteroids is regulated remains unclear. Steroid production in peripheral tissues is acutely regulated by the steroidogenic acute regulatory (StAR) protein, which mediates the rate-limiting step in steroid biosynthesis: the intramitochondrial delivery of cholesterol to cytochrome P450scc for conversion to steroid. We recently demonstrated that StAR is present in discrete cell types in the adult brain, suggesting that neurosteroid production is mediated by StAR. Nevertheless, little is known regarding the presence of StAR in the developing brain. In the present study, the presence of StAR and for the first time, its homolog, the putative cholesterol transport protein metastatic lymph node 64 (MLN64), were defined in the neonatal mouse brain using immunocytochemical techniques. Both StAR and MLN64 were found to be present in the brain with staining patterns characteristic to each protein, indicating the authenticity of StAR and MLN64 immunoreactivity. Furthermore, we found MLN64 to be expressed in the adult brain as well, apparently at higher levels than StAR. Importantly, StAR protein is present in cells that also express P450scc. These data suggest that, as with the adult, neurosteroid production during development occurs through a StAR-mediated pathway.

Methylation and expression of the Myo D1 determination gene

1990 ◽  
Vol 326 (1235) ◽  
pp. 277-284 ◽  
Keyword(s):  
De Novo ◽  
Cell Types ◽  
Model System ◽  
Parental Line ◽  
Molecular Control ◽  
Embryo Cells ◽  
End Stage ◽  
Derivatives Of

Mouse embryo cells induced to differentiate with the demethylating agent 5- azacytidine represent an excellent model system to investigate the molecular control of development. Clonal derivatives of 10T1/2 cells that have become determined to the myogenic or adipogenic lineages can be isolated from the multipotential parental line after drug treatment. These determined derivatives can be cultured indefinitely and will differentiate into end-stage phenotypes on appropriate stimulation. A gene called Myo D1, recently isolated from such a myoblast line, will confer myogenesis when expressed in 10T1/2 or other cell types (Davis et al. 1987). The cDNA for Myo D1 contains a large number of CpG sequences and the gene is relatively methylated in 10T1/2 cells and an adipocyte derivative, but is demethylated in myogenic derivatives. Myo D1 may therefore be subject to methylation control in vitro . On the other hand, preliminary observations suggest that Myo D1 is not methylated at CCGG sites in vivo so that a de novo methylation event may have occurred in vitro . These observations may have significance in the establishment of immortal cell lines and tumours.

scholarly journals Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC)

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3112-3119
Author(s):  
RJ Jr Kelm ◽  
GA Hair ◽  
KG Mann ◽  
BW Grant
Keyword(s):  
De Novo ◽  
Blood Platelets ◽  
Bone Matrix ◽  
Cell Types ◽  
Sensitivity Analyses ◽  
Complex Type ◽  
Oligosaccharide Structure ◽  
The Matrix

Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.

scholarly journals Birth and delineation of the intestinal stem cell niche

2021 ◽  
Author(s):  
Neil McCarthy ◽  
Guodong Tie ◽  
Shariq Madha ◽  
Adrianna Maglieri ◽  
Judith Kraiczy ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Critical Period ◽  
Stem Cell Niche ◽  
De Novo ◽  
Cell Types ◽  
Muscle Layer ◽  
Intestinal Stem Cells ◽  
Morphogenetic Protein ◽  
Cell Niche

Wnt and Rspondin (RSPO) signaling triggers proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here we report that the functional ISC niche is a complex, multi-layered mesenchymal structure that includes distinct smooth muscle populations and describe how that niche organizes early in mouse life. Diverse sub-cryptal cells provide redundant supportive factors, with distinct BMPi and the most potent Wnt agonist, RSPO2, restricted to single cell types. Two functionally opposing elements arise in tandem during a critical period of crypt morphogenesis: a prominent shelf of BMP+ sub-epithelial myofibroblasts that promote epithelial differentiation and the muscularis mucosae, a specialized muscle layer generated de novo to supplement other RSPO and BMPi sources. In vivo ablation of smooth muscle, while preserving trophocytes, raises crypt BMP activity and potently limits crypt expansion. Thus, distinct and progressively refined mesenchymal components together create the milieu necessary to propagate crypts during rapid organ growth and to sustain ISCs in the adult niche.

scholarly journals Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC)

Blood ◽  
1992 ◽  
Vol 80 (12) ◽  
pp. 3112-3119 ◽  
Author(s):  
RJ Jr Kelm ◽  
GA Hair ◽  
KG Mann ◽  
BW Grant
Keyword(s):  
De Novo ◽  
Blood Platelets ◽  
Bone Matrix ◽  
Cell Types ◽  
Sensitivity Analyses ◽  
Complex Type ◽  
Oligosaccharide Structure ◽  
The Matrix

Abstract Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.

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