scholarly journals 5-bromo-2'-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells

1977 ◽  
Vol 164 (3) ◽  
pp. 645-652 ◽  
Author(s):  
G A Coetzee ◽  
W Gevers
Keyword(s):  
Adenosine Triphosphatase ◽  
Primary Cultures ◽  
Cardiac Cells ◽  
Cell Protein ◽  
Heart Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Active Centre ◽  
Cellular Dna ◽  
Hamster Heart

1. Treatment of hamster heart cells in primary culture with 5-bromo-2'-deoxyuridine resulted in the greatly increased activity of a particulate Ca2+- or Mg2+-dependent ATPase (adenosine triphosphatase). 2. 5-Bromo-2'-deoxyuridine exerted these effects only when it was incorporated into cellular DNA, and then in a concentration-dependent manner. 3. Serially replated cells contained less of the activity (expressed as a function of total cell protein) than did the primary cultures, but the stimulation caused by 5-bromo-2'-deoxyuridine addition was much greater. 4. The affected enzyme was apparently localized in the plasma membrane of the cells with its active centre exposed to the outer environment [ecto-(ATPase) dependent on Ca2+ or Mg2+].5. The activity was unaffected by treatment with p-chloromercuriphenylsulphonate, ouabain andverapamil. 6. Ecto (5'-nucleotidase) activity was not increased by 5-bromo-2'-deoxyuridine treatment of cells, and ecto-(p-nitrophenyl phosphatase) activity was only slightly enhanced.

scholarly journals Adrenal Cell Aldosterone Production Is Stimulated by Very-Low-Density Lipoprotein (VLDL)

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 721-731 ◽  
Author(s):  
Yewei Xing ◽  
William E. Rainey ◽  
John W. Apolzan ◽  
Omar L. Francone ◽  
Ruth B. S. Harris ◽  
...  
Keyword(s):  
Adrenal Gland ◽  
Primary Cultures ◽  
Signal Transduction Pathway ◽  
Free Calcium ◽  
Low Density ◽  
Dependent Manner ◽  
Very Low Density Lipoproteins ◽  
Concentration Dependent Manner ◽  
Adrenal Cells

Very low-density lipoproteins (VLDL) are a class of large lipoprotein synthesized in the liver. The key function of VLDL, in vivo, is to carry triglyceride from the liver to adipose tissue. As a steroidogenic organ, the adrenal gland mainly uses lipoproteins as sources of cholesterol. Although VLDL receptors have been detected in the human adrenal, the function of VLDL in the adrenal gland remains unknown. Herein, we used primary cultures of human and bovine adrenal cells and the adrenocortical cell line H295R as models to determine the effects of VLDL on adrenal steroidogenesis. Our studies revealed that VLDL significantly increased aldosterone synthesis in all of the models tested. This increase was largely due to VLDL's stimulation of the expression of steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2). VLDL increased CYP11B2 mRNA expression in a concentration-dependent manner. Effects of VLDL on CYP11B2 transcript levels were not additive with angiotensin II or potassium but were additive with the cAMP pathway agonists ACTH and forskolin. Nifedipine completely inhibited the effects of VLDL on CYP11B2 mRNA, suggesting that calcium is the main signal transduction pathway used by VLDL in adrenal cells. Indeed, VLDL increased cytosolic free calcium levels. An in vivo study conducted in sucrose-fed rats showed a positive correlation between elevated triglyceride (VLDL) levels in plasma and CYP11B2 expression in the adrenal. In conclusion, we have shown that VLDL can stimulate aldosterone synthesis in adrenocortical cells by increasing StAR and CYP11B2 expression, an event likely mediated by a calcium-initiated signaling cascade.

Regulation of glucose transport in aortic smooth muscle cells by cAMP and cGMP

2001 ◽  
Vol 353 (3) ◽  
pp. 513-519 ◽  
Author(s):  
Christopher J. MacKENZIE ◽  
Jill M. WAKEFIELD ◽  
Fiona CAIRNS ◽  
Anna F. DOMINICZAK ◽  
Gwyn W. GOULD
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Primary Cultures ◽  
Muscle Cells ◽  
Mek Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Common Element ◽  
Dependent Manner ◽  
Concentration Dependent Manner

We have studied the ability of cGMP and cAMP to modulate platelet-derived growth factor (PDGF)-stimulated 2-deoxy-d-glucose (deGlc) transport in primary cultures of vascular smooth muscle cells (VMSC) from rat aorta. PDGF stimulated deGlc transport in a time- and concentration-dependent manner. 8-Bromo-cGMP and atrial natriuretic peptide(1–28) [ANP(1–28)] were found to reduce PDGF-stimulated deGlc transport without affecting basal (unstimulated) transport activity. In contrast, 8-bromo-cAMP and dibutyryl-cAMP stimulated basal deGlc transport 2-fold and were without effect on PDGF-stimulated deGlc transport. 8-Bromo-cGMP also inhibited 8-bromo-cAMP-stimulated deGlc transport. The stimulation of deGlc transport by PDGF was sensitive to the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) inhibitor PD98059, and we show that ERK1/2 was activated by PDGF. Neither 8-bromo-cGMP nor ANP(1–28) inhibited PDGF-stimulated ERK activation, suggesting that the effects of cGMP and ANP(1–28) were not mediated by inhibition of this kinase. Our data also argue against a role for cGMP-dependent protein kinase in mediating the effects of cGMP or ANP(1–28). Collectively, our data suggest that in VSMC: (i) cGMP and cAMP have opposing effects on deGlc transport; (ii) PDGF and cAMP have common elements in the pathways by which they activate deGlc transport; and (iii) a common element may be the target of the cGMP-mediated inhibition of deGlc transport.

Uncoupling of cardiac cells by fatty acids: structure-activity relationships

1991 ◽  
Vol 260 (3) ◽  
pp. C439-C448 ◽  
Author(s):  
J. M. Burt ◽  
K. D. Massey ◽  
B. N. Minnich
Keyword(s):  
Fatty Acids ◽  
Oleic Acid ◽  
Unsaturated Fatty Acids ◽  
Saturated Fatty Acids ◽  
Decanoic Acid ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Dependent Manner ◽  
Rat Heart Cells

The permeability and conductance of gap junctions between pairs of neonatal rat heart cells were rapidly and reversibly decreased by oleic acid in a dose- and time-dependent manner. Other unsaturated fatty acids (C-18: cis 6, 9, or 11, and C-18, 16, and 14, cis 9), saturated fatty acids (C-10, 12, and 14), and saturated fatty alcohols (C-8, 10, and 12) also caused uncoupling. The most effective compounds of the unsaturated and saturated fatty acid and saturated fatty alcohol series caused essentially complete uncoupling at comparable aqueous concentrations. However, oleic acid uncoupled cells at membrane concentrations as low as 1 mol%, whereas decanoic acid required upwards of 35 mol%. The channels that support the action potential remained functional at these same membrane concentrations. The data are discussed in terms of the possible mechanism by which these compounds cause uncoupling and the possible role of uncoupling by nonesterified free fatty acids in the initiation of arrhythmias during and after ischemic insults.

scholarly journals Adenosine formation and release from neonatal-rat heart cells in culture

1985 ◽  
Vol 229 (3) ◽  
pp. 799-805 ◽  
Author(s):  
P Meghji ◽  
C A Holmquist ◽  
A C Newby
Keyword(s):  
Rat Heart ◽  
Neonatal Rat ◽  
Heart Cell ◽  
Heart Cells ◽  
Dependent Manner ◽  
Nucleoside Transporter ◽  
Concentration Dependent Manner ◽  
Cells In Culture ◽  
Neonatal Rat Heart ◽  
Rat Heart Cells

The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM-2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter.

Tumor necrosis factor induces matrix metalloproteinases in cardiomyocytes and cardiofibroblasts differentially via superoxide production in a PI3Kγ-dependent manner

2010 ◽  
Vol 298 (3) ◽  
pp. C679-C692 ◽  
Author(s):  
Ahmed E. Awad ◽  
Vijay Kandalam ◽  
Subhadeep Chakrabarti ◽  
Xiuhua Wang ◽  
Josef M. Penninger ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Matrix Metalloproteinases ◽  
Tumor Necrosis ◽  
Primary Cultures ◽  
Oxidase Inhibitor ◽  
Cardiac Cells ◽  
Superoxide Production ◽  
Dependent Manner ◽  
Necrosis Factor

Tumor necrosis factor (TNF) is an inflammatory cytokine that is upregulated in a number of cardiomyopathies. Adverse cardiac remodeling and dilation result from degradation of the extracellular matrix by matrix metalloproteinases (MMPs). We investigated whether TNF can directly trigger expression and activation of MMPs in cardiac cells. We compared MMP expression profile and activities between primary cultures of mouse neonatal cardiomyocytes and cardiofibroblasts and in cellular and extracellular compartments. In response to recombinant TNF (rTNF, 20 ng/ml), cardiomyocytes exhibited faster and more pronounced superoxide production compared with cardiofibroblasts, concomitant with increased expression of several MMPs. MMP9 levels increased more rapidly and about twofold more in cardiomyocytes than in cardiofibroblasts. TNF did not induce MMP2 expression. Expression of collagenases (MMP8, MMP12, MMP13, and MMP14) increased significantly, while total collagenase activity increased to a greater degree in conditioned medium of cardiomyocytes than in cardiofibroblasts. rTNF-mediated MMP expression and activation were dependent on superoxide production and were blocked by apocynin, an NADPH oxidase inhibitor. We identified phosphatidylinositol 3-kinase (PI3K)γ as a key factor in TNF-mediated events since TNF-induced superoxide production, MMP expression, and activity were significantly suppressed in cardiomyocytes and cardiofibroblasts deficient in PI3Kγ. We further demonstrated that the TNF-superoxide-MMP axis of events is in fact activated in heart disease in vivo. Wild-type and TNF−/− mice subjected to cardiac pressure overload revealed that TNF deficiency resulted in reduced superoxide levels, collagenase activities, PI3K activity, and fibrosis leading to attenuated cardiac dilation and dysfunction. Our study demonstrates that TNF triggers expression and activation of MMPs faster and stronger in cardiomyocytes than in cardiofibroblasts in a superoxide-dependent manner and via activation of PI3Kγ, thereby contributing to adverse myocardial remodeling in disease.

Nociceptin (1–13)NH2 Inhibits Stimulated Calcitonin-Gene-Related-Peptide Release From Primary Cultures of Rat Trigeminal Ganglia Neurones

Cephalalgia ◽  
2007 ◽  
Vol 27 (8) ◽  
pp. 868-876 ◽  
Author(s):  
A Capuano ◽  
D Curró ◽  
C Dello Russo ◽  
G Tringali ◽  
G Pozzoli ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Calcitonin Gene Related Peptide ◽  
Neonatal Rat ◽  
Trigeminal Ganglia ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Related Peptide ◽  
Cgrp Release ◽  
Calcitonin Gene ◽  
Consistent Increase

In this work we have developed and characterized primary cultures of neonatal rat trigeminal ganglia neurones; calcitonin-gene-related-peptide (CGRP) released from cells was taken as a marker of neuronal function. A significant and consistent increase in CGRP secretion was elicited by non-specific (56 mM KCl or veratridine) or specific (capsaicin) depolarizing stimuli. This paradigm was subsequently used to investigate the effects of nociceptin, an opioid-like peptide involved in central and peripheral control of nociception. We found that the nociceptin analogue nociceptin (1–13)NH2 (NOC) did not affect baseline CGRP release, but it reduced in a concentration-dependent manner CGRP release induced by all tested stimuli. NOC-induced reduction was statistically significant from 0.01 nM onward and achieved maximal effects at 10 nM. Such effects of NOC were seemingly mediated by the activation of specific ORL1 receptors, as a well-known nociceptin antagonist, N(Phe1)nociceptin (1–13)NH2, was able to completely revert NOC inhibition of capsaicin-stimulated CGRP release.

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture

2002 ◽  
Vol 283 (6) ◽  
pp. R1354-R1361 ◽  
Author(s):  
Paul L. Dudas ◽  
J. Larry Renfro
Keyword(s):  
Proximal Tubule ◽  
Primary Cultures ◽  
Renal Proximal Tubule ◽  
Proximal Tubules ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Electrophysiological Properties ◽  
Ussing Chambers ◽  
And Control ◽  
Inorganic Sulfate

The mechanisms and control of transepithelial inorganic sulfate (Si) transport by primary cultures of chick renal proximal tubule monolayers in Ussing chambers were determined. The competitive anion, S2O3 2− (5 mM), reduced both unidirectional reabsorptive and secretory fluxes and net Sireabsorption with no effect on electrophysiological properties. The carbonic anhydrase (CA) inhibitor ethoxzolamide decreased net Si reabsorption ∼45%. CAII protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Cortisol reduced net Sireabsorption up to ∼50% in a concentration-dependent manner. Thyroid hormone increased net Si reabsorption threefold in 24 h, and parathyroid hormone (PTH) acutely stimulated net Sireabsorption ∼45%. These data indicate that CA participates in avian proximal tubule active transepithelial Si reabsorption, which cortisol directly inhibits and T3 and PTH directly stimulate.

Effects on the ATP Content of Cultured Cells after Radiographic Contrast Media Exposure

1989 ◽  
Vol 30 (5) ◽  
pp. 541-547 ◽  
Author(s):  
A. Nordby ◽  
K. Thorstensen ◽  
J. Halgunset ◽  
O. A. Haugen ◽  
S. Solberg
Keyword(s):  
Contrast Media ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Established Cell Line ◽  
Dependent Manner ◽  
Atp Content ◽  
Concentration Dependent Manner ◽  
Short Period ◽  
Radiographic Contrast Media ◽  
Established Cell

The ATP content of cultured cells after exposure to meglumine-calcium metrizoate, sodium metrizoate, iohexol, iopamidol and saline was studied. Initially, the ATP content diminished rapidly for a short period and thereafter slowly during the incubation. After incubation with contrast media or saline, the ATP content slowly increased to normal when the cells were reincubated with fresh nutrient medium. Different contrast media and saline with the same final osmolality produced a similar effect on the ATP content of the cultured cells. Cellular association of meglumine-sodium diatrizoate, sodium metrizoate, sodium-iothalamate, iohexol and iopamidol was also examined. The established cell line NHIK 3025 as well as primary cultures of human umbilical endothelium were found to accumulate contrast media in a time-and concentration-dependent manner. When the incubation was carried out at 4°C, the cellular accumulation of contrast medium was less than 35 per cent of that seen at 37°C. It therefore seems that energy-dependent processes are involved to some degree.

scholarly journals Caseinolytic Proteins (Clp) in the Genus Klebsiella: Special Focus on ClpK

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 200
Author(s):  
Tehrim Motiwala ◽  
Blessing Oluebube Akumadu ◽  
Sbahle Zuma ◽  
Mbalenhle Sizamile Mfeka ◽  
Wanping Chen ◽  
...  
Keyword(s):  
Environmental Conditions ◽  
In Silico ◽  
Protein Quality ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Cell Protein ◽  
Special Focus ◽  
Dependent Manner ◽  
Dynamic Simulations ◽  
Concentration Dependent Manner

Caseinolytic proteins (Clp), which are present in both prokaryotes and eukaryotes, play a major role in cell protein quality control and survival of bacteria in harsh environmental conditions. Recently, a member of this protein family, ClpK was identified in a pathogenic strain of Klebsiella pneumoniae which was responsible for nosocomial infections. ClpK is linked to the thermal stress survival of this pathogen. The genome wide analysis of Clp proteins in Klebsiella spp. indicates that ClpK is present in only 34% of the investigated strains. This suggests that the uptake of the clpk gene is selective and may only be taken up by a pathogen that needs to survive harsh environmental conditions. In silico analyses and molecular dynamic simulations show that ClpK is mainly α-helical and is highly dynamic. ClpK was successfully expressed and purified to homogeneity using affinity and anion exchange chromatography. Biophysical characterization of ClpK showed that it is predominantly alpha-helical, and this is in agreement with in silico analysis of the protein structure. Furthermore, the purified protein is biologically active and hydrolyses ATP in a concentration- dependent manner.

Uncoupling of cardiac cells by doxyl stearic acids specificity and mechanism of action

1989 ◽  
Vol 256 (4) ◽  
pp. C913-C924 ◽  
Author(s):  
J. M. Burt
Keyword(s):  
Single Channel ◽  
Methyl Esters ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Head Group ◽  
Fatty Acyl Chain ◽  
Heart Cells ◽  
Dependent Manner

The influence of doxyl stearic acids (DSAs) on gap junctional conductance (gj) between pairs of neonatal rat heart cells was studied. DSAs are spin probes that perturb the membrane at different depths depending on position of the doxyl group on the fatty acyl chain. 16-DSA and 12-DSA rapidly and reversibly reduced gj to unmeasureable levels in a dose- and time-dependent manner. Single channel events observed when gj was low were of the same unitary size as those observed under control conditions. The methyl esters of 16- and 12-DSA, stearic acid itself, and TEMPO, an analogue of the doxyl group that has no fatty acyl chain, had no effect on gj. Protonation of the carboxyl head group (by acidifying the solution) reduced the potency of 16- or 12-DSA. Spontaneous beating activity and action potentials were observed at concentrations of the DSAs 15-20 times that necessary for uncoupling. These results indicate that uncoupling by the DSAs requires the presence of the charged carboxyl group and localized perturbation of the channel at the lipid-channel interface by the doxyl group. Furthermore, they predict that unsaturated free fatty acids, which accumulate during ischemia, may exert their arrhythmogenic effect by reducing gj, and thereby slowing conduction.

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