scholarly journals Effect of 5′-deoxy-5′-isobutylthioadenosine on formation and release of adenosine from neonatal and adult rat ventricular myocytes

1993 ◽  
Vol 291 (3) ◽  
pp. 833-839 ◽  
Author(s):  
P Meghji ◽  
A C Skladanowski ◽  
A C Newby ◽  
L L Slakey ◽  
J D Pearson
Keyword(s):  
Neonatal Rat ◽  
Nucleoside Transport ◽  
Minor Role ◽  
Heart Cells ◽  
Polymorphonuclear Leucocytes ◽  
Intact Cells ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Atp Concentration ◽  
Rat Myocytes

1. Studies in rat polymorphonuclear leucocytes have suggested that 5′-deoxy-5′-isobutylthioadenosine (IBTA), an inhibitor of the IMP-selective cytosolic 5′-nucleotidase, may be used to test its role in adenosine formation in intact cells. We investigated adenosine formation in neonatal and adult rat cardiomyocytes. 2. 2-Deoxyglucose (30 mM) with oligomycin (2 micrograms/ml) induced a 90-100% fall in ATP concentration in 10 min in neonatal and 60 min in adult heart cells. Adenosine accumulation was substantially increased, accounting for 13% of the fall in ATP concentration in neonatal cells and 56% in adult cells. 3. Anti-(rat liver ecto-5′-nucleotidase) serum did not inhibit adenosine accumulation. Furthermore, dipyridamole (10 microM), a nucleoside-transport blocker, inhibited by 80% the appearance of the newly formed adenosine in the medium, showing that adenosine is produced intracellularly by both adult and neonatal-rat myocytes in response to inhibition of oxidative metabolism. 4. IBTA (3 mM) inhibited by 80% the appearance of adenosine in the medium, but did not inhibit total adenosine accumulation by neonatal-rat myocytes and only modestly inhibited total adenosine accumulation by adult myocytes. 5. IBTA, like dipyridamole, inhibited incorporation of extracellular adenosine (10 microM) into neonatal and adult ventricular myocyte nucleotides by 60-70%. Transport of IBTA (100 microM) into the cells did not appear to be inhibited by dipyridamole (30 microM). 6. We conclude that IBTA acted primarily to inhibit adenosine release from myocytes. The small effect on adenosine formation rates implies that the IMP-selective cytosolic 5′-nucleotidase plays a minor role in this tissue.

scholarly journals The control of adenosine concentration in polymorphonuclear leucocytes, cultured heart cells and isolated perfused heart from the rat

1983 ◽  
Vol 214 (2) ◽  
pp. 317-323 ◽  
Author(s):  
A C Newby ◽  
C A Holmquist ◽  
J Illingworth ◽  
J D Pearson
Keyword(s):  
Adenine Nucleotide ◽  
Maximal Activity ◽  
Adenosine Kinase ◽  
Neonatal Rat ◽  
Heart Cells ◽  
Polymorphonuclear Leucocytes ◽  
Perfused Heart ◽  
Intact Cells ◽  
Adenosine Concentration ◽  
Rat Heart Cells

Rat polymorphonuclear leucocytes or neonatal-rat heart cells in culture were treated with 2′-deoxycoformycin and 5-iodotubercidin at concentrations that inhibited adenosine deaminase (EC 3.5.4.4) and adenosine kinase (EC 2.7.1.20) inside the intact cells, and the rate of adenosine accumulation was determined. The basal rate of adenosine formation was 2% (polymorphonuclear leucocytes) or 9% (heart cells) of the maximal activity of adenosine kinase also measured in intact cells. Greatly increased rates of adenosine formation were observed during adenine nucleotide catabolism. This condition also led to a decrease in adenosine kinase activity. When isolated rat hearts were perfused with 5-iodotubercidin alone at a concentration which inhibited adenosine kinase, no increase in tissue or perfusate adenosine or inosine concentration was observed. However, perfusion with hypoxic buffer or infusion of adenosine into the coronary circulation at a rate (20 nmol/min) equivalent to 40% of the activity of adenosine kinase caused large increases in effluent perfusate adenosine and inosine concentrations. These data argue unanimously against the existence of a substrate cycle controlling adenosine concentration. They suggest instead that an increase in the rate of adenosine formation is the principal cause of elevations in adenosine concentration during ATP catabolism.

Mechanical stimulation of organogenic cardiomyocyte growth in vitro

1996 ◽  
Vol 270 (5) ◽  
pp. C1284-C1292 ◽  
Author(s):  
H. H. Vandenburgh ◽  
R. Solerssi ◽  
J. Shansky ◽  
J. W. Adams ◽  
S. A. Henderson
Keyword(s):  
Mechanical Load ◽  
Mechanical Stretch ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Heart Cells ◽  
Mechanical Stimuli ◽  
Rat Cardiomyocytes ◽  
Morphological Alterations

Adherent cultures of neonatal rat cardiomyocytes were subjected to progressive, unidirectional lengthening for 2-4 days in serum-containing medium. This mechanical stretch (25% increase in initial length each day) simulates the eccentric mechanical load placed on in vivo heart cells by increases in postnatal blood pressure and volume. The in vitro mechanical stimuli initiated a number of morphological alterations in the confluent cardiomyocyte population which were similar to those occurring during in vivo heart growth. These include cardiomyocyte organization into parallel arrays of rod-shaped cells, increased cardiomyocyte binucleation, and cardiomyocyte hypertrophy by longitudinal cell growth. Stretch stimulated DNA synthesis in the noncardiomyocyte population but not in the cardiomyocytes. Myosin heavy chain (MHC) content increased 62% over 4 days of stretch and included increased accumulation of both fetal beta-MHC and adult alpha-MHC isoforms. This new model of stretch-induced cardiomyocyte hypertrophy may assist in examining some of the complex mechanogenic growth processes that occur in the rapidly enlarging neonatal heart.

scholarly journals Silica nanoparticles induce cardiotoxicity interfering with energetic status and Ca2+ handling in adult rat cardiomyocytes

2017 ◽  
Vol 312 (4) ◽  
pp. H645-H661 ◽  
Author(s):  
Carlos Enrique Guerrero-Beltrán ◽  
Judith Bernal-Ramírez ◽  
Omar Lozano ◽  
Yuriana Oropeza-Almazán ◽  
Elena Cristina Castillo ◽  
...  
Keyword(s):  
Silica Nanoparticles ◽  
Glutathione Depletion ◽  
Energy Status ◽  
Adrenergic Stimulation ◽  
Rat Cardiomyocytes ◽  
Atp Production ◽  
Adult Rat ◽  
Intracellular Ca ◽  
Rat Myocytes ◽  
Potential Risks

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO2) has emerged as a promising therapy vector for the heart. However, its potential toxicity and mechanisms of damage remain poorly understood. This study provides the first exploration of SiO2-induced toxicity in cultured cardiomyocytes exposed to 7- or 670-nm SiO2 particles. We evaluated the mechanism of cell death in isolated adult cardiomyocytes exposed to 24-h incubation. The SiO2 cell membrane association and internalization were analyzed. SiO2 showed a dose-dependent cytotoxic effect with a half-maximal inhibitory concentration for the 7 nm (99.5 ± 12.4 µg/ml) and 670 nm (>1,500 µg/ml) particles, which indicates size-dependent toxicity. We evaluated cardiomyocyte shortening and intracellular Ca2+ handling, which showed impaired contractility and intracellular Ca2+ transient amplitude during β-adrenergic stimulation in SiO2 treatment. The time to 50% Ca2+ decay increased 39%, and the Ca2+ spark frequency and amplitude decreased by 35 and 21%, respectively, which suggest a reduction in sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity. Moreover, SiO2 treatment depolarized the mitochondrial membrane potential and decreased ATP production by 55%. Notable glutathione depletion and H2O2 generation were also observed. These data indicate that SiO2 increases oxidative stress, which leads to mitochondrial dysfunction and low energy status; these underlie reduced SERCA activity, shortened Ca2+ release, and reduced cell shortening. This mechanism of SiO2 cardiotoxicity potentially plays an important role in the pathophysiology mechanism of heart failure, arrhythmias, and sudden death. NEW & NOTEWORTHY Silica particles are used as novel nanotechnology-based vehicles for diagnostics and therapeutics for the heart. However, their potential hazardous effects remain unknown. Here, the cardiotoxicity of silica nanoparticles in rat myocytes has been described for the first time, showing an impairment of mitochondrial function that interfered directly with Ca2+ handling.

Abstract 358: Hypoxia and Hyperglycemia Induces M 6 A Mrna Methylation in Major Heart Cells

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Rajesh Kumari ◽  
Prabhat Ranjan ◽  
Zainab Suleiman ◽  
Jing Li ◽  
Suresh K Verma
Keyword(s):  
Heart Failure ◽  
Endothelial Cells ◽  
Heart Disease ◽  
Cardiovascular Diseases ◽  
Neonatal Rat ◽  
Diabetic Heart ◽  
Diabetic Patients ◽  
Heart Cells ◽  
Rat Cardiomyocytes ◽  
Mrna Methylation

Background: According to American heart association, over 70 % diabetic patients die from heart and stroke related diseases. The term “diabetic heart diseases” includes coronary heart disease, heart failure and diabetic cardiomyopathy in diabetic patients. Epigenetic and epitranscriptomic modifications play critical roles in progress of diabetic heart disease. Recent evidences indicated that m 6 A methylation involved in ischemic cardiomyopathy. But, the role of m 6 A mRNA methylation in cardiovascular diseases along with diabetic co-morbidity factors has not been studied in details. Thus, here we hypothesize that alterations in m 6 A mRNA methylation under hypoxic and hyperglycemic conditions contributes to severity of ischemic heart disease. Method and Results: To address our hypothesis, we have determined the levels of m 6 A mRNA methylation in NRVM, NRVF and HUVEC, HMVE and mouse primary endothelial cells under hypoxic and hyperglycemic conditions. We also examined the m 6 A levels in mice hearts post 5 days of MI. m 6 A mRNA methylation was significantly upregulated both in human and mouse ischemic hearts. Furthermore, hypoxia and hyperglycemia significantly induced m 6 A methylation in neonatal rat cardiomyocytes, fibroblasts, and mouse primary endothelial cells (isolated from WT and db/db mice). Next, we measured the methylation machinery both at RNA and protein levels. Interestingly, in corroboration with our methylation data, the expression of both m 6 A writers (Mettl3 and WTAP) and Readers (YTHDF2) was significantly increased. To determine the target transcripts which were highly methylated post-ischemia, we performed deep sequencing of methylated RNA after their immunoprecipitation using MeRIP-sequencing protocol. Our MeRIP-seq data has suggested a differentially m 6 A methylated targets both in-vitro and in-vivo ischemic sample. Conclusion: Over all, for the first time our data showed that hypoxia and hyperglycemia alters m 6 A mRNA methylation which may contribute to enhance the severity of cardiovascular diseases under hyperglycemic conditions. Further understanding of the mechanisms, may present a novel approach to potentially regulate m 6 A methylation, which may help in preventing/reducing heart failure in diabetic patients.

Augmentation of sarcolemmal Ca by anionic amphiphile: contractile response of three ventricular tissues

1986 ◽  
Vol 250 (2) ◽  
pp. H247-H254 ◽  
Author(s):  
G. A. Langer ◽  
T. L. Rich
Keyword(s):  
Progressive Increase ◽  
Neonatal Rat ◽  
Adult Rabbit ◽  
Head Group ◽  
Intact Cells ◽  
Whole Cells ◽  
Adult Rat ◽  
Ventricular Tissue ◽  
Dodecyl Sulfate ◽  
Rat Ventricle

The anionic amphiphile dodecyl sulfate was used at the concentration of 100 microM, below the concentration at which membrane permeability is compromised. In rat myocardial tissue culture dodecyl sulfate induced a large increase in Ca uptake in the intact cells, of which 84% was distributed in a rapidly exchangeable (t1/2 less than 18 s) and 16% in a slowly exchangeable (t1/2 = 17 min) compartment. Dodecyl sulfate induced a large increase in Ca bound to isolated sarcolemmal membrane of these cells. This increase in membrane binding and the distribution in whole cells is consistent with insertion of dodecyl sulfate in the sarcolemma (SL) with subsequent Ca binding to its anionic head group. With perfusate extracellular calcium concentration ([Ca]o) equal to 1 mM, dodecyl sulfate produced greater than 60% increase in active force in ventricular tissue from adult rabbit and neonatal rat but virtually no increase in adult rat ventricle. Preperfusion with 0.1 mM [Ca]o or 10(-6) M ryanodine markedly increased the relative response of adult rat ventricle to dodecyl sulfate. After a quiescent period in rabbit ventricle, dodecyl sulfate caused a progressive increase of force of each beat compared with control; i.e., the treppe response was increased at each successive beat. This did not occur in adult rat ventricle. These results further clarify the different quantitative role of SL-bound versus sarcotubular Ca in the hearts of different species.

Ginseng: Cardiotonic in adult rat cardiomyocytes, cardiotoxic in neonatal rat cardiomyocytes

Life Sciences ◽  
2006 ◽  
Vol 79 (25) ◽  
pp. 2337-2344 ◽  
Author(s):  
Brian J. Poindexter ◽  
Ashley W. Allison ◽  
Roger J. Bick ◽  
Amitava Dasgupta
Keyword(s):  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Neonatal Rat Cardiomyocytes

IL-1β alters the expression of the receptor tyrosine kinase gene r-EphA3 in neonatal rat cardiomyocytes

1998 ◽  
Vol 274 (1) ◽  
pp. H331-H341 ◽  
Author(s):  
Yun You Li ◽  
Charles F. McTiernan ◽  
Arthur M. Feldman
Keyword(s):  
Tyrosine Kinase ◽  
Cardiac Myocytes ◽  
Differential Display ◽  
Receptor Tyrosine Kinase ◽  
Neonatal Rat ◽  
Kinase Gene ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Protein Levels ◽  
Tnf Α

To identify proinflammatory cytokine responsive genes in the myocardium, we used differential display to study RNA isolated from neonatal rat cardiac myocytes treated with tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Sequence analysis of differential display products confirmed by reverse Northern blots revealed one clone as the partial sequence of an Eph-related receptor tyrosine kinase (r-EphA3). In cardiac myocytes, 36-h exposure to TNF-α and IL-1β reduced r-EphA3 transcripts to 59.9% ( P < 0.01) of control levels; this effect was largely dependent on IL-1β. Western blot analysis showed that changes in r-EphA3 protein levels reflect that seen for transcripts. Cardiac nonmyocytes expressed substantially lower levels of r-EphA3. Full-length r-EphA3 cDNA clone (3,077 base pair) yielded an amino acid sequence with 90–98% homology to the Eph receptor human EphA3, chick EphA3, and mouse EphA3. In the adult rat, r-EphA3 transcripts were most abundant in the heart, brain, and lung. These results suggest that IL-1β may exert its effect on cardiac myocytes at least in part by altering r-EphA3 expression.

Na+/Ca2+exchange in neonatal rat heart cells: antisense inhibition and protein half-life

1998 ◽  
Vol 275 (2) ◽  
pp. C459-C467 ◽  
Author(s):  
Martin K. Slodzinski ◽  
Mordecai P. Blaustein
Keyword(s):  
Half Life ◽  
Cyclopiazonic Acid ◽  
Neonatal Rat ◽  
Start Codon ◽  
Heart Cells ◽  
Rat Cardiomyocytes ◽  
Antisense Knockdown ◽  
Low Concentrations ◽  
Neonatal Rat Heart ◽  
Rat Heart Cells

Cardiac Na+/Ca2+exchanger (NCX) protein half-life ( t ½) and antisense knockdown were studied in primary cultured neonatal rat cardiomyocytes. Protein t ½ was determined using [35S]methionine with a pulse-chase protocol. The 35S signal in NCX was identified by immunoprecipitation and Western blotting. The t ½ of NCX protein was 33 h. Low concentrations (0.5 μM) of chimeric, phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted to the region around the start codon of NCX1 transcript were used to knock down NCX protein and activity. Control myocytes (no oligos or scrambled oligos for at least 4 days) exhibited spontaneous Ca2+ transients (measured with fura 2). The sustained (“diastolic”) Ca2+ concentration in the cytosol ([Ca2+]cyt) of control cells was unaffected by cyclopiazonic acid (CPA) plus caffeine (Caf), which promote depletion of sarcoplasmic reticular Ca2+ stores, but [Ca2+]cytrose in control cells when external Na+ was removed. In contrast, ∼60% of cells treated with AS-oligos for at least 4 days did not exhibit spontaneous Ca2+transients or respond to Na+-free medium; however, CPA + Caf did induce a prolonged elevation in [Ca2+]cytin these cells. In all cells, 50 mM K+ increased [Ca2+]cyt. NCX protein was reduced by ∼50% in cells treated with AS-oligos for 7 days but was not reduced after only 2 days. These biochemical data are consistent with the physiological evidence of NCX knockdown in ∼60% of cells.

Localization of the Na/Ca exchange-dependent Ca compartment in cultured neonatal rat heart cells

1995 ◽  
Vol 268 (1) ◽  
pp. C119-C126 ◽  
Author(s):  
G. A. Langer ◽  
S. Y. Wang ◽  
T. L. Rich
Keyword(s):  
Total Content ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
High Dose ◽  
Heart Cells ◽  
Cell Content ◽  
Intact Cells ◽  
Content Ratio ◽  
On Line ◽  
Rat Heart Cells

It has been previously established, in both adult and cultured neonatal cardiac cells, that there is a discrete Na/Ca exchange-dependent Ca compartment. It has been proposed that a component of junctional sarcoplasmic reticulum (JSR) Ca and Ca bound to the apposed inner sarcolemmal leaflet represent together the subcellular locus of the compartment. The present study examines this proposal. The amount of Ca in the total compartment is measured isotopically in intact functional cells (using the on-line "scintillation disk" technique) under a variety of perfusion conditions. Under identical labeling conditions, sarcolemmal membranes are rapidly (within a few hundred milliseconds) isolated from another set of intact cells by "gas dissection," and the amount of Ca bound to the membranes is measured. Probes that specifically decrease SR Ca content (thapsigargin, caffeine, low-dose ryanodine) decrease total cell content and sarcolemmal binding proportionally. High-dose ryanodine (producing closure of SR channels) markedly reduces sarcolemmal binding relative to total content of the compartment. The sarcolemmal sites saturate between 1 and 2 mM extracellular Ca ([Ca]o), whereas the total compartment saturates between 4 and 6 mM [Ca]o. Below 1 mM [Ca]o, sarcolemmal binding is maintained relative to total compartment content. Finally, the total compartment increases after reversal of the intracellular Na to extracellular Na ([Na]i/[Na]o) gradient with sarcolemmal content-to-total content ratio dependent on the method used to reverse the [Na]i/[Na]o ratio. The results are consistent with localization of the Na/Ca exchange-dependent compartment to the subsarcolemmal region ("cleft") where JSR Ca is in equilibrium with anionic inner sarcolemmal leaflet Ca binding sites.

scholarly journals Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor

2018 ◽  
Vol 115 (10) ◽  
pp. E2419-E2428 ◽  
Author(s):  
Magdalena Korczynska ◽  
Mary J. Clark ◽  
Celine Valant ◽  
Jun Xu ◽  
Ee Von Moo ◽  
...  
Keyword(s):  
Acetylcholine Receptors ◽  
Muscarinic Acetylcholine Receptor ◽  
Dissociation Rate ◽  
Muscarinic Acetylcholine Receptors ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Muscarinic Acetylcholine ◽  
Adult Rat ◽  
Subtype Selectivity ◽  
Subtype Selective

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound ’628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, ’628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of ’628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between ’628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like ’628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

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