Organ culture of rat heart: maintained high sensitivity of fetal atria before innervation to norepinephrine

1988 ◽  
Vol 66 (7) ◽  
pp. 901-906 ◽  
Author(s):  
Hikaru Tanaka ◽  
Yutaka Kasuya ◽  
Hiroshi Saito ◽  
Koki Shigenobu
Keyword(s):  
Organ Culture ◽  
Culture Medium ◽  
Sympathetic Innervation ◽  
High Sensitivity ◽  
Fold Increase ◽  
Neonatal Rat ◽  
Fetal Period ◽  
Sensitivity Changes ◽  
Β Receptor ◽  
Right Atria

Changes in sensitivity to norepinephrine (NE) of fetal and neonatal rat right atria placed in organ culture were examined. The high sensitivity to NE of the 17-day fetal atria was maintained during organ culture for 5 days. The pD2 value for NE at the 17th day of gestation was 8.66 ± 0.09, and that after organ culture for 5 days was 8.62 ± 0.09. The sensitivity of 1-day-old neonatal artia was significantly lower than that of fetal atria; but when they were cultured for 24 h, there was a 10-fold increase in sensitivity. The pD2 value before culture was 7.59 ± 0.05, and that after culture was 8.54 ± 0.04. NE added to the culture medium prevented this increase in sensitivity. Similar changes were observed in the sensitivity to isoproterenol, but not in the sensitivity to forskolin, indicating that these sensitivity changes were of a postjunctional nature and most likely due to some changes in the β-receptor and (or) its coupling to adenylate cyclase. Therefore, the decrease in myocardial sensitivity to NE observed during the late fetal period is most likely to be caused by factor(s) related to sympathetic innervation.

Rat heart organ culture for the study of trophic substances involved in the maintenance of normal sensitivity to norepinephrine: possible involvement of cAMP and search for other factors

1990 ◽  
Vol 68 (7) ◽  
pp. 898-902 ◽  
Author(s):  
Hikaru Tanaka ◽  
Koki Shigenobu
Keyword(s):  
Organ Culture ◽  
Sympathetic Innervation ◽  
High Sensitivity ◽  
Calcitonin Gene Related Peptide ◽  
Right Atrial ◽  
Intracellular Camp ◽  
Rat Serum ◽  
Related Peptide ◽  
Calcitonin Gene

In organ culture conditions, in the absence of in vivo factors, the newborn rat right atria acquire a high sensitivity to agonists similar to that seen before sympathetic innervation and after denervation. In the present study, we examined the effects of various extracts and substances on the development of supersensitivity to norepinephrine (NE) to obtain information on the in vivo factors that regulate myocardial sensitivity. Addition of rat serum, right atrial extract, superior cervical ganglionic extract, vas deferens extract, carbachol, insulin, cortisone, thyroxin, and neuropeptide Y in the culture medium did not prevent the development of supersensitivity. Addition of NE completely inhibited the development of supersensitivity. This effect of NE was blocked by sotalol but not by phentolamine. Addition of calcitonin gene related peptide, forskolin, and 8-bromo-cAMP partially inhibited the development of supersensitivity. These results are consistent with the view that NE released from sympathetic nerve terminals in the newborn atria maintains myocardial sensitivity at normal level by acting on β-adrenergic receptors, and that the effect may be partially mediated by a rise in intracellular cAMP concentration.Key words: rat neonate cardiac muscle, organ culture, sympathetic innervation, norepinephrine, calcitonin gene related peptide, sensitivity, trophic control.

scholarly journals Responses of hypertrophied myocytes to reactive species: implications for glycolysis and electrophile metabolism

2011 ◽  
Vol 435 (2) ◽  
pp. 519-528 ◽  
Author(s):  
Brian E. Sansbury ◽  
Daniel W. Riggs ◽  
Robert E. Brainard ◽  
Joshua K. Salabei ◽  
Steven P. Jones ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxygen Consumption ◽  
Energy Demand ◽  
Lactate Production ◽  
Fold Increase ◽  
Cellular Protein ◽  
Reactive Species ◽  
Neonatal Rat ◽  
Glycolytic Flux ◽  
Rat Cardiomyocytes

During cardiac remodelling, the heart generates higher levels of reactive species; yet an intermediate ‘compensatory’ stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischaemia or in the failing heart. For this, we measured energetics in control and PE (phenylephrine)-treated NRCMs (neonatal rat cardiomyocytes) under basal conditions and when stressed with HNE (4-hydroxynonenal). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a 2-fold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homoeostasis under conditions of increased oxidative stress.

The Culture in vitro of Urogenital Organs of Pleurodeles waltlii

Development ◽  
1962 ◽  
Vol 10 (4) ◽  
pp. 465-470
Author(s):  
Charles L. Foote ◽  
Florence M. Foote
Keyword(s):  
Organ Culture ◽  
Germ Cells ◽  
Culture Medium ◽  
Developmental Stages ◽  
Rana Catesbeiana ◽  
Sex Differentiation ◽  
Organ Cultures ◽  
Tissue And Organ Culture ◽  
Pleurodeles Waltlii

Earlier reports (Foote & Foote, 1958a, b, 1959) describe growth and maintenance in vitro of larval organs, particularly gonads, of Rana catesbeiana and Xenopus laevis. Immature germ cells of both testes and ovaries are well maintained in vitro, especially if the culture medium is supplemented with watersoluble sex-hormonal substances, although germ cells in process of maturation become necrotic. Recently some urogenital organs from the salamander, Pleurodeles waltlii, have been grown in vitro. Tissues and organs from this amphibian might prove to be more suitable for tissue and organ culture investigations than those of Anurans. Animals at three different ages were used in this study: recently hatched larvae, metamorphosing animals, and adults. To determine whether sex differentiation would occur in vitro, trunk portions of young larvae of Pleurodeles waltlii of developmental stages 37–38 (Gallien & Durocher, 1957) were placed in organ cultures.

Abstract 208: Mitochondrial-targeted Grk2 Increases Superoxide Formation And Impairs Cardiomyocyte Respiratory Reserve Capacity

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Priscila Y Sato ◽  
J K Chuprun ◽  
Jessica Ibetti ◽  
John W Elrod ◽  
Walter J Koch
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Permeability Transition ◽  
Fold Increase ◽  
Confocal Imaging ◽  
Neonatal Rat ◽  
Redox Stress ◽  
Mitochondrial Oxidative Stress ◽  
Neonatal Rat Ventricular Myocytes ◽  
Cellular Dysfunction

β-adrenergic receptors (βARs) are powerful regulators of cardiovascular function and are impaired in heart failure (HF). Signal transduction of βARs is canonically shut down by phosphorylation via G protein-coupled receptor kinase 2 (GRK2) and the subsequent binding of β-arrestins. This process of receptor desensitization is enhanced in HF via the up-regulation of GRK2 and contributes to disease progression. We have recently reported non-canonical actions of GRK2, which contribute to the development of HF independent of βAR desensitization. We have previously shown that GRK2 can act as a pro-death kinase in cardiomyocytes bytranslocating to mitochondria and activating mitochondria permeability transition. This study was designed to gain more understanding of the mitochondrial function of GRK2. We isolated adult cardiomyocytes from cardiac-specific transgenic mice overexpressing GRK2 at levels found in human HF (TgGRK2), and examined superoxide production using the redox sensitive reporter MitoSox Red. Confocal imaging revealed a 4.6 fold increase in superoxide levels in cardiomyocytes overexpressing Grk2 as compared to non-transgenic (NLC) cardiomyocytes (corrected total cell fluorescence 11.59±1.06, TgGRK2 (n= 3 hearts, 88 cells) vs 2.54±0.02 NLC (n=3 hearts, 52 cells), (p<0.001). This indicates that the chronic elevation of GRK2 induces mitochondrial oxidative stress priming the myocyte for enhanced injury. To further explore the mitochondrial actions of GRK2 and consequences of redox stress we examined oxidative phosphorylation by performing oxygen consumption measurements in neonatal rat ventricular myocytes overexpressing GRK2 or GFP-expressing control myocytes. Seahorse analysis showed that cells overexpressing GRK2 have a significant decrease in spare respiratory capacity indicating that cells with elevated GRK2 levels have an impaired capacity to generated ATP during times of stress. Further studies with mutants that limit GRK2 kinase activity or mitochondrial localization demonstrate that mitochondrial GRK2 may be a significant contributor to cellular dysfunction as seen in heart failure.

Development of embryonic rat eyes in organ culture

Development ◽  
1968 ◽  
Vol 19 (3) ◽  
pp. 407-414
Author(s):  
R. Christy Armstrong ◽  
Joel J. Elias
Keyword(s):  
Organ Culture ◽  
Culture Medium ◽  
Folic Acid Deficiency ◽  
Experimental Conditions ◽  
Acid Deficiency ◽  
Series Of Experiments ◽  
Development In Vitro ◽  
Ocular System

Abnormalities of the ocular system which appear in organ culture in Waymouth's medium with freshly added glutamine (Armstrong & Elias, 1968) resemble those caused by transitory pteryolglutamic acid (PGA or folic acid) deficiency in vivo (Armstrong & Monie, 1966). The configurations of such malformations as lens herniations, retinal diverticula, and rosette-like formations of the retina are remarkably similar in both cases. The experiments reported in this paper were undertaken in an effort to understand the mechanisms involved in the production of similar abnormalities by two very different experimental conditions: the addition of glutamine in vitro and the transitory deficiency of PGA in vivo. One series of experiments involved the effects of manipulation of the PGA and glutamine content of the culture medium on eye development in vitro. Parallel studies on PGA-deficiency in vivo were undertaken in conjunction with organ-culture experiments in order to compare the effects on abnormal eye morphogenesis.

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype

1990 ◽  
Vol 259 (6) ◽  
pp. L415-L425 ◽  
Author(s):  
P. E. Roberts ◽  
D. M. Phillips ◽  
J. P. Mather
Keyword(s):  
Epithelial Cell ◽  
Molecular Mechanisms ◽  
Basal Medium ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Number ◽  
Neonatal Rat ◽  
Rat Lung ◽  
Cell Type

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

Abstract 3660: Cardioprotective Effect of Eicosapentaenoic Acid, an Important Fish Oil, through Suppression of Endothelin-1-induced Cardiomyocyte Hypertrophy via PPAR-α

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Nobutake Shimojo ◽  
Subrina Jesmin ◽  
Masaaki Soma ◽  
Seiji Maeda ◽  
Takashi Miyauchi ◽  
...  
Keyword(s):  
Eicosapentaenoic Acid ◽  
Fish Oil ◽  
Fold Increase ◽  
Neonatal Rat ◽  
Cardiomyocyte Hypertrophy ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Pre Treatment ◽  
Cardiovascular Benefit ◽  
Hypertrophic Changes

A growing body of evidences report the cardiovascular benefit of fish oil including eicosapentaenoic acid (EPA) in humans and experimental animals. While many studies link EPA to cardiac protection, the effect of EPA on endothelin (ET)-1-induced cardiomyocyte hypertrophy is unknown. On the other hand, the previous study demonstrated peroxisomal proliferator-activated receptor (PPAR) -α ligand (fenofibrate) prevents ET-1-induced cardiomyocyte hypertrophy. Though EPA is one of the lignads of PPAR-α, there was no study linking relationship between EPA and PPAR-α on hypertrophied cadiomyocyte. The present study investigated whether ET-1-induced cardiomyocyte hypertrophy could be prevented by the pre-treatment of EPA. Cardiomyocytes were accumulated from neonatal rat heart, cultured and at day 4 of culture, the cardiomyocytes were divided into three groups: control, ET-1 (0.1nM) treated and EPA-pre-treated (10μM) ET-1 groups. A 90% increase in cardiomyocyte surface area, a 75% increase in protein synthesis rate and an elevated actinin expression in cardiomyocyte were observed after ET-1 administration and these changes were greatly prevented by EPA pre-treatment. ET-1-induced hypertrophied cardiomyocytes showed a 2.3-fold and 2.1-fold increase in ANP and BNP mRNA expression, respectively, which were also suppressed by EPA pre-treatment. Pre-treatment of EPA could also attenuate phosphorylated JNK (an important component of MAPK cascade), c-Jun and PPAR-α in ET-1-induced hypertrophied cardiomyocytes. In conclusion, the present study showed that ET-1 can induce significant hypertrophic changes in cardiomyocytes with upregulation of important hypertrophic markers, and that this remodeling was effectively prevented by the pre-administration of EPA through suppressing PPAR-α, phosporylated JNK, and c-Jun.

Abstract P215: β3-Adrenergic Receptor Mediated NOS Signaling in Cardiomyocytes

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Vabren L Watts ◽  
Xiaolin Niu ◽  
Karen L Miller ◽  
Lili A Barouch
Keyword(s):  
Molecular Mechanisms ◽  
Pressure Overload ◽  
Fold Increase ◽  
Left Ventricular ◽  
Neonatal Rat ◽  
Regulatory Site ◽  
Ventricular Cardiomyocytes ◽  
Unstimulated Control ◽  
Key Factor ◽  
Nos Isoforms

Beta3 -adrenergic receptors play a pivotal role in modulating cardiac function, though their precise role in the heart remains controversial. We have recently demonstrated alterations in Ca 2+ -dependent NOS isoforms and decreased NOS activity in left ventricular tissue of beta3 -/- mice after pressure overload. However, the exact manner in which beta3-AR signaling regulates these isoforms to stimulate NOS activity at the cardiomyocyte level is not well understood. In this study we used a specific beta3-AR agonist, BRL37344 (BRL), to assess the role of beta3-AR in eNOS and nNOS regulation in hypertrophied isolated neonatal rat ventricular cardiomyocytes (NRVM). To induce hypertrophy we pretreated cells with norepinephrine for 72 hours, which resulted in a 70% increase in cell size and a 25% increase in beta3-AR mRNA expression as compared with non-hypertrophied cells, analyzed by immunocytochemistry and real-time PCR. In hypertrophied cardiomyocytes, BRL administration lead to a time-dependent 5-fold increase in NOS activity, measured by the arginine-to-citrulline conversion assay. beta3-activation also caused a 1.5-fold increase in nNOS phosphorylation at positive regulatory site Ser1416, and dephosphorylation of negative regulatory site Ser847 as compared with unstimulated control. NOS activity and nNOS phosphorylation overlapped in time. In addition BRL induced phosphorylation eNOS-Ser114, which indicates eNOS deactivation. Pretreatment with pertussis toxin (PTX) suppressed BRL-induced nNOS-Ser1416 phosphorylation, nNOS-Ser847 dephosphorylation, and NOS activity, suggesting G i/o dependency. Taken together, our data suggest that BRL regulates NOS signaling in ventricular cardiomyocytes via phosphorylation regulation of nNOS. To our knowledge this is first study to demonstrate a role for nNOS phosphorylation as a key factor in beta3-AR signaling. These results contribute significantly to our understanding the negative inotropic properties of myocardial beta3-AR at cellular levels during cardiac sympathetic overstimulation, and will ultimately aid in drug discoveries that target the molecular mechanisms associated heart failure.

Simplex Optimized Acetylacetone Method for Formaldehyde

1973 ◽  
Vol 56 (6) ◽  
pp. 1496-1502
Author(s):  
Fred P Czech
Keyword(s):  
Standard Deviation ◽  
Experimental Design ◽  
Design Process ◽  
Analytical Methods ◽  
High Sensitivity ◽  
Fold Increase ◽  
Chromotropic Acid ◽  
High Quality ◽  
Relative Standard ◽  
Aoac Method

Abstract The AOAC colorimetric acetylacetone method for formaldehyde in sugar products is optimized by means of the simplex experimental design process. The resultant method is almost 4 times as sensitive as the original AOAC method. It is about 33% more sensitive than the simplex optimized J-acid procedure and 45% more sensitive than the simplex optimized chromotropic acid method and, thus, represents one of the most sensitive methods now available. The average relative standard deviation is about ±2.7%. The limit of detectability is estimated to be 30 ppb. The didactic exposition of the simplex optimization process reported earlier is applied to the AOAC acetyl-acetone method. Further insights into simplex operations are provided and certain advantages of simplex application are pointed out. It is shown that, by sacrificing the high sensitivity of the optimized method the parameters of the new method result in about a 10-fold increase in analytical speed. Further application of kinetic considerations and the advantages of high quality, productivity, and economy in chemical analytical methods by means of the simplex experimental design process are described.

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