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 Insertion of isolated insulin receptors into placental membrane vesicles

1992 ◽  
Vol 281 (2) ◽  
pp. 425-430 ◽  
Author(s):  
K Christiansen ◽  
J Carlsen
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Insulin Receptors ◽  
Membrane Vesicles ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Receptor Number ◽  
Triton X

Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.

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.

scholarly journals Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells

1984 ◽  
Vol 220 (1) ◽  
pp. 165-172 ◽  
Author(s):  
C F H Van Schravendijk ◽  
E L Hooghe-Peters ◽  
P De Meyts ◽  
D G Pipeleers
Keyword(s):  
Mouse Brain ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Cell Types ◽  
Receptor Interaction ◽  
Target Cells ◽  
Scatchard Analysis ◽  
Cortical Cells ◽  
Foetal Brain ◽  
Binding Cell

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.

Kinetic analysis of clearance of epidermal growth factor in isolated perfused rat kidney

1991 ◽  
Vol 261 (6) ◽  
pp. F988-F997
Author(s):  
D. C. Kim ◽  
M. Hanano ◽  
Y. Sawada ◽  
T. Iga ◽  
Y. Sugiyama
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Rat Kidney ◽  
In Vivo Studies ◽  
Scatchard Analysis ◽  
Renal Handling ◽  
Apparent Dissociation Constant ◽  
Epidermal Growth

Our previous in vivo studies identified the saturable uptake of epidermal growth factor (EGF) by rat kidney (D.C. Kim, Y. Sugiyama, H. Sato, T. Fuwa, T. Iga, and M. Hanano, J. Pharm. Sci. 77: 200-207, 1988). In the present study, renal handling of EGF in filtering and nonfiltering isolated perfused rat kidneys was investigated. At designated times after the recirculatory perfusion of 125I-EGF in the nonfiltering kidney, the surface-bound and internalized EGF were separately determined by an acid-washing technique. Time profiles of cell-surface-bound and internalized EGF obtained at the perfusion of a tracer 125I-EGF were fitted to the pharmacokinetic model, and kinetic parameters obtained were as follows: konRs = 0.49 ml.min-1.g-1, koff = 0.87 min-1, kint = 0.20 min-1 where konRs is the binding clearance of EGF with its receptor and koff and kint represent the dissociation and internalization rate constants of the EGF-receptor complex, respectively. The Scatchard analysis of the concentration-dependent EGF binding in the nonfiltering kidney suggests the presence of two binding components, one with high affinity [the apparent dissociation constant (Kd1 = 0.1 nM) and the other with low affinity (Kd2 = 30 nM]. By comparing the internalization clearance (CLi) with filtering and nonfiltering kidneys, we concluded that the renal uptake of EGF occurs mainly from the antiluminal side via receptor-mediated endocytosis in a saturable manner and that the nonsaturable reabsorption of EGF from the luminal membrane after glomerular filtration is relatively small. The contribution of reabsorption, however, becomes larger with the increase in EGF concentration.

scholarly journals GATA4 is a survival factor in adult cardiac myocytes but is not required for α1A-adrenergic receptor survival signaling

2008 ◽  
Vol 295 (2) ◽  
pp. H699-H707 ◽  
Author(s):  
Yuan Huang ◽  
Casey D. Wright ◽  
Satoru Kobayashi ◽  
Chastity L. Healy ◽  
Megan Elgethun ◽  
...  
Keyword(s):  
Cell Death ◽  
Cardiac Myocytes ◽  
Adrenergic Receptor ◽  
Double Knockout ◽  
Survival Factor ◽  
Downstream Effector ◽  
Adult Cardiac Myocytes ◽  
Survival Signaling ◽  
Downstream Mediator

Recently, we defined an α1A-adrenergic receptor-ERK (α1A-AR-ERK) survival signaling pathway in adult cardiac myocytes. Previous studies in neonatal cardiac myocytes indicated that the cardiac-specific transcription factor GATA4 is a downstream mediator of α1-ERK signaling and that phosphorylation of GATA4 by ERK increases DNA binding and transcriptional activity. Therefore, we examined GATA4 as a potential downstream effector of α1A-ERK survival signaling in adult cardiac myocytes. We measured norepinephrine (NE)-induced cell death in cultured cardiac myocytes lacking α1-ARs (cultured from α1A/B-AR double-knockout mice, α1ABKO mice) that are susceptible to cell death induced by several proapoptotic stimuli, including NE. Our results show that overexpression of GATA4 is sufficient to protect α1ABKO cardiac myocytes from NE-induced cell death. However, we found that the α1A-subtype did not induce phosphorylation or increase the activity of GATA4 in adult mouse cardiac myocytes in culture or in vivo. Furthermore, we examined the effect of siRNA-mediated knockdown of GATA4 on α1A-survival signaling. In α1B-knockout cardiac myocytes, which express only the α1A-subtype and are protected from NE-induced cell death, GATA4 knockdown did not reverse α1A-survival signaling in response to NE. In summary, we found that GATA4 acted as a survival factor by preventing cell death in α1ABKO cardiac myocytes, but GATA4 was not activated by α1-AR stimulation and was not required for α1A-survival signaling in adult cardiac myocytes. This also identifies an important mechanistic difference in α1-signaling between adult and neonatal cardiac myocytes.

scholarly journals Measuring α4β2∗ Nicotinic Acetylcholine Receptor Density in Vivo with [18F]nifene PET in the Nonhuman Primate

2013 ◽  
Vol 33 (11) ◽  
pp. 1806-1814 ◽  
Author(s):  
Ansel T Hillmer ◽  
Dustin W Wooten ◽  
Maxim S Slesarev ◽  
Elizabeth O Ahlers ◽  
Todd E Barnhart ◽  
...  
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Scatchard Analysis ◽  
Kinetic Properties ◽  
Receptor Density ◽  
Apparent Dissociation Constant ◽  
Nicotinic Acetylcholine ◽  
Compartment Modeling ◽  
Modeling Analysis

[18F]Nifene is an agonist PET radioligand developed to image α4β2∗ nicotinic acetylcholine receptors (nAChRs). This work aims to quantify the receptor density ( Bmax) of α4β2∗ nAChRs and the in vivo (apparent) dissociation constant ( KDapp) of [18F]nifene. Multiple-injection [18F]nifene experiments with varying cold nifene masses were conducted on four rhesus monkeys with a microPET P4 scanner. Compartment modeling techniques were used to estimate regional Bmax values and a global value of KDapp. The fast kinetic properties of [18F]nifene also permitted alternative estimates of Bmax and KDapp at transient equilibrium with the same experimental data using Scatchard-like methodologies. Averaged across subjects, the compartment modeling analysis yielded Bmax values of 4.8 ± 1.4, 4.3 ±1.0, 1.2 ± 0.4, and 1.2 ± 0.3 pmol/mL in the regions of antereoventral thalamus, lateral geniculate, frontal cortex, and subiculum, respectively. The KDapp of nifene was 2.4 ± 0.3 pmol/mL. The Scatchard analysis based on graphical evaluation of the data after transient equilibrium yielded Bmax estimations comparable to the modeling results with a positive bias of 28%. These findings show the utility of [18F]nifene for measuring α4β2∗ nAChR Bmax in vivo in the rhesus monkey with a single PET experiment.

Pubescence-related changes in hepatocyte insulin dynamics in female rats

1989 ◽  
Vol 256 (6) ◽  
pp. E780-E787
Author(s):  
G. R. Krakower ◽  
A. H. Kissebah
Keyword(s):  
Sexual Dimorphism ◽  
Insulin Binding ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Insulin Degradation ◽  
Subsequent Increase ◽  
Androgen Treatment ◽  
Age Related ◽  
Receptor Number ◽  
Related Increase

Insulin binding and receptor-mediated insulin processing were investigated in isolated hepatocytes from sexually maturing female rats and from age-matched animals that had undergone prepubertal ovariectomy or whose sexual dimorphism had been disrupted by neonatal androgen treatment. Equilibrium insulin binding was determined after 18 h of incubation with 125I-TyrA14-monoiodoinsulin at 4 degrees C. Receptor-mediated insulin processing was studied after overnight binding with 100 pM insulin at 4 degrees C and subsequent incubation at 37 degrees C. Insulin binding at tracer concentrations increased 50% from sexual immaturity at 3-4 wk through pubescence at 6-8 wk and was further increased into young adulthood at 10-12 wk. Scatchard analysis indicated that the altered binding was due primarily to an increase in receptor number. Increased binding with sexual maturation resulted in correspondingly higher levels of insulin in each of the four compartments of processing studied (cell surface bound, internalized, degraded, and released). A corresponding increase in receptor-mediated insulin degradation after 10 min at 37 degrees C was observed, as changes in insulin degradation were proportional to the increase in insulin receptor binding. The age-related increase in insulin binding and subsequent increase in degradation were abolished by prepubertal ovariectomy. Furthermore, disrupting sexual dimorphism by perinatal androgen treatment resulted in a reduction of the age-related increase in insulin binding, but the percentage of insulin degraded was significantly greater than that accounted for by the increase in receptor number. As a result, insulin degradation per unit of receptor-bound hormone was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

The Infection of Cells by Bullet-Shaped Viruses

1969 ◽  
Vol 27 ◽  
pp. 372-373
Author(s):  
Frederick A. Murphy ◽  
Alyne K. Harrison ◽  
Sylvia G. Whitfield
Keyword(s):  
Electron Microscopy ◽  
Physical Properties ◽  
Host Cell ◽  
Thin Section ◽  
Cultured Cells ◽  
Cell Infection ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Section Electron Microscopy

The bullet-shaped viruses are currently classified together on the basis of similarities in virion morphology and physical properties. Biologically and ecologically the member viruses are extremely diverse. In searching for further bases for making comparisons of these agents, the nature of host cell infection, both in vivo and in cultured cells, has been explored by thin-section electron microscopy.

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