Comparative study on the effects of exogenous palmitate and erucate on intracellular electric properties of cultured beating heart cells.
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Biomedical subjects
Publications and source records attributed to C Frelin.
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Electrical properties of cultured newborn rat heart cells are investigated by the use of microelectrophysiological methods. Amplitudes of resting and action potentials appear close to those of in situ heart cells. Elevated spike rate of rise reveals functional fast sodium channels. An inconstant ratio of cells exhibit pacemaker-like activity but no relationship can be established between this automaticity and the tissular origin of the cultured cells. The pulsation rate appears to be linked to the action potential duration and to the pace-maker potential slope. Spontaneous arrhythmias may occur; they are mainly caused by anomalous conduction and (or) erratic pacemaker driving. Thus heart cell cultures may be considered as a precious tool in the field of the cardiac electrophysiologal and physiopathological studies.
The metabolism of glucose and palmitate by new born rat heart cell in tissue culture was investigated to ascertain the usefulness of this model system for metabolic studies. Heart cells in culture oxidize palmitate at a rate which is close to the rate reported in vivo. The fate of glucose is shown to differ from in vivo. High rates of lactate release are observed although there are many evidences that cells are not hypoxic. This impaired regulation is likely due to an impaired uptake of glucose.
Each medium renewal of confluent primary heart cell cultures derived from new born rats induces a pleiotypic response which leads to active proliferation. The presence of serum in the culture medium is essential for this activation of growth. Nutrient starvation prior to the activation decreases the response of the cells to serum. Serum starvation prior to the activation increases the serum dependence of the incorporation of labelled leucine but leaves the serum dependence of DNA synthesis unchanged. Ageing in culture decreases the serum dependence of the incorporation of labelled thymidine and amino acids but maintains it for alpha amino isobutyric acid transport. Several active components in human serum were distinguished by fractionated dialysis. A single dialyzable component stimulates both thymidine and amino acid incorporations. The transport of 2 deoxy-D-glucose is activated by another rapidly dialyzing component. The activation of alpha amino isobutyric acid transport may result from several components that are distanct from the previous ones. These results imply that a multiplicity of controls underly the pleiotypic activation of heart cell cultures by medium changes.
The transport of alpha-aminoisobutyric acid has been studied as a function of age in heart cell cultures derived from new-born Rats. The specific rate of alpha-aminoisobutyric acid transport decreased slightly once confluency had been reached and then, from day 8 on, increased abruptly. This last phase may be related to the expression of specialized functions encountered in older cultures.
Intracellular investigation of bioelectrical properties is performed on cultivated rat myocardial cells. Electrophysiological parameters of cells dissociated from rat myocardium are very similar to those of in vivo cells. Fast sodium channels are found to be fonctional. Spontaneous arrhythmia are recorded from some impaled cells.
The pleiotypic effects of medium replacement were studied in rat heart cell cultures. After each medium change alpha-aminoisobutyric acid and glucose transport are increased, RNA and protein syntheses are activated. DNA synthesis did not begin before 12 hours and was followed by a wave of mitoses. This sequence of events suggests that the stimulated cells were in early G 1 phase. DNA synthesis, following the shift to a fresh medium, is linearly related to the amount of serum used as is protein synthesis. However when serum concentrations higher than 20 percent were used no increased protein synthesis could be observed suggesting the existence of another limiting factor, which was probably the isoleucine content of the medium. The serum stimulating factor is heat stable, dialysable and was found in both human and fetal calf sera.
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Improvement in the method of heart cell cultures is described and justified in relation to environmental factors. The validity of such cardiac cell culture for cardiac research is discussed in light of particular cellular activities: differentiation of lipoprotein lipase, myoglobin biosynthesis, glucose and fatty acid metabolism pleiotypic responses (rotein, RNA, and DNA biosyntheses, substrates transports) to serum stimulation, and the architectonic growth of muscle and nonmuscle cells.
Palmitic acid oxidation has been studied in cultures of beating heart cells. The data reported suggest: 1. The main source of fatty acids for oxidation in cell cultures is provided by lypolysis of preexisting intracellular fatty acids. Most fatty acids that have just entered the cell are not oxidized immediately. Thus, aged heart cells in culture seem to retain their ability to utilize fatty acids as their main energy source. 2. In cultured heart cells, the amount of CO2 obtained from the carboxyl group was much higher than that obtained from the methyl group. On the other hand, beta-hydroxybutrate has been shown to be formed mainly from the methyl group. This may indicate that beta-oxidation terminates at the beta-hydroxybutyrate stage because of premature dissociation of this last intermediate from the enzyme complex.
The multiple effects of serum on metabolic activities and macromolecular syntheses of heart cell cultures are discussed in this chapter. This pleiotropic response, which is linearly related to the amount of serum used, allowed us to quantitatively test the growth-promoting activity of different hormones. In addition, the possible mediation of the serum effects by cyclic nucleotides is considered.