Oxidation of carbohydrates and palmitate by intact cultured neonatal rat heart cells.

The rates of uptake and oxidation of glucose, lactate, pyruvate, and palmitate were measured for "mixed" cultures of rat heart cells that exhibit a myocyte-to-fibroblast ratio similar to that observed in vivo. Glucose uptake and conversion to lactate were also measured using enriched cultures of myocytes and fibroblasts. The metabolism of mixed cultures, which contain 70-80% myocytes, closely resembles that of enriched myocyte cultures. The energy production and substrate oxidation rates of cultured neonatal heart cells, adult myocytes, and perfused hearts are compared. It appears that the energy requirements of cultured heart cells are much lower than that of whole tissue. The results suggest that the metabolism of cultured heart cells may be basically the same as those in vivo but appears to be different because of reduced energy requirements in culture.

Effect of calcium on halothane-depressed beating in heart cells in culture.

Heart cells in culture need no external stimulation to contract; they beat rhythmically at a rate and intensity dependent on culture conditions. These cells respond to the general anesthetic 2-bromo-2-chloro-1,1,1-trifluorethane (halothane), with a loss of beating intensity and a lessening of beating rate. Increased calcium concentrations in growth medium reversed the halothane-depressed beating intensity of heart cells in culture; however, increased calcium concentrations had no effect on the halothane-depressed beating rate. Calcium uptake and release took place in two phases, fast and slow. Only the fast calcium uptake was affected by halothane. Like halothane-depressed beating intensity, the halothane-depressed fast calcium uptake also can be reversed by increased calcium in the growth medium of beating heart cells in culture. Data in this manuscript support the theory that general anesthetics dissolve in membranes and thus disrupt membrane function. The anesthetic halothane appears to affect myocardial beating intensity through its ability to disrupt fast calcium uptake. Halothane also depresses the cardiac beating rate, but the data collected do not relate beating rate with calcium metabolism.

Glucose metabolism, insulin effects, and developmental age of cultured neonatal rat heart cells.

Cultured heart cells from 2-3 day old and 5-6 day old neonatal rats have been used as a model system for the characterization of carbohydrate metabolism in developing cardiac tissue. The rate of depletion of glucose from the growth medium was dependent on 1) the age of the animals from which the cultured cells were obtained, and 2) the presence and absence of serum and/or insulin in the growth medium. The glucose depletion rate in insulin and serum-containing medium was 9.63 +/- 0.96 nmol/min/mg protein for heart cell cultures from 2 day old rats and 3.51 +/- 0.68 nmol/min/mg protein in heart cell cultures from 5 day old rats. Appearance of lactate in the medium during these experiments occurred at the rates of 18.6 +/- 7.9 nmol/min/mg and 6.4 +/- 1.2 nmol/min/mg, respectively. In the absence of serum and insulin, the medium glucose depletion rates were 5.7 +/- 1.6 and 2.2 +/- 0.5 nmol/min/mg for cells derived from 2-day-old and 5-day-old rats, respectively. It is apparent from these data that immature cardiac cells depend upon glucose as a primary source of energy for muscle contraction and cellular growth, and that less-efficient energy-yielding metabolic pathways are used to obtain ATP.

Effect of halothane on cardiac sarcoplasmic reticulum Ca2+-ATPase at low calcium concentrations.

The effect of halothane on cardiac sarcoplasmic reticulum Ca2+-ATPase activity was studied at low calcium concentrations (0.4-20 microM). Clinical concentrations of halothane (1%-3%, v/v) were found to depress Ca2+-ATPase activity more severely at lower calcium levels than at the higher calcium levels previously reported (greater than 0.1 mM). An increase in calcium concentration in the external medium of a preparation of isolated cardiac sarcoplasmic reticulum vesicles antagonized the halothane-induced depression of the Ca2+-ATPase activity. The depression of calcium-activated ATPase activity by halothane appears to take place by a competitive-type inhibition. The Ca2+-ATPase Vmax remained constant at 0.175 mumole/min/mg of protein with an increasing Km (0.47 microM-4.09 microM). Halothane depression of sarcoplasmic reticulum function may in part explain the ability of halothane to depress myocardial function.

A comparative study of myosin and its subunits in adult and neonatal-rat hearts and in rat heart cells from young and old cultures.

A possible explanation for the decrease in myosin Ca2+-dependent ATPase activity as rat heart cells age in culture is presented. The subunit structure and enzyme kinetics of myosin from adult and neonatal rat hearts and from rat heart cells of young and old cultures are compared. These studies indicate that the loss in Ca-ATPase activity of myosin from older cultures was an intrinsic property of the myosin itself. Myofibrillar fractions from the indicated four sources showed no qualitative or quantitative differences in electrophoretic patterns. Myosin from older cultures was more sensitive to alkaline denaturation than was myosin from younger cultures, as indicated by its more accelerated loss of K+(EDTA)-dependent ATPase activity after 10 min of incubation at pH 10. Furthermore, myosin from older cultures was more temperature-sensitive, as indicted by a more rapid loss of Ca-ATPase with decrease in assay temperature. It is suggested that there is either a change in conformation of myosin molecules at or near the active site of the enzyme or alternatively there is a change in light chain 1-light chain 2 and/or light-chain-heavy-chain interaction(s) in the myosin molecules under study.

Suppression of milk secretion (exocytosis) by concanavalin a in vitro.

The observation that concanavalin A can inhibit milk secretion was evaluated in an in vitro system employing minced mammary gland or isolated alveoli from lactating rats. Release of milk constituents (casein, lactose and fat globules) into the medium in the presence and absence of concanavalin A was measured during 1 or 2 h incubations. The effect of concanavalin A on glucose uptake and CO2 production of the minced tissue was also studied. Concanavalin A suppressed release of milk components at a concentration as low as 80 micrograms/ml of medium. Respiration of minced mammary tissue in the presence of concanavalin A (100 micrograms/ml of medium) was essentially the same as that of the control. The data are evidence that concanavalin A acts directly on the mammary cell in suppressing milk secretion and that the effect is not due to cytotoxicity.

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors.

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were not dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.

Evaluation of a proteolytic enzyme mixture isolated from crude trypsins in tissue disaggregation.

Trypsin, chymotrypsin and elastase are the pancreatic enzymes required for neonatal rat heart tissue disaggregation. We have previously developed a procedure for the isolation of a mixture of these three enzymes from commercial crude-trypsin samples. Toxic materials present in certain crude-trypsin samples are removed during purification. An evaluation of this mixture was conducted for its ability to disaggregate neonatal rat heart tissue for cell culture. Large numbers of cells were released with minimal cellular damage as determined by their ability to survive and function in culture. Rat lung and kidney tissue also were disaggregated successfully and cultured with this preparation. It is apparent that this enzyme preparation has a potential for disaggregating a wide variety of tissues.

Halothane and the beating response and ATP turnover rate of heart cells in tissue culture.

The effects of halothane on the beating response of rat heart cells in tissue culture were studied using an optical-electronic monitoring device. A dose-response curve was obtained over a concentration range to as much as 5 vol per cent halothane. The clinical dosage of 1 vol per cent halothane decreased the inotropic response of 4-10-day-old cells to 59 plus or minus 10 per cent of the original beating strength; no significant decrease in beating strength was seen in 25-30-day-old cells. One volume per cent halothane caused no significant change in the chronotropic response of the heart cells. Higher concentrations of halothane caused significant negative chronotropic and negative inotropic responses in a dose-related manner. When glycolysis was inhibited by 2-deoxyglucose in the growth medium, the cells became dependent on fatty-acid oxidation and oxidative phorphorylation for energy and showed increased sensitivity to halothane; for example, the chronotropic response to 5-8-day old cells treated with 2-deoxyglucose was decreased approximately 70 per cent by exposure to 3 vol per cent halothane, whereas 4-10-day-old cells maintained on a complete growth medium showed only a 40 per cent decrease. Increasing concentrations of halothane decreased the rate of ATP turnover. This supports evidence suggesting that halothane blocks electron transport in the NADH-coenzyme Q reductase level. The model described provides a means for determining anesthetic potency in a mammalian system in terms of functional as well as metabolic responses. It also provides a means for study of metabolic effects of anesthetics and other drugs.

Effects of cortisol on cultured rat heart cells: lipase activity, fatty acid oxidation, glycogen metabolism, and ATP levels as related to the beating phenomenon.

This paper reports the determination of the ability of rat heart cells in culture to release [(14)C]palmitate from its triglyceride and to oxidize this fatty acid and free [(14)C]palmitate to (14)CO(2) when the cells are actively beating and when they stop beating after aging in culture. In addition, the levels of glucose, glycogen, and ATP were determined to relate the concentration of these metabolites with beating and with cessation of beating. When young rat heart cells in culture are actively beating, they oxidize free fatty acids at a rate parallel with cellular ATP production. Both fatty acid oxidation and ATP production remain constant while the cells continue to beat. Furthermore, glucose is removed from the growth medium by the cells and stored as glycogen. When cultured cells stop beating, a decrease is seen in their ability to oxidize free fatty acids and to release them from their corresponding triglycerides. Concomitant with decreased fatty acid oxidation is a decrease in cellular levels of ATP until beating ceases. Midway between initiation of cultures and cessation of beating the cells begin to mobilize the stored glycogen. When the growth medium is supplemented with cortisol acetate and given to cultures which have ceased to beat, reinitiation of beating occurs. Furthermore, all decreases previously observed in ATP levels, fatty acid oxidation, and esterase activity are restored.