Maria W. Seraydarian

Oxygen consumption of mammalian myocardial cells in culture: Measurements in beating cells attached to the substrate of the culture dish

A Lucite attachment which permitted the measurement of oxygen consumption in cells in culture without manipulating the cells was constructed. The attachment fit over commercially available dishes for cell culture and had an oxygen electrode built into it. Oxygen uptake of cells in culture was thus measured. Cells were attached to the substrate of the culture dish during the measurements and could be observed in an inverted phase microscope. Cells did not show any morphological changes, e.g., cell shapes or beating rate in case of myocardial cells, before and after the measurements of oxygen consumption. Using this method the rate of oxygen consumption was determined in rat myocardial and heart non-muscle cells in culture and also in HeLa and L6 cell lines. Myocardial cells in culture had an approximately four times higher rate of oxygen uptake compared with heart non-muscle, HeLa, and L6 cells. The oxygen uptake of beating myocardial cells was higher by about 50% compared with quiescent myocardial cells.

In vitro studies of beating heart cells in culture XII. The utilization of ATP and phosphocreatine in oligomycin and 2-deoxyglucose inhibited cells

Abstract 1. The ATP concentration in the cultured rat heart cells did not change significantly when either glycolysis or oxidative phosphorylation was inhibited with 2-deoxyglucose and oligomycin, respectively. When both glycolysis and oxidative phosphorylation inhibitors were present simultaneously, the ATP level dropped significantly. The combined effect of both inhibitors exceeded the sum of the effects of the individual inhibitors. 2. The utilization of ATP in the presence of either oligomycin or 2-deoxyglucose and in the presence of both inhibitors simultaneously was reflected in a decrease in phosphocreatine concentration. 3. The rate of high energy phosphate utilization in quiescent cells was calculated to be 0.289 ± 0.040 nmole/mg protein per min. The utilization of energy for contraction was calculated to be 0.055 ± 0.010 nmole/mg protein per contraction. 4. Taking into consideration the energy requirements of beating cells and the change in ATP + phosphocreatine when the inhibitors were present singly and simultaneously, it was concluded that a control of metabolism involving glycolysis and oxydative phosphorylation is operating under normal oxidative conditions. The sum to the rates of the two pathways, when operating alone, is greater than the sum of their rates when operating simultaneously.

In vitro studies of beating heart cells in culture XIII. The effect of 1-fluoro-2,4-dinitrobenzene

Abstract 1-Fluoro-2,4-dinitrobenzene (FDNB) caused the cessation of beating of heart cells in tissue culture. The beating stopped after 5 to 10 min in 0.19 m m FDNB. The cessation of beating in FDNB-treated cells correlated with a decrease in ATP to 70–80% of the control level, without any change in phosphocreatine concentration. FDNB appeared therefore to inhibit ATP:creatine phosphotransferase. In a time period equivalent to that used in the FDNB experiments (5–10 min), cells incubated with 2-deoxyglucose (2-DG) + oligomycin continued to beat, and the change in ATP concentration was very small. The total high energy phosphate was greatly depleted and reflected in phosphocreatine decrease. The total change in high-energy phosphate was the same in the presence of oligomycin + 2-DG as in FDNB + oligomycin + 2-DG. In the former case the energy utilization was reflected in a phosphocreatine depletion, while in the latter in the ATP level. It was concluded that the spontaneous contractions of heart cells utilize ATP directly and are independent of the phosphocreatine level as long as the ATP level is essentially undiminished. Possible effects of FDNB on metabolism, contractile proteins, and 45Ca uptake and washout were investigated.

Studies on the frog's sartorius at different stages of activity.

Abstract Myosin was prepared from I g of frogs sartorius muscle and the yield was 20 mg to 30 mg of purified protein per gram wet weight tissue. The extractability of myosin was the same whether the muscle was at rest, in contraction or in fatigue. The adenosine triphosphate concentration was not changed in activity nor in fatigue and thus the extractability of myosin could be related to the unchanged concentration of adenosine triphosphate, but not to the state of activity of the muscle. The adenosine triphosphate activity and the concentration of sulfhydryl groups were the same in pairs of muscles either at rest and in contraction or at test and in fatigue. A fifteen fold decrease in the rate of relaxation of a muscle fatigued by single twitches suggests a progressive change in the relaxing factor system during fatigue.

Regulation of energy metabolism and intracellular energy transport in muscle

Abstract Energy production in contractile tissues is coupled to energy utilization, so that in the myocardium for example enough energy is available for the heart to function over a wide range of activity from sleep to exhaustive exercise. The coupling is precise and therefore the production of energy must be regulated precisely. It is proposed that the creatine-phosphoryl - creatine-mitochondrial - creatine-kinase system plays an essential role in such regulation and provides for energy shuttle from the mitochondria to the site of energy utilization.

The effect of inhibitors on the calcium exchange of heart cells in tissue culture

The effect of oligomycin + 2-deoxyglucose (2-DG), 1-fluoro-2,4-dinitrobenzene (FDNB) and 2,4-dinitrophenol (DNP) on the 45Ca exchange of rat cardiac cells in culture was investigated. All three inhibitors caused the cessation of spontaneous beating of the cells with a concomitant decrease of ATP to 70 to 80% of control value. It is not known which step in the spontaneous rhythmic contraction is sensitive to the decrease of ATP and therefore it was of interest to study the 45Ca exchange as the possible senstive site. A previously described method for direct counting of cellular activity was used. In the absence of the inhibitors the cellular washout curves were resolved into three components. The most rapid component represents residual labeling solution, the next component (λ = 1.02 min−1) represents a portion of cellular Ca2+, while the last slow component seems to involve mitochondrial binding of Ca2+. In the presence of oligomycin + 2-DG and FDNB, the washout patterns were the same as in the control cells. The cessation of beating in the presence of these inhibitors therefore cannot be interpreted in terms of any effect on Ca2+ exchange. However, in the presence of DNP, the washout curve could be resolved into two phases only, the fast extracellular and the fast cellular phases were present but the slowest third phase was missing and no 45Ca uptake corresponding to the slowly exchanging phase of the cell was detected. This result gives support to the interpretation that the third phase of the washout pattern represents predominantly mitochondrial exchange.

Isozymes of Creatine Kinase in Mammalian Myocardial Cell Culture

The high resting metabolism of the myocardium, a precise coupling of energy production to energy utilization and an efficient access to the available energy in the process of contraction are the functional characteristics of adult myocardium. The mechanisms which subserve these parameters are investigated.

PHOSPHAGEN IN MYTILUS CALIFORNIANUS.

Abstract 1. 1. N-phosphorylarginine of muscle extracts of Mytilus californianus, and some other species investigated, gives three distinct spots on two-dimensional chromatography. 2. 2. The three fractions, AP1, AP2 and AP3, had a 1:1 ratio of arginine to phosphate (P). One minute hydrolysis in 0·5 N HCl at 100°C resulted in splitting to arginine and inorganic P. None of the fractions contained either carbohydrate, amino acids other than arginine, or acetyl. AP2 was found to contain Ca2+. 3. 3. All three fractions were substates for arginine phosphoryltransferase; however, when Mg2+ was omitted from the assay system only AP1 showed a liberation of free arginine. 4. 4. AP1, AP2 and AP3 differed in the rate of incorporation of either arginine-C14 or P32. AP1 became labeled first, followed by AP2, and AP3, in P32 experiments. AP1 and often AP1 and AP2 were labeled with arginine-C14. 5. 5. In several experiments, when AP1 was the only fraction labeled with arginine-C14, AP2 became radioactive upon stimulation of the ABRM. A possibility of selective participation, of the three fractions, in muscle contraction is discussed.