Oxidative phosphorylation properties of mitochondria isolated from transplanted hepatoma.

Mitochondria were isolated from Morris hepatomas with rapid (types 3683, 7777, and 3924A) and intermediate (types 5123D and 7800) growth rates, using proteolytic digestion of minced tumor tissue to release the particles. Mitochondria isolated by the same procedure from rat liver were employed as controls. All the hepatoma mitochondria were capable of coupled respiration with normal phosphorylation yields (ADP/O) and respiratory control ratios ranging from 2 to considerably more than 10. Particles from hepatomas 7777 and 7800 exhibited properties closest to liver mitochondria, while those from hepatomas 3683 and 3924A showed the greatest difference. All the hepatoma mitochondria were capable of oxidizing succinate, 3-hydroxybutyrate and monoamines. However, the oxidation rates of the latter two substrates by mitochondria from hepatomas 3683 and 3924A were only a fraction of the control rates. These differences appeared to be due, at least in part, to the structural instability of the isolated hepatoma mitochondria. In contrast to the reports of others, all hepatoma mitochondria exhibited considerable stimulation of ATPase activity by uncouplers. Maximal stimulation of ATPase activity by representatives of three classes of uncouplers was in all instances comparable to the values obtained for rat liver mitochondria.     

The effect of corticotropin on phospholipid metabolism in isolated adrenocortical cells.

Trypsin-dispersed cat adrenocortical cells were incubated at 37 degrees C in modified Eagle's medium containing [14C]arachidonic acid of sodium [14C]-acetate and then in non-radioactive medium. Radioactive incorporation was obtained in all phospholipids, with the greatest amount of radioactivity in phosphatidylcholine, followed by phosphatidylethanolamine, phosphatidyl-serine, and phosphatidylinositol. Concentrations of individual phospholipids generally paralleled the relative amounts of corresponding radiolabeled phospholipids, although the percentage of phosphatidylinositol was considerably lower than its radioactive counterpart, resulting in a high specific activity of this particular phospholipid. Although a potently steroidogenic concentration of corticotropin failed to enhance release of label from any particular phospholipid, analysis of specific activity showed that corticotropin stimulation was accompanied by an increased turnover of phosphatidylinositol and phosphatidic acid. These studies demonstrate that isolated cortical cells have the capacity to synthesize phospholipids from radioactive precursors. The finding that the acute effects of corticotropin are associated with changes in specific phospholipids, including phosphatidylinositol and phosphatidic acid, conforms to the general pattern observed in other secretory systems.     

Early reactions of cadmium with Ehrlich cells.

Ehrlich ascites tumor cells accumulate cadmium against a concentration gradient in a bisphasic uptake process. There is little efflux of the metal from preloaded cells into a cadmium-free medium. Incorporation of 3H-thymidine into DNA is markedly inhibited by cadmium ion at 5-100 ng atoms of Cd/mg of cell protein, but uptake of the nucleoside label into cells is not depressed in this concentration range. Cell respiration is much less affected by cadmium ion despite the sensitivity of isolated mitochondria to the metal. Model experiments using several cadmium complexes with known conditional formation constants show that bovine heart mitochondria have strong affinity for cadmium ion. The contrast between this result and the resistance of cells to respiratory inhibition with cadmium ion is discussed to illustrate the difficulty in relating in vitro studies to the cell. The behavior of cadmium ion with the Ehrlich cell is compared with data for zinc ion to reveal similarities in inhibition of nucleoside metabolism and respiration but a sharp difference in transport properties.     

Oxidative and phosphorylative activities of mitochondria isolated from cotton hypocotyls

Mitochondria isolated from 2 strains of cotton plant hypocotyls (Gossypium hirsutum L. var. Rex smooth leaf and Rex glandless) were examined for their oxidative phosphorylation activities. Bovine serum albumin at a relatively high concentration was essential in the extraction medium for the isolation of oxidatively active mitochondria from both strains of cotton. Phosphorylation was obtained only with Rex glandless cotton mitochondria. This activity was low in comparison to the mitochondria isolated from soybeans (Glycine max L. var. Lee). The endogenous gossypol content was found to be much higher in the Rex smooth leaf tissue than in the Rex glandless tissue. In turn, comparable gossypol differences were found associated with their respective mitochondrial fractions. Exogenous gossypol uncoupled succinate oxidation with active mitochondria isolated from soybeans. Gossypol as a possible uncoupler is discussed and compared to carbonyl cyanide, m-chlorophenyl hydrazone and 2,4-dinitrophenol.     

Lung oxygen consumption and mitochondria of alveolar epithelial and endothelial cells.

We examined oxygen consumption by lung slices and measured the volume density of mitochondria of granular pneumocytes, alveolar type I cells, and alveolar capillary endothelial cells in several species. We found that lung oxygen consumption (mu-1 02 times h-1 times mg DNA-1) varies inversely with the log of animal body weight and with the species alveolar diameter and directly with the species respiratory rate. The volume density of granular pneumocyte mitochondria show a direct linear correlation with the lung's oxygen consumption and the species respiratory rate, and an inverse linear correlation with the species alveolar diameter. The volume density of mitochondria in type I alveolar epithelial cells and capillary endothelial cells, considered together, did not differ in the two species studied (mouse and rat). We conclude that there are interspecies differences in oxygen consumption by lung cells and that granular pneumocytes contribute to these differences. We suggest that, at least part of these differences, are related to interspecies differences in surfactant secretory activity.     

Sodium ion transport in isolated intestinal epithelial cells. II. Comparison of the effect of actively transported sugars on sodium ion efflux in cells isolated from jejunum and ileum.

Na+ transport studies in intestinal epithelial cells indicate that enterocytes from different regions of the small intestine differ in their response to actively transported sugars. 1. Compared with sugar-free medium total Na+ efflux rate constants from isolated rat jejunal cells were significantly increased when medium contained actively transported sugars, glucose and galactose, but not when medium contained fructose. 2 In contrast total Na+ efflux rate constants from isolated rat ileal cells did not respond to actively transported sugars, glucose and galactose. 3. Similar results for the effect of actively transported sugars on Na+ ellux were obtained for isolated rabbit jejunal and ileal epithelial cells. 4. Passive Na+ efflux rate constants for isolated jejunal and ileal enterocytes are not significantly different, indicating similiar permeability characteristics.     

In vitro effect of ethanol on polyphosphoinositide metabolism in bulk isolated brain cells.

Metabolism of polyphosphoinositide was studied in bulk isolated brain cells. Cells were isolated by a rapid method using mechanical disruption followed by molecular seiving and centrifugation. Incorporation of [32Pi]orthophosphate into phosphatidyl inositol-4-phosphate and phosphatidylinositol-4,5 bis-phosphate was optimum at 30 and 60 min, respectively, in the isolated cells. Breakdown studies showed maximum loss of 32Pi after 60 min. Addition of ethanol at and above 10 mM concentration in vitro significantly decreased the incorporation of 32Pi into both phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5 bis-phosphate by the isolated cells. However, the spontaneous breakdown of polyphosphoinositide was not altered in the presence of ethanol in vitro.     

The effect of insulin on macrophage metabolism and function

This study examined the effect of insulin on rat macrophage metabolism and function. The following parameters were studied: cell migration in response to thioglycollate and BCG stimuli, macrophage phagocytic capacity, H₂O₂ production, glucose and glutamine metabolism as indicated by the measurement of enzyme activities, the utilization of metabolites and production and oxidation of substrates. The results indicate that insulin: (1) did not affect cell migration in response to thioglycollate and BCG; (2) enhanced the phagocytic capacity of macrophages and the production of H₂O₂ by macrophages; (3) increased the metabolism of glucose and reduced that of glutaminase.     

Sodium ion transport in isolated intestinal epithelial cells. The effect of actively transported sugars on sodium ion efflux

A technique to measure Na⁺ efflux from isolated intestinal epithelial cells has permitted us to examine the mechanisms responsible for Na⁺ transport in absorptive cells without contamination by other cell types. We examined the effect of actively transported sugars on Na⁺ efflux from isolated rat jejunal epithelial cells to evaluate the mechanism by which actively transported non-electrolytes stimulate Na⁺ absorption. Glucose, galactose and 3-O-methylglucose, sugars known to be actively transported by the small intestine, stimulate total Na⁺ efflux from isolated epithelial cells. This stimulation results from an increase of active Na⁺ transport, since it is inhibited by ouabain. Glucose stimulation is significantly greater than that produced by galactose or 3-O-methylglucose, 2-Deoxyglucose, a sugar that is not actively transported, has no effect on total Na⁺ efflux from isolated cells. Phloridzin, which has no effect on Na⁺ efflux in a sugar-free medium, completely abolishes the effect of galactose. These findings (a) support the hypothesis that the increase in intestinal absorption of Na⁺ in the presence of actively transported non-electrolytes occurs by a transcellular route; and (b) are consistent with the ion-gradient model. The results are not compatible with the direct energy-coupling model.     

Calcium ion accumulation and volume changes of isolated liver mitochondria. Calcium ion-induced swelling

1. Liver mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing an oxidizable substrate and phosphate accumulated added Ca²⁺. During this process H⁺ appeared in the medium and the mitochondrial suspension showed increased light-scattering. Respiration was markedly stimulated. 2. The addition of excess of Ca²⁺, respiratory inhibitors or uncoupling agents caused extensive mitochondrial swelling associated with release of Ca²⁺ into the suspending medium. When the suspension became anaerobic extensive swelling also occurred. Only under conditions when the addition of uncoupling agents would have produced high rates of electron transport, e.g. in the presence of succinate, was the structural integrity of the mitochondrion maintained after Ca²⁺ accumulation. 3. Conditions that prevented respiration-dependent Ca²⁺ accumulation also prevented Ca²⁺-induced swelling. Bovine plasma albumin was without effect, indicating that U-factor was not involved. Oligomycin together with ADP or ATP partially stabilized the mitochondria against Ca²⁺-induced swelling. 4. It is suggested that a `high-energy' intermediate generated by coupled electron transport is required to prevent the mitochondrial swelling that results as a consequence of Ca²⁺ accumulation.     

Top-down elasticity analysis and its application to energy metabolism in isolated mitochondria and intact cells

This paper reviews top-down elasticity analysis, which is a subset of metabolic control analysis. Top-down elasticity analysis provides a systematic yet simple experimental method to identify all the primary sites of action of an effector in complex systems and to distinguish them from all the secondary, indirect, sites of action. In the top-down approach, the complex system (for example, a mitochondrion, cell, organ or organism) is first conceptually divided into a small number of blocks of reactions interconnected by one or more metabolic intermediates. By changing the concentration of one intermediate when all others are held constant and measuring the fluxes through each block of reactions, the overall kinetic response of each block to each intermediate can be established. The concentrations of intermediates can be changed by adding new branches to the system or by manipulating the activities of blocks of reactions whose kinetics are not under investigation. To determine how much an effector alters the overall kinetics of a block of reactions, the overall kinetic response of the block to the intermediate is remeasured in the presence of the effector. Blocks that contain significant primary sites of action will display altered kinetics; blocks that change rate only because of secondary alterations in the concentrations of other metabolites will not. If desired, this elasticity analysis can be repeated with the primary target blocks subdivided into simpler blocks so that the primary sites of action can be defined with more and more precision until, with sufficient subdivision, they are mapped onto individual kinetic steps. Top-down elasticity analysis has been used to identify the targets of effectors of oxygen consumption in mitochondria, hepatocytes and thymocytes. Effectors include poisons such as cadmium and hormones such as tri-iodothyronine. However, the method is more general than this; in principle it can be applied to any metabolic or other steady-state system.     

Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production

The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity.