Oxidative phosphorylation and ultrastructural transformation in mitochondria in the intact ascites tumor cell.

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90 degrees light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox --> condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90 degrees light-scattering increase, which also occurs at this time, showed a t (1/2) of only 25 sec which agreed temporally with a slower orthodox --> maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox --> condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.

Oxygen consumption of human blood platelets. I. Effect of thrombin.

The effect of thrombin on the oxygen consumption of washed human platelets was measured polarographically with the Clark oxygen electrode. The average basal respiratory rate was 18 plus or minus 1.6 (mean plus or minus S.E.) natoms oxygen per min per 10-9 platelets. Thrombin (1.9 units/ml) caused a 4-13-fold increase in the rate of oxygen consumption (138 plus or minus 14 (mean plus or minus S.E.) natoms oxygen per min per 10-9 platelets). The thrombin-stimulated increase of oxygen consumption was transient, lasting from 1 to 1.5 min before returning to the respiratory rate observed before the thrombin addition. Release of platelet constituents appeared to precede the stimulation of oxygen consumption. These results may provide a basis for explaining the discrepancy in the literature concerning the effects of thrombin on platelet respiration.

Oxygen consumption of human blood platelets. II. Effect of inhibitors on thrombin-induced oxygen burst.

The effect of selected inhibitors on the thrombin-stimulated burst and the basal oxygen consumption of washed human platelets were investigated and compared with inhibition of the release reaction. Cyanide (0.2 mM) caused complete inhibition of the basal respiration, but only 15% inhibition of the thrombin-stimulated burst of oxygen consumption. Similar differential inhibitory effects were observed with oligomycin, antimycin, rotenone and N-ethylmaleimide. Prostaglandin E1 (0.03 mM) and acetylsalicylic acid (0.8 mM) had little effect on basal respiration, but inhibited the thrombin-stimulated burst of oxygen consumption. N-Ethylmaleimide (0.4 mM) inhibited the release of calcium from platelets by 90%, while prostaglandin E1, acetylsalicylic acid and the above mitochondrial inhibitors caused no more than 30% inhibition of the release reaction. Our results provide evidence that basal respiration and a portion of the thrombin-stimulated burst of oxygen consumption are involved in respiratory chain phosphorylation, and that this component of the thrombin-stimulated burst may be coupled to the maintenance of the release reaction.

Effects of succinylacetone on growth and respiration of L1210 leukemia cells.

4,6-Dioxoheptanoic acid (succinylacetone, SA), a potent inhibitor of heme biosynthesis, suppressed growth and decreased respiration of L1210 leukemia cells in vitro. Growth of cells incubated in the presence of 2--4 mM SA for the first 2 days declined, and after 3 days virtually ceased. L1210 cells in the logarithmic growth phase exhibited active respiration (40 +/- 9.3 nanoatoms oxygen/min X 10(7) cells at 37 degrees C) which was inhibited by and released by uncouplers of oxidative phosphorylation. These and other inhibitors of mitochondrial function clearly demonstrate a mitochondrial basis for the cellular respiration in both control and SA-treated cells. L1210 cells in the stationary phase exhibited a marked decrease in oxygen consumption compared to cells in logarithmic growth. At the concentrations used in this study, SA was not immediately toxic to L1210 cells, but inhibited growth at 2 days without lowering levels of cellular heme. Thus, it appears unlikely that inhibition of growth of L1210 cells by SA can be ascribed either to heme depletion or to impairment of respiration.

Reserpine as an uncoupler of oxidative phosphorylation and the relevance to its psychoactive properties.

Many drugs differing widely in chemical structure uncouple mitochondrial oxidative phosphorylation in vitro. This observation has led to the hypothesis that in vivo uncoupling is the basis of their pharmacological activity. Serpasil, a parenteral preparation of reserpine, recently has been shown to uncouple oxidative phosphorylation in vervet monkey kidney mitochondria. Although the drug exhibits some properties of a "classical" uncoupler, our studies show that it has a dual effect on energy conservation. Reserpine released respiratory control in rat liver mitochondria only when dissolved in organic solvents (as in Serpasil) or when deprotonated. Reserpine also released the oligomycin-induced respiratory control in beef heart submitochondrial particles, and inhibited energized uptake of Ca2- by rat liver mitochondria. Reserpine had a dual effect on mitochondrial ATPase: It (a) enhanced ATP hydrolysis by intact liver mitochondria, and (b) inhibited ATP hydrolysis by submitochondrial particles of beef heart. On a molar basis, reserpine was less effective than carbonyl cyanide 3-chlorophenylhydrazone in all bioenergetic reactions examined. Homogenates and mitochondria isolated from brain and liver of rats stuporous from intraperitoneally injections of Serpasil exhibited no detectable abnormalities in respiratory states and responded to known uncouplers in the expected manner. There was no evidence of in vivo uncoupling of oxidative phosphorylation as a basis of the pharmacological activity of reserpine, although interference with energy transfer may be involved in toxic manifestations of the drug. The results indicate the need for caution in interpreting the action of drugs formulated in complex pharmaceutical preparations and based solely on in vitro experiments.

Effects of tricyclic antidepressant drugs on energy-linked reactions in mitochondria.

The effects of impramine and chlorimipramine on energy-linked reactions in mitochondria were characterized. Both compounds exhibited some characteristics of classical uncouplers of oxidative phosphorylation, i.e. they released respiratory control, hindered ATP synthesis, and enhanced ATPase activity of isolated rat liver mitochondria. Unlike classical uncouplers, however, these compounds only weakly stimulated proton uptake in intact mitochondria. They also exhibited unusual effects on energy-linked reactions in beef heart submitochondrial particles (SMP). Both compounds inhibited NADH oxidation in SMP in an "oligomycin-like" manner, and inhibited ATPase activity of SMP and the soluble F1-ATPase. In contrast, the drugs weakly inhibited ATPase activities of bovine adrenal gland chromaffin granules and resealed granule ghosts. The mechanisms responsible for the multiple effects on mitochondrial energy-linked processes are unclear. They may be related to the hydrophobicity of the drugs, as has been shown for other hydrophobic amines.

Incorporation of calcium into the soft tissues and calcareous corpuscles of larval Taenia taeniaeformis.

Larval Taenia taeniaeformis in vivo accumulates 45Ca2+ in soft tissues and calcareous corpuscles. Radioactivity was demonstrable in the corpuscles six months after a single dose of 45Ca2+ was administered to the host by means of a stomach tube. Ca2+ also was taken up by isolated larvae. Accumulation in vitro was more rapid then in vivo and was correlated with the external Ca2+ concentration. Temperature variation, oxygen availability, and metabolic inhibitors had little effect on the Ca2+ uptake, indicating that active transport of Ca2+ is unlikely in this parasite. Variations in the external Pi concentrations had no effect on Ca2+ accumulation or on its distribution. Addition of 5% CO2 increased the uptake of Ca2+ by the calcareous corpuscles under anaerobic conditions. Radioactivity from NaH14CO3 also was accumulated in soft tissues and corpuscles of T. taeniaeformis. Assuming that the 14C taken up by the corpuscles was in the form of 14CO3(2-), the ratio of Ca2+ to CO3(2-) accumulation in the corpuscles approximates the ratio of these constituents in dolomite: CaMg(CO3)2.

Respiratory activities associated with mesosomal vesicles and protoplast membranes of Staphylococcus aureus.

Analysis of oxidase and dehydrogenase activities, cytochrome content of mesosomal vesicles, and protoplast membranes showed that the respiratory chain in Staphylococcus aureus is associated predominantly with the protoplast membranes.

The activity of tricyclic antidepressant drugs against Trypanosoma cruzi.

Imipramine and related derivatives were tested as possible chemotherapeutic agents against Trypanosoma cruzi parasites in vitro. The IC50 values and the lethal concentrations for two cloned stocks of the parasite were determined. 2-Nitrodesmethylimipramine was the most effective compound tested (IC50 = 4-7 microM). Parasites that were able to grow and to complete the intracellular cycle in mammalian cells in the presence of the drug could be selected. Differences in susceptibility to some imipramine analogs between T. cruzi-cloned stocks were found. The study also shows that modification of the imipramine molecule by electron-withdrawing groups greatly enhances its biological activity.

Structural changes in mitochondria induced by uncoupling reagents. The response to proteolytic enzymes.

Interaction of uncoupling reagents with bovine serum albumin markedly inhibited its hydrolysis by proteolytic enzymes. The inhibition presumably is due to conformational transitions in the protein substrate induced by the binding of the ligand-uncoupling reagents. The proteolysis of casein, a protein that does not bind these reagents, was not affected, indicating that the proteinases themselves were not inactivated. In contrast, interaction of uncoupling reagents with freshly isolated rat liver mitochondria enhanced their susceptibility to proteolytic enzymes. This was shown by an increase in the release of ninhydrin-reacting material, by an increase in free acid groups and by a decrease in the turbidity of the mitochondrial suspensions. These effects, although opposite in direction to those obtained with albumin, are also presumed to indicate structural changes in the mitochondrial proteins and a disorganization of the protein-phospholipid complex. It is suggested that such structural alterations are expressed functionally as the uncoupling of oxidative phosphorylation.

Antidepressant drugs suppress growth of the human pathogenic protozoan Giardia lamblia.

The tricyclic antidepressant drug chlorimipramine, some of its congeners, and its mammalian metabolites inhibited the growth in vitro of the human intestinal pathogen Giardia lamblia. The inhibitory action may reside in their ability to inhibit the membrane-associated ATPase characteristic of eukaryotes. Synthesis of other compounds of the tricyclic class may produce drugs of potential therapeutic use in the treatment of giardiasis and possibly other protozoal diseases.

Binding of tricyclic antidepressant drugs to trophozoites of Giardia lamblia.

1. Parameters affecting the binding of the tricyclic drugs imipramine and 3-chloroimipramine to Giardia lamblia trophozoites were studied. 2. Two to three times more chlorimipramine than imipramine was bound, consistent with a similar difference in suppressing parasite growth (Weinbach et al., 1985). 3. Kinetic analysis and the ease with which bound drugs can be washed out of the parasites indicate that noncovalent forces are involved in the drug-parasite interaction. 4. Evidence is presented that such drugs probably bind to parasite protein at common binding sites. 5. The data relate to our earlier observation that chlorimipramine is about ten times more effective than metronidazole (Crouch et al., 1986) in suppressing G. lamblia growth in vitro. Tricyclic drugs, therefore, merit serious consideration as novel therapeutic agents against giardiasis.

Calcium transport and catabolism of adenosine triphosphate in the protozoan parasite Giardia lamblia.

1. Calcium uptake by washed trophozoites of Giardia lamblia was dependent on inorganic orthophosphate and stimulated by glucose. Uptake was both rapid and substantial: 224 +/- 73 nmoles Ca2+/mg protein/min. 2. Known inhibitors of Ca2+ uptake in mammalian cells also impeded Ca2+ influx into G. lamblia. 3. The inhibitor studies indicated that Ca2+ transport in G. lamblia was an active process. Energy for such a process could be provided by the action of ATPases. 4. Two types of ATPases were found in the parasite; one, a membrane-associated enzyme activated by Ca2+; the other, a soluble, cytosolic enzyme activated by Mg2+. 5. These enzymes differed not only in their intracellular distribution and divalent cation requirements, but also in their sensitivity to calmodulin antagonists. The particulate enzyme was sensitive to these inhibitors whereas the soluble ATPase was not. 6. Our data indicate that Ca2+ transport in G. lamblia is mediated by a membrane-bound, calmodulin-regulated, Ca2+-ATPase.

Complementation of an Escherichia coli glycolysis mutant by Giardia lamblia triosephosphate isomerase.

The Giardia lamblia triosephosphate isomerase (TIM) gene was cloned using a probe generated by a polymerase chain reaction that employed primers complementary to highly conserved regions of TIM. The nucleotide sequence predicts a protein that is 38 and 47% identical to TIM from prokaryotic and eukaryotic sources, respectively. Like all other Giardia protein-coding genes studied thus far, the TIM gene lacks introns and is transcribed to yield a polyadenylated mRNA with an extremely short 5' untranslated region. The Giardia TIM gene complemented an Escherichia coli triosephosphate isomerase deletion mutant. The simplicity and success of complementation suggests its general utility in cloning Giardia genes of known function.