Prominent glutamine oxidation activity in mitochondria of hematopoietic tumors.

Well-coupled mitochondria of hematopoietic tumors were isolated from mouse erythroleukemia and rat chloroma tumors grown in male DBA/2J mice and Long-Evans rats, respectively. We used erythroleukemia and chloroma mitochondria to determine their ability to utilize glutamine as an energy source for adenosine triphosphate formation. Oxypolarographic tests showed the following. (a) Presence of a prominent glutaminase activity in erythroleukemia and chloroma mitochondria is evidenced by their active glutamine-supported respiratory state 3. (b) Glutamine oxidation is mediated through a nicotinamide adenine dinucleotide-linked reaction inhibited by rotenone. (c) Under similar conditions, mitochondria isolated from rabbit bone marrow have shown a feeble glutamine oxidation activity, while in mitochondria from rat liver the activity was not detectable and in those from rat kidney it was prominent as expected. (d) The determination of apparent Km and Vmax values for substrate-supported adenosine triphosphate formation has shown 8- to 10-fold lower Km values for glutamine oxidation as compared to that of glutamate, with virtually the same Vmax for each substrate in each mitochondria. These results clearly show the presence of a high glutamine oxidation activity in erythroleukemia and chloroma mitochondria and suggest that one of the glutamine hydrolysis products in those mitochondria may have an important role in supplying adenosine triphosphate in the corresponding malignant cells.

Special features of Pi transport in pig heart and rat liver mitochondria as revealed by 6,6'-dithiodinicotinic acid (CPDS).

CPDS (6,6'-dithiodinicotinic acid), a non permeant thiol agent which affects several mitochondrial functions in a way different to that of mersalyl [18-19] revealed striking differences between the phosphate translocating systems of pig heart and rat liver mitochondria. Pi entry was measured either by swelling in 0.12 M ammonium phosphate or by rapid centrifugation in 32Pi medium. Pi efflux was measured after preloading of mitochondria with 32Pi, by exchange against Pi or malate; the "ATP-FCCP" system has been tested previously [19]. In pig heart mitochondria, Pi entry seems to proceed exclusively via the Pi/OH- carrier; CPDS completely inhibits this transport and the energy-linked functions. In contrast n-butyl-malonate does not affect the Pi-entry and the energy-linked functions. The Pi efflux is not affected either by CPDS or mersalyl, which do not produce a swelling in the "ATP-uncoupler system". In rat liver mitochondria, CPDS inhibits only the Pi/OH- carrier; both CPDS and n-butylmalonate are necessary to inhibit completely Pi entry. CPDS as well as mersalyl provokes a swelling in the presence of the "APT-uncoupler system". The results suggest two distinct functions of phosphate transport in both types of mitochondria.

Mechanism of interaction of ticlopidine and its analogues with the energy-conserving mechanism in mitochondria.

It was shown recently that the antiaggregating agent ticlopidine and some of its analogues inhibit the energy-conserving mechanism in mitochondria [Abou-Khalil et al., Biochem. Pharmac. 33, 3893 (1984)]. In the present investigation, the mechanism of inhibition by these drugs was investigated by studying their effects on key reactions of oxidative phosphorylation. Liver mitochondria were isolated from Sprague-Dawley male rats, and the interactions of ticlopidine and six of its analogues with those key reactions were tested. We found: The transport of phosphate, glutamate and succinate into mitochondria was not affected significantly by ticlopidine or any of its analogues; however, it was inhibited by both mersalyl and N-ethylmaleimide as expected. There was no inhibitory effect of the tested drugs on the mitochondrial [3H]ADP translocation activity; rather, ticlopidine produced a concentration-dependent increase of that activity, reaching 54% with 20 micrograms/ml. Ticlopidine and its analogue, PCR 5325, increased the latent ATPase activity by about 400% and the DNP-dependent ATPase by about 50%. Also, PCR 4099 caused a 115% increase in the latent activity, whereas the effects of the remaining analogues varied from slight activation to slight inhibition. Under nonphosphorylation conditions, the mitochondrial H+ extrusion resulting from succinate oxidation was inhibited by ticlopidine in a concentration-dependent manner reaching a quasi total inhibition with 40 micrograms/ml. While PCR 5325 gave results similar to ticlopidine, PCR 4099 was less inhibitory and the other analogues were ineffective. These data indicate that the inhibitory action caused by ticlopidine and some of its analogues on oxidative phosphorylation does not reside at one particular site in the mitochondrial membrane; rather, the inhibition seems to be the outcome of profound alterations in mitochondrial ADP translocase, latent ATPase, and proton translocation in the respiratory chain.

Swelling of mitochondria by the platelet antiaggregating agent ticlopidine.

Our studies on the effects of ticlopidine on mitochondrial functions led us to an intriguing observation related to its interaction with mitochondrial membranes. Liver mitochondria were isolated from Sprague-Dawley rats and assayed for swelling by spectrophotometry. When ticlopidine was added to mitochondria preincubated in an isotonic test medium, an induced-swelling activity was observed. This activity was time and concentration dependent and occurred in different isosmotic solutions. Several analogues of ticlopidine, assayed under identical conditions, produced only a minor effect. Respiratory chain inhibitors, uncouplers, ATP, and phosphate protected the mitochondria against the ticlopidine-induced swelling, whereas oligomycin did not. Comparative studies with the drugs chloramphenicol, nitroso-chloramphenicol, and salicylate (known for their association with mitochondrial injury) showed the first two to have little effect while the third one caused swelling as expected. On the other hand, oxypolarographic tests of respiring mitochondria in the presence of ticlopidine showed that the drug is not an uncoupling agent. These results indicate that the antiaggregating agent ticlopidine interacts with mitochondrial membranes causing swelling which, in turn, may alter mitochondrial permeability; however, unlike some other swelling agents, it does not act as a classical uncoupler.

Effects of ticlopidine, a new platelet antiaggregating agent, and its analogues on mitochondrial metabolism. Oxidative phosphorylation, protein synthesis and DNA polymerase activity.

The effects of ticlopidine and six of its analogues on mitochondrial functions were studied in isolated rat liver mitochondria. The influence of ticlopidine and each of the following analogues: PCR 5325, PCR 4099, PCR 3787, PCR 2362, PCR 4499 and PCR 0665 was evaluated by determining their interaction with three major mitochondrial activities. (A) Oxidative phosphorylation, measured by oxypolarography, was assayed in the presence of glutamate or succinate as source of energy, and both State 4 and State 3 were recorded. Ticlopidine, at 20 micrograms/ml, slightly increased glutamate State 4, whereas it was without effect on that of succinate. At higher concentration (40 micrograms/ml), ticlopidine caused 40-45% inhibition of State 4 with both substrates. All the other analogues tested at either 20 or 40 micrograms/ml were virtually without effect on the respiration. However, at 20 micrograms/ml, ticlopidine and some of its analogues inhibited mitochondrial State 3, while under similar conditions other analogues had little or no effect on this state. (B) Mitochondrial protein synthesis, measured by [14C]-L-leucine incorporation, was not affected significantly by any of these drugs. Whereas chloramphenicol at 10 micrograms/ml caused 80% inhibition, ticlopidine and its analogues in concentrations inhibitory to State 3 did not inhibit mitochondrial protein synthesis. (C) Mitochondrial DNA polymerase activity, determined by [3H] thymidine 5'-triphosphate incorporation, was not inhibited by these drugs. We conclude that, while ticlopidine and analogues have little or no effect on either mitochondrial protein synthesis or mitochondrial DNA polymerase activity, ticlopidine and some of its analogues are inhibitory of the energy conserving mechanism in mitochondria.

Application of thermospray liquid chromatography-mass spectrometry for the analysis of chloramphenicol and three related compounds.

The thermospray (TS) liquid chromatographic-mass spectrometric analysis of the antibiotic chloramphenicol and three related compounds is presented. The three additional compounds are dehydrochloramphenicol, aminodehydrochloramphenicol, and nitrophenylaminopropanediol. Baseline separation of the four compounds is achieved. The TS mass spectrum of each of the four compounds includes a prominent [MH]+ ion plus some fragment ion peaks.

Energy-linked Sulfate Uptake by Corn Mitochondria via the Phosphate Transporter.

Corn shoot mitochondria possess an energy-linked transport system for sulfate uptake as demonstrated by osmotic swelling and [(35)S]SO(4) (2-) accumulation. Maximum uptake is secured in the presence of Mg(2+) and oligomycin with sucrose for osmotic support. Neither phosphate nor dicarboxylate anions are required. When added simultaneously, millimolar concentrations of phosphate block [(35)S]SO(4) (2-) uptake after the initial minute. Mersalyl, N-ethylmaleimide, and 2,4-dinitrophenol are strong inhibitors of sulfate uptake; n-butylmalonate is a weak inhibitor. These inhibitors act in the same fashion on phosphate uptake. It is concluded that sulfate uptake in the absence of phosphate is by the phosphate transporter.

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: II. Phosphate and Divalent Cation Requirement.

The requirement for phosphate and Mg(2+) in energy-linked [(3)H] ADP accumulation by corn mitochondria has been studied. Arsenate will fully substitute for phosphate; sulfate partially substitutes; acetate, bicarbonate, and pyrophosphate are ineffective. Phosphate is also taken up by the mitochondria, but the ADP/Pi ratio varies widely with experimental treatments. ADP does not exchange with endogenous labeled phosphate, although Pi/(32)Pi exchange occurs.Mg(2+) is also accumulated during ADP uptake. Mg(2+) can be substituted with varying efficiency by other divalent cations, but not monovalent cations. Effective cations typically increase phosphate uptake, particularly Ca(2+) . Ca(2+) -activated ADP accumulation is insensitive to carboxyatractyloside over a wide range of Ca(2+) concentrations. When Ca(2+) is substituted for Mg(2+) it is not necessary to block ATP formation to secure high levels of ADP accumulation, since Ca(2+) will divert energy from ATP formation into ion uptake.It is suggested that the transport mechanism may carry out a concerted transport of ADP and phosphate with bound divalent cation. The phosphate transporter may be involved, or alternatively a special mechanism for trivalent anion transport may exist which acts cooperatively with the phosphate transporter.

Energy-linked Adenosine Diphosphate Accumulation by Corn Mitochondria: I. General Characteristics and Effect of Inhibitors.

Corn mitochondria show respiration-linked net accumulation of [(3)H]ADP in the presence of phosphate and magnesium, especially if the formation of ATP is blocked with oligomycin. Inhibition of ADP-ATP exchange by carboxyatractyloside also activates ADP accumulation, and addition of carboxyatractyloside or palmitoyl-coenzyme A to oligomycin-blocked mitochondria produces additional ADP uptake. With carboxyatractyloside the accumulated ADP is phosphorylated to ATP. With oligomycin, only a little ATP is formed. Millimolar concentrations of ADP are required for maximum uptake, and the K(m) (3.77 millimolar) for ADP translocation is independent of whether oligomycin or carboxyatractyloside is used. This is not true for ADP concentrations in the 0.05 to 0.25 millimolar range. Accumulated [(3)H]ADP rapidly exchanges with unlabeled AMP, ADP, or ATP, but not with other diphosphate nucleotides or 2 millimolar substrate anions. [(3)H]AMP is not accumulated, but [(3)H]ATP is accumulated to about one-half the extent of [(3)H]ADP. Tricarboxylate substrates inhibit ADP net uptake, and inhibition by citrate is competitive with K(i) = 10 millimolar. The evidence suggests the presence of a pathway, carboxyatractyloside-insensitive and different from the translocase, which operates to maintain adenine nucleotides in the matrix.

Mitochondrial metabolism in normal, myeloid, and erythroid hyperplastic rabbit bone marrow: effect of chloramphenicol.

Protein synthesis, respiration, and oxidative phosphorylation were studied in mitochondria isolated from normal, myeloid, and erythroid hyperplastic bone marrow of rabbits. Mitochondria from normal and myeloid hyperplastic marrow showed a similar degree of 14C-leucine incorporation while a threefold higher activity was demonstrated in mitochondria from erythroid marrow. The incorporation was mitochondrial-concentration-dependent and in all cases was inhibited by low concentrations of chloramphenicol (CAP). The respiratory activity of mitochondria isolated from either myeloid or erythroid marrow was twice as high as the normal, but in all cases respiration and phosphorylation were unaffected by concentrations of CAP which totally inhibited protein synthesis.

Pyruvate-dependent oxidative phosphorylation in erythroid and myeloid tumor mitochondria.

The pyruvate-supported oxidative phosphorylation activity was determined in mitochondria isolated from the fast-growing erythroid and myeloid tumors of hematopoietic origin. Normal bone marrow and liver mitochondria were used for comparison. In the absence of primers, both tumor mitochondria exhibited a pyruvate-dependent respiratory state 4/state 3 transition, which was totally inhibited by either alpha-cyanocinnamate or arsenite. The transition rate increased in a concentration-dependent manner from 5 to 100 microM pyruvate, where the maximum activity was reached. Increasing the concentration to 500 microM and beyond, however, resulted in decreasing state 3 respiratory jump with little or no jump demonstrable at concentrations above 5 mM. Moreover, the addition of high concentrations of pyruvate during the respiratory state 3 caused a blockage of that state which was reestablished by the addition of succinate or alpha-ketoglutarate. These results clearly show the capacity of erythroid and myeloid tumor mitochondria to actively utilize low concentrations of pyruvate to support their oxidative phosphorylation activity. The reason for the absence of activity found with the high concentration, however, is not readily apparent.

An oligomycin-resistant Mg2+-dependent ATPase in rabbit bone marrow mitochondria.

Rabbit bone marrow mitochondria isolated by differential centrifugation showed typical oxypolarographic tracings with glutamate oxidation with ADP:O ratio of 2.9. Similar results were obtained with liver mitochondria of the same animal. When marrow mitochondria were oxydizing a substrate such as glutamate, added MgCl2 markedly stimulated state-4 respiration giving a respiratory rate identical to that of state-3. In contrast, no Mg2+-stimulation was observed with liver mitochondria. Oligomycin completely blocked the stimulation by Mg2+ but further addition of 2,4-dinitrophenol reactivated the oxygen consumption by uncoupling. Further purification of marrow mitochondria by density gradient centrifugation in Percoll provided identical oxypolarographic results. Moreover, when marrow mitochondria were incubated without Mg2+, they showed a low ATPase activity that was stimulated by 2,4-dinitrophenol and blocked by oligomycin. The presence of Mg2+ in the incubation medium uncovered an additional ATPase activity which was resistant to oligomycin and apparently unaffected by 2,4-dinitrophenol. It is concluded that bone marrow mitochondria possess two types of ATPase activity distinguished on the basis of their reactivity with oligomycin, 2,4-dinitrophenol and Mg2+.

Inhibition by rhodamine 123 of protein synthesis in mitochondria of normal and cancer tissues.

The effect of the mitochondrial dye rhodamine 123 (Rho 123) on protein synthesis (PS) activity was investigated in mitochondria isolated from liver and from both chloroma and erythroleukemia tumors. Incorporation of labelled leucine into mitochondrial protein was used to measure the rate of PS. While PS specific activity was much higher in hematopoietic tumors mitochondria as compared to that of liver, the addition of increased concentration of Rho 123 in all tested organelles resulted in increased inhibition of PS to reach 75-82% with 10 micrograms/ml of the dye. Similar results were obtained with 10 micrograms/ml of chloramphenicol, the specific inhibitor of mitochondrial PS. Moreover, under the conditions of the study, the addition of Rho 123 to mitochondria did not trigger any ATPase activity, thus eliminating any competition for the energy source ATP between PS and ATPase. These results demonstrate that, in addition to its known inhibitory action on oxidative phosphorylation, the mitochondrial dye Rho 123 has a potent inhibitory effect on PS in both liver and hematopoietic tumors mitochondria.

Interaction of rhodamine 123 with mitochondria isolated from drug-sensitive and -resistant Friend leukemia cells.

Mitochondria isolated from Friend leukemia cell lines sensitive (FS) and resistant (FR) to rhodamine 123 (Rho123), showed respiratory control and ADP/O ratios indicative of well-coupled oxidative phosphorylation activity. When Rho123 was added to mitochondria from both cell lines, respiratory State 4 increased. The increase was higher in mitochondria isolated from resistant than from sensitive cells. Respiratory State 3 was slightly more inhibited by Rho123 in resistant than in sensitive cell mitochondria (98 and 82% inhibition, respectively). While it is not clear how the uncoupling-like effects of Rho123 on State 4 contribute to cellular toxicity, our results indicate that differential cellular sensitivity to the drug does not correlate with inhibition of oxidative phosphorylation in mitochondria isolated from drug-sensitive and -resistant cells.

Importance of the mitochondrial amino acid pool in the sensitivity of erythroid cells to chloramphenicol: role of glycine and serine.

The amino acid pool of mitochondria has been recently implicated in the sensitivity of erythroid cells to chloramphenicol (CAP) [8]. In the present study, we have analyzed the composition of that pool in the sensitive erythroleukemia mitochondria (EM) and compared it with that of the resistant chloroma mitochondria (CM). We have also tested the effect of every major component in each pool on the sensitivity to CAP. The study of endogenous amino acid composition of EM and CM was performed with a JEOL 5AH amino acid analyzer, and the sensitivity to the drug was assayed by determining its interaction with the mitochondrial protein synthesis activity. Analysis of the total endogenous pool showed about 20% more amino acids in CM compared to EM. However, some amino acids were present in significantly higher (e.g. glycine, serine and histidine) or lower (e.g. proline, leucine, arginine, glutamic acid and threonine) quantity within EM. When compensating for each low amino acid in either CM or EM by addition of that particular acid to the incubation medium, only glycine and serine had a significant effect. Thus, the addition of glycine or serine enhanced the sensitivity to CAP from 14 to 49-52% in CM, but were without effect in EM. Addition of the other acids to either mitochondria gave little or no effect. Since serine could be interconvertible intramitochondrially to glycine, and because the latter with succinate are the first reactants in heme biosynthesis which is initiated inside the mitochondria, it would appear that erythroid cell sensitivity to CAP is predetermined by the mitochondrial glycine-serine pool and might be somehow related to the pathway of heme biosynthesis in these cells.

High-performance liquid chromatographic determination of chloramphenicol and four analogues using reductive and oxidative electrochemical and ultraviolet detection.

A high-performance liquid chromatographic procedure is described for the separation, quantitation and identification of chloramphenicol, dehydrochloramphenicol, nitrophenylaminopropanedione, nitrosochloramphenicol and aminochloramphenicol. An isocratic reversed-phase system with ultraviolet and electrochemical detectors in tandem was assembled and used. The system was constructed with special accommodation to enable us to use the electrochemical detector in both reductive and oxidative modes. Retention characteristics, hydrodynamic voltammograms under reductive and oxidative conditions and ultraviolet absorbance are reported. Applicability of the procedure to biological fluids was demonstrated by separation and detection of chloramphenicol after incubation with human blood.