The cyclosporins inhibit lymphocyte activation at more than one site

Cyclosporin A (CsA), a potent immunosuppressive agent, acts primarily by inhibiting T cell function. Although several potential sites of action have been identified, the mechanisms whereby CsA mediates its immunosuppressive properties have not been fully delineated. We have examined the effects of the immunosuppressive cyclosporins, CsA, dihydrocyclosporin D, and cyclosporin G, and a nonimmunosuppressive analog, cyclosporin H, on early events associated with activation of human T cells. Interleukin 2 (IL 2) receptor expression, as measured by immunofluorescence, was unaffected by CsA. Despite this, in the continuous presence of CsA, exogenous IL 2 did not bypass CsA inhibition of phytohemagglutinin (PHA)-induced proliferation. Thus, one site of activity of CsA is on IL 2-induced proliferation of IL 2 receptor-expressing cells. In addition, several potential mechanisms for inhibiting IL 2 secretion were identified. Changes in cytosolic free Ca2+ ([Ca2+]i), an obligatory event for PHA-induced IL 2 secretion, were inhibited by a 30-min preincubation with the immunosuppressive cyclosporins but not the inactive analog. In this action, the drug effects cannot be distinguished from that of Ca2+ channel blockers. The active compounds also resulted in membrane depolarization, an effect which may, in part, explain the reduction in PHA-induced changes in [Ca2+]i. These results identify multiple sites of action of the immunosuppressive cyclosporins, the combination of which likely accounts for their selective inhibition of T cell function in vitro and in vivo.     

Glucagon treatment of rats inhibits the accumulation of lysophospholipids by liver mitochondria during preparation and subsequent incubation

1) Rat liver mitochondria isolated from rats exposed to³²P_i for 24 h and treated with glucagon for 15 min before sacrifice contained less lysophosphatidylcholine and lysophosphatidylethanolamine than those from control animals. 2) Incubation of the mitochondria at 37°C for 15 min increased the lysophosphatidylcholine concentration of control mitochondria but not of those from glucagon-treated animals. 3) Lysophosphatidylethanolamine showed little change during in vitro incubation of mitochondria and this was not affected by glucagon treatment. 4) These results are discussed in relation to the effects of glucagon treatment on mitochondrial function in situ and in vitro.     

Interleukin 2-mediated events in gamma-interferon production are calcium dependent at more than one site

Both leukotrienes and dibutyryl cyclic GMP can replace the interleukin 2 (IL 2) requirement for gamma-interferon (gamma-IFN) production. In this study, the Ca dependence of the IL 2 help was demonstrated by blockage of gamma-IFN production by the Ca blocker Mn, and the competitive reversal of this block by Ca. Neither leukotriene C4 nor dibutyryl cyclic GMP could reverse the Mn block, which suggests that arachidonic acid release from phospholipids is not the only Ca-dependent event in IL 2 help for gamma-IFN production. A role for calmodulin or protein kinase C in the IL 2-mediated events was suggested by the blockage of gamma-IFN production by chlorpromazine. Relatively high concentrations of Ca were able to replace the IL 2 helper effects. Consistent with this were Ca influx experiments that showed that IL 2 helper signals for gamma-IFN production involved activation of a Ca channel.     

Stimulation of the respiratory chain of rat liver mitochondria between cytochrome c1 and cytochrome c by glucagon treatment of rats

Mitochondria from glucagon-treated rats oxidize succinate, but not ascorbate plus tetramethylphenylenediamine, faster in the uncoupled state than do control mitochondria. The rate of O(2) uptake in the presence of both substrates is equal to the sum of the rates of the O(2) uptake in the presence of either substrate alone. It is concluded that the mitochondrial respiratory chain is limited at some point between cytochromes b and c and that this step is regulated by glucagon. Measurement of the cytochrome spectra under uncoupled conditions in the presence of succinate and rotenone demonstrates a crossover between cytochromes c and c(1) when control mitochondria are compared with those from glucagon-treated rats, cytochrome c being more oxidized and cytochrome c(1) more reduced in control mitochondria. Under conditions where pyruvate metabolism is studied the control mitochondria are generally more oxidized than those from glucagon-treated rats, the redox state of cytochrome b-566 correlating with the rate of pyruvate metabolism in sucrose medium. However, when the redox state of the mitochondria is taken into account, a crossover between cytochromes c and c(1) is again apparent. The spectra of the b cytochromes are complex, but cytochrome b-562 appears to become more reduced relative to cytochrome b-566 in mitochondria from glucagon-treated rats than in control mitochondria. This can be explained by the existence of a more alkaline matrix in glucagon-treated rats, the redox potential for cytochrome b being pH-sensitive. It is concluded that glucagon stimulates electron flow between cytochromes c(1) and c. The physiological significance of these findings is discussed.     

Inhibition of gluconeogenesis in rat liver by lipoic acid. Evidence for more than one site of action

Hepatocytes obtained from starved rats were incubated in oxygenated Krebs bicarbonate buffer containing 2% defatted bovine serum albumin. DL-alpha-Lipoic (dithio-octanoic) acid (1.0 mM) caused striking reductions in hepatic glucose output in the presence of each of the following substrates: pyruvate, lactate, alanine, dihydroxyacetone, glycerol, and fructose. With lactate as substrate, 0.1-1.0 mM-lipoate caused a concentration-dependent inhibition of gluconeogenesis. With the same substrate, e.g. lactate, 0.25-2.0 mM-octanoate abolished the effect of lipoate in a dose-dependent manner. Additional experimental data are presented which support the concept that the antigluconeogenic effects of lipoic acid in liver can be attributed largely, if not entirely, to sequestration of intramitochondrial coenzyme. A, presumably as lipoyl-CoA, bisnorlipoyl-CoA, or tetranorlipoyl-CoA.     

Evidence for more than one Ca2+ transport mechanism in mitochondria.

The active transport and internal binding of the Ca2+ analogue Mn2+ by rat liver mitochondria were monitored with electron paramagnetic resonance. The binding of transported Mn2+ depended strongly on internal pH over the range 7.7-8.9. Gradients of free Mn2+ were compared with K+ gradients measured on valinomycin-treated samples. In the steady state, the electrochemical Mn2+ activity was larger outside than inside the mitochondria. The observed gradients of free Mn2+ and of H+ could not be explained by a single "passive" uniport or antiport mechanism of divalent cation transport. This conclusion was further substantiated by observed changes in steady-state Ca2+ and Mn2+ distributions induced by La3+ and ruthenium red. Ruthenium red reduced total Ca2+ or Mn2+ uptake, and both inhibitors caused release of divalent cation from preloaded mitochondria. A model is proposed in which divalent cations are transported by at least two mechanisms: (1) a passive uniport and (2) and active pump, cation antiport or anion symport. The former is more sensitive to La3+ and ruthenium red. Under energized steady-state conditions, the net flux of Ca2+ or Mn2+ is inward over (1) and outward over (2). The need for more than one transport system inregulating cytoplasmic Ca2+ is discussed.     

On the mechanism of action of polyether XXVIII at site I of the electron-transfer chain in rat liver mitochondria

In rat liver mitochondria, the macrocyclic polyether, dibenzo-18-crown-6 (polyether XXVIII) inhibits the oxidation of NAD-dependent substrates, as stimulated by ADP, uncouplers and valinomycin plus K⁺. It does not inhibit the oxidation of succinate. It is concluded that polyether XXVIII inhibits electron transfer in the NADH-CoQ span of the respiratory chain. This is a process that is reversed by menadione. Inhibition of oxidation of NAD-dependent substrates in K⁺-depleted mitochondria induced by the polyether is reversed by concentrations of K⁺ higher than 60 mM, and also by Li⁺, a cation that does not complex with polyether XXVIII. As assayed by swelling mitochondria, reversal of the inhibition of electron transfer is accompanied by influx of monovalent cations. Polyether XXVIII also inhibits in submitochondrial particles the aerobic oxidation of NADH, but not that of succinate; this inhibition is also reversed by K⁺ at high concentrations, and Li⁺. The data are consistent with the hypothesis that a monovalent cation is required for maximal rates of electron transport in the NADH-CoQ span of the respiratory chain.     

Interaction of fluorescent berberine alkyl derivatives with respiratory chain of rat liver mitochondria

The cationic fluorescent dyes, berberines, have been observed to inhibit NAD-linked respiration in rat liver mitochondria. Low concentrations inhibit electron transport in the NAD-ubiquinone span after penetration into mitochondria. More hydrophobic alkyl derivatives proved to be stronger inhibitors showing more rapid onset of inhibition. The inhibition was totally dependent on the energization of the membrane; however, the addition of a hydrophobic anion stimulated the inhibition effects in uncoupled mitochondria. Substantially higher concentrations of berberines are needed for the inhibition of the oxidation of succinate. The excess of dye interacting with surface dipoles in the energized state can inhibit the energy transduction through the complexbc ₁. On the basis of the difference in the rate of fluorescence response when berberines are added to coupled mitochondria and the corresponding inhibition effects, the presence minimally of two binding sites was suggested. The dye bound on the outer surface is highly fluorescent and inhibits the energy transduction if added in excess. The remaining dye interacting with NADH dehydrogenase does not fluoresce. The accumulation of alkylberberine in mitochondria results in additional effects in the region of cytochromeb the nature of which is not fully understood.     

Succinate-ubiquinone reductase site of the respiratory chain

Data on succinate-ubiquinone reductase are critically reviewed. The structural and catalytic properties of succinate dehydrogenase and succinate-ubiquinone reductase are compared. The redox components, active centers and proteins involved in the enzyme interaction with ubiquinone are described. Some structural and kinetic features of the succinate-ubiquinone reductase as the respiratory chain component and feasible mechanisms of regulation of the succinate-ubiquinone reductase activity are discussed.     

Ceramide interaction with the respiratory chain of heart mitochondria

A study is presented on the interaction of ceramide with the respiratory chain of rat heart mitochondria, and a comparison is made between the effects elicited by short- and long-chain ceramides. N-Acetylsphingosine (C(2)-ceramide) and N-palmitoylsphingosine (C(16)-ceramide) inhibited to the same extent the pyruvate+malate-dependent oxygen consumption. Succinate-supported respiration was also inhibited by ceramides, but this activity was substantially restored upon the addition of cytochrome c, which, on the contrary, was ineffective toward the ceramide-inhibited NADH-linked substrate oxidation. Direct measurements showed that short- and long-chain ceramides caused a large release of cytochrome c from mitochondria. The ceramide-dependent inhibition of pyruvate+malate and succinate oxidation caused reactive oxygen species to be produced at the level of either complex I or complex III. The activity of the cytochrome c oxidase, measured as ascorbate/TMPD oxidase activity, was significantly stimulated and inhibited by C(2)- and C(16)-ceramide, respectively. Similar effects were observed on the activity of the individual respiratory complexes isolated from bovine heart. Short- and long-chain ceramides had definitely different effects on the mitochondrial membrane potential. C(2)-ceramide caused an almost complete collapse of the respiration-dependent membrane potential, whereas C(16)-ceramide had a negligible effect. Similar results were obtained when the potential was generated in liposome-reconstituted complex III respiring at the steady-state. Furthermore, C(2)-ceramide caused a drop of the membrane potential generated by ATP hydrolysis instead of respiration, whereas C(16)-ceramide did not. Finally, only short-chain ceramides inhibited markedly the reactive oxygen species generation associated with membrane potential-dependent reverse electron flow from succinate to complex I. The emerging indication is that the short-chain ceramide-dependent collapse of membrane potential is a consequence of their ability to perturb the membrane structure, leading to an unspecific increase of its permeability.     

The respiratory chain components of higher plant mitochondria

Tightly coupled mitochondria have been prepared from a variety of plant sources: white potato (Solanum tuberosum), Jerusalem artichoke (Heliantus tuberosus), cauliflower buds (Brassica oleracea), and mung bean hypocotyls (Phaseolus aureus). Mitochondria with no appreciable coupling were also prepared from skunk cabbage spadices (Symplocarpus foetidus).

Room temperature difference spectra show that these mitochondria are very similar in the qualitative and quantitative composition of their electron carriers. The different cytochromes are present in the amounts of 0.1 to 0.3 mμmole per mg of mitochondrial protein. The molar ratios of the different electron carriers are, on the average: 0.7:0.7:1.0:3 to 4:10 to 15 respectively for cytochrome aa₃, cytochromes b, cytochromes c, flavoproteins, and pyridine nucleotides.

From low temperature difference spectra carried out under particular experimental conditions, it can be deduced that these mitochondria contain 3 b cytochromes whose α bands are located at 552, 557, and 561 mμ, and 2 c cytochromes, one of which, a c₁-like cytochrome, is firmly bound to the mitochondrial membrane. Cytochrome oxidase can be optically resolved into its 2 components a and a₃.

For all kinds of mitochondria, the rates of oxidation of succinate are similar as well as the turnover of cytochrome oxidase (50-70 sec⁻¹), regardless of the metabolic activities of the tissues. The number of mitochondria per cell appears to be the controlling factor of the intensity of tissue respiration.

     

Measurement of the proton-motive stoichiometry of the respiratory chain of rat liver mitochondria: the effect of N-ethylmaleimide

The apparent proton-motive stoichiometry as measured by the oxygen-pulse technique in KCl medium is depressed by the rapid uptake of inorganic phosphate, unless endogenous phosphate is depleted or uptake is inhibited. In sucrose or choline chloride media, where the internal pH is more acid than in KCl media, uptake may be greatly diminished. In the absence of significant phosphate uptake, the observed stoichiometry of around 8, obtained with no added substrate or respiratory inhibitors, appears to be characteristic of NADH oxidation without significant participation of the proton-translocating NAD(P) transhydrogenase. A mechanistic stoichiometry of at least 8 is indicated.