Analysis of cell-mediated mineralization in culture of bone-derived embryonic cells with neurofibromatosis

von Recklinghausen neurofibromatosis (NF1) is an autosomal dominant genetic disorder associated with congenital pseudoarthrosis and with short stature. To examine whether the NF1 phenotype includes functional osteogenic defects, embryonic bone-derived cells affected with NF1 were tested in culture for specific alkaline phosphatase (ALP) activity and cell-mediated mineralization and compared with other embryonic bone derived cells. NF1 showed a relatively higher specific ALP activity, which has further increased in response to dexamethasone + β-glycerophosphate (βGP) (Dex medium) coordinately with a decrease in cell proliferation. In the control group, two samples showed increased ALP activity, one showed decreased activity and the forth one did not show any change in ALP. NF1 cells were distinguished from other cells regarding day 21 mineralization, they did not mineralize when cultured with ascorbate alone in the absence of Dex medium, whereas control cells did mineralize. Adding βGP resulted in mineralization by NF1 cells but less than in other cells. In addition, NF1 cells responded to dexamethasone by increasing the βGP-induced mineralization, as opposed to cells from other embryonic bones, which either did not respond or have even decreased mineralization under dexamethasone. Upon cis-hydroxyproline exposure, Dex medium has also distinguished NF1 cell ALP activity from that of other cell origins. Inhibition of respiratory complex II by malonate showed that most embryonic bone-derived cells of 12 weeks gestation are malonate resistant; thus, malonate selection was ineffective. This is in contrast to rat marrow stromal cells previously shown to undergo mineralizing cell enrichment in response to malonate. Exposure to levamisole, of Dex-treated cells, at days 0–11 has inhibited day 21 mineralization in all tested cultures in spite of the increase in day 11-specific ALP activity. Both malonate and levamisole did not distinguish NF1 cells from the osteogenic phenotype of other cells. Essentially embryonic bone-derived cells from 12 weeks gestation, cultured in the absence of βGP, retained their mineralization capacity, which does not increase under dexamethasone, as distinguished from NF1 cells which require βGP for mineralization and positively respond to dexamethasone. Therefore, bone-derived NF1 cells may be useful for studying the regulation of the mineralization process.     

Cyclosporin A and its non-immunosuppressive derivative exhibit a differential effect on cell-mediated mineralization in culture

Chronic immunosuppressive treatment with cyclosporin A (CsA) is associated with decreased bone density. However, in culture, CsA inhibits osteoclast differentiation and bone resorption. This raises the question as to whether CsA also affects osteoblast function. Immunophilin, one of the CsA-binding cyclophilins that is implicated in the immunosuppressive action of CsA via calcineurin, is a peptidyl prolyl cis-trans isomerase (PPI). CsA also binds a mitochondrial membrane PPI which is implicated in controlling permeability transition pores. Therefore, in the present study we tested the effect of CsA on cell mediated mineralization in parallel with mitochondrial rhodamine retention as an indicator of mitochondrial membrane potential. Rat marrow stromal cells were grown in dexamethasone (DEX) medium to stimulate mineralization in culture, and CsA was added to various cultures using different treatment schedules. Low dose (0.1 μM) CsA inhibited mineralization, compared to controls, when present in the cultures during days 3–11 of DEX stimulation. Contrarily, high dose CsA (1.0 μM) resisted the inhibitory effect of the low dose. SDZ 220–384 (SDZ), a non-immunosuppressive derivative of CsA which is known, like CsA, to bind to mitochondrial cyclophilin but does not inhibit calcineurin, was also tested. Both high and low doses of SDZ decreased mineralization when present in the cultures from day 3 or from day 0. The similar effect of the low CsA dose and SDZ on mineralization is in accord with their ability to block permeability transition pores. The differential effect, on day 21 mineralization, between high CsA dose and SDZ took place in parallel to their opposing effects on mitochondrial membrane potential. On days 4–8, mitochondrial rhodamine retention was higher under CsA than under SDZ. Under these conditions there was no significant difference between the effects of these drugs on cell proliferation measured on day 11; there was a minor decrease in specific alkaline phosphatase activity by SDZ, too small to explain the extent of mineralization inhibition by SDZ. These results suggest that permeability transition pores might be involved in controlling mineralization. Unlike SDZ, CsA exhibits an additional effect on the mitochondrial membrane potential and on mineralization when applied at a high dose on day 3. Therefore identifying the additional activity of high dose CsA, which is missing in SDZ, may be beneficial. Such activity is expected to resist changes in rhodamine retention and decreased mineralization induced by SDZ, and yet enable preservation of immunosuppressive activity of CsA. J. Cell. Biochem. 64:209–216. © 1997 Wiley-Liss, Inc.     

Cell-mediated mineralization in culture at low temperature associated with subtle thermogenic response

In both the growth plate and in marrow stromal cell cultures cell-mediated mineralization is preceded by characteristics of anaerobic and low efficiency energy metabolism. Reagents that increase mineralization like malonate and dexamethasone (DEX) also increase the mitochondrial membrane potential (MtMP) especially 1 week after DEX stimulation. Contrarily, levamisole, which decreases mineralization, also decreases MtMP. Modulation of MtMP and energy metabolism could be linked to regulation of mineralization by the uncoupling of oxidative phosphorylation. This uncoupling should be associated with thermogenesis in cells that induce mineralization. We examined whether cold temperature affects mineralization, and whether cellular thermogenesis takes place at cold temperature in parallel to changes in MtMP. Osteoprogenitor cells (OPC) induced, in DEX stimulated rat marrow stroma, higher mineralization at 33°C than at 37°C. Increased mineralization by cold temperature required long incubation since incubation in the cold during short intervals, 3–4 days, did not increase mineralization relative to (37°C) controls. Marrow stromal cells in the presence of valinomycin responded to incubation at 33°C by retaining all the vital dye after 4 h, unlike the cells at 37°C; however, after 24 h the level of dye retention at 33°C was the same as at 37°C. The delayed response of the temperature-dependent (> 37°C) K⁺ ionophor to incubation in the cold indicated that certain cells may respond to low temperature by local intracellular heating, and by heat conduction to the plasma membrane. DEX-stimulated stromal cells, unlike unstimulated cells, showed increased mitochondrial rhodamine 123 retention in the presence of valinomycin after 24 h in the cold, which corresponds to day 4 of OPC induction. This is consistent with the concept that valinomycin-induced cell damage is mediated by (cold-induced) local heating. The mechanism of this cell damage should selectively prefer non-thermogenic (rhodamine retaining) over thermogenic (rhodamine leaking) cells such as OPC. At cold temperature DEX-stimulated stromal cells showed the best anti-OPC selection under exposure to valinomycine between days 3–7, concurrent with the period of rhodamine leakage from the mitochondria. These results indicate that thermogenesis is enhanced during the period of low MtMP in mineralizing cells, and prolonged exposure to cold increases mineralization also due to induction of subtle thermogenesis. © 1996 Wiley-Liss, Inc.     

Propofol Prevents Oxidative Stress by Decreasing the Ischemic Accumulation of Succinate in Focal Cerebral Ischemia–Reperfusion Injury

Oxidative stress caused by mitochondrial dysfunction during reperfusion is a key pathogenic mechanism in cerebral ischemia–reperfusion (IR) injury. Propofol (2,6-diisopropylphenol) has been proven to attenuate mitochondrial dysfunction and reperfusion injury. The current study reveals that propofol decreases oxidative stress injury by preventing succinate accumulation in focal cerebral IR injury. We evaluated whether propofol could attenuate ischemic accumulation of succinate in transient middle cerebral artery occlusion in vivo. By isolating mitochondria from cortical tissue, we also examined the in vitro effects of propofol on succinate dehydrogenase (SDH) activity and various mitochondrial bioenergetic parameters related to oxidative stress injury, such as the production of reactive oxidative species, membrane potential, Ca²⁺-induced mitochondrial swelling, and morphology via electron microscopy. Propofol significantly decreased the ischemic accumulation of succinate by inhibiting SDH activity and inhibited the oxidation of succinate in mitochondria. Propofol can decrease membrane potential in normal mitochondria but not in ischemic mitochondria. Propofol prevents Ca²⁺-induced mitochondrial swelling and ultrastructural changes to mitochondria. The protective effect of propofol appears to act, at least in part, by limiting oxidative stress injury by preventing the ischemic accumulation of succinate.     

Characterization of the Excitotoxic Potential of the Reversible Succinate Dehydrogenase Inhibitor Malonate

Abstract: Although the mechanism of neuronal death in neurodegenerative diseases remains unknown, it has been hypothesized that relatively minor metabolic defects may predispose neurons to N -methyl- d -aspartate (NMDA) receptor-mediated excitotoxic damage in these disorders. To further investigate this possibility, we have characterized the excitotoxic potential of the reversible succinate dehydrogenase (SDH) inhibitor malonate. After its intrastriatal stereotaxic injection into male Sprague-Dawley rats, malonate produced a dose-dependent lesion when assessed 3 days after surgery using cytochrome oxidase histochemistry. This lesion was attenuated by coadministration of excess succinate, indicating that it was caused by specific inhibition of SDH. The lesion was also prevented by administration of the noncompetitive NMDA antagonist MK-801. MK-801 did not induce hypothermia, and hypothermia itself was not neuroprotective, suggesting that the neuroprotective effect of MK-801 was due to blockade of the NMDA receptor ion channel and not to any nonspecific effect. The competitive NMDA antagonist LY274614 and the glycine site antagonist 7-chlorokynurenate also profoundly attenuated malonate neurotoxicity, further indicating an NMDA receptor-mediated event. Finally, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline) was ineffective at preventing malonate toxicity at a dose that effectively reduced S -AMPA toxicity, indicating that non-NMDA receptors are involved minimally, if at all, in the production of the malonate lesion. We conclude that inhibition of SDH by malonate results in NMDA receptor-mediated excitotoxic neuronal death. If this mechanism of "secondary" or "weak" excitotoxicity plays a role in neurodegenerative disease, NMDA antagonists and other "antiexcitotoxic" strategies may have therapeutic potential for these diseases.     

Polarized electric field effects on the regulation of succinate dehydrogenase activity in amphibian muscle and liver: kinetic study

Electropolarity treatment (0.8 V/DC/Cm) was given to the gastrocnemius muscle of Bufo melanostictus every day for 5 min. for 5 days, and kinetic study of succinate dehydrogenase (SDH) in muscle and liver was conducted with different effectors - sodium malonate, ethylene diamine tetra acetic acid (EDTA), calcium chloride (CACl2) and sodium citrate. Of the four modulators tested, the malonate and EDTA inhibited while sodium citrate and CACl2 activated the enzyme. The significance of the modulation in SDH activity to different extents was discussed.     

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).     

Effect of intermittent hypoxic hypoxia on energy supply of rat skeletal muscle during adaptation to physical load

The experiment, on Wistar male rats was carried out to investigate influence of endurance training (swimming with load 7.0 +/- 1.3% body weight, 30 min a day, during 4 weeks) and additional intermittent hypoxic training (12% O2 in N2 - 15 min, 21% O2 - 15 min, 5 sessions a day, during the first 2 weeks) on the following parameters: ADF-stimulated mitochondrial respiration, lactate/pyruvate ratio, succinate dehydrogenase activity, and lipid peroxidation in skeletal muscle. The next oxidation substrates were used: 1 mmol/l succinate and 1 mmol/l alpha-ketoglutarate as well as the next inhibitor succinate dehydrogenase 2 mmol/l malonate. It was shown that physical work combined with intermittent hypoxic training led to the increase of mitochondrial respiration effectiveness in muscle energy supply under alpha-ketoglutarate oxidation in comparison with succinate oxidation as well as to the decrease of succinate dehydrogenase activity and lipid peroxidation. The study suggested that these changes may correct mitochondrial dysfunction under intensive muscular work.     

Selection of malonate-resistant stromal cell-derived osteoprogenitor cells in vitro

Bone marrow stromal cells give rise to osteoprogenitor cell (OPC) colonies, with characteristic mineralized bone nodules in vitro. During differentiation, OPCs in the culture are surrounded by heterogeneous populations of various cell lineages and by different OPC differentiation stages. In the present study, attempts were made to increase the homogeneity of OPCs in culture. The reliance on energy metabolism restricted to glycolysis, which is specific to the premineralizing skeletal cells, was tested as a selectable marker for cells in this stage. Day 12 alkaline phosphatase (ALP) and day 20–21 calcium precipitates were used as early and late OPC differentiation markers. Malonate, a competitive inhibitor of succinate dehydrogenase, was added to the OPC stimulation medium, to interfere with the Krebs cycle-dependent energy metabolism operating in most of the stromal cells. OPCs that entered the stage of energy metabolism restricted to glycolysis were expected to become malonate resistant. Malonate showed dose and time dependence, 10 mM malonate added on day 3, decreased day 12 ALP activity/well to the lowest level. Variations in time and length of exposure to malonate used during the first 12 days of differentiation showed an inverse correlation between specific ALP activity and cell yield. Malonate-treated variations of specific ALP and of cell yield indices were up to 30- to 40-fold larger than variations within day 21 calcium precipitates. Thus, calcifying cells were almost unchanged relatively to noncalcifying cells. These results indicate that malonate-resistant cells are mostly selected, rather than induced, to differentiate by malonate. The results also show that stromal derived OPCs undergo a similar biochemical stage as in chondrocytes. © 1993 Wiley-Liss, Inc.     

Manipulation of Membrane Potential Modulates Malonate-Induced Striatal Excitotoxicity In Vivo

Abstract: Malonate is a reversible inhibitor of succinate dehydrogenase (SDH) that produces neurotoxicity by an N -methyl- d -aspartate (NMDA) receptor-dependent mechanism. We have examined the influence of pharmacological manipulation of membrane potential on striatal malonate toxicity in rats in vivo by analysis of lesion volume. Depolarization caused by coinjection of the Na⁺,K⁺-ATPase inhibitor ouabain or a high concentration of potassium greatly exacerbated malonate toxicity; this combined toxicity was blocked by the noncompetitive NMDA antagonist MK-801. The toxicity of NMDA was also exacerbated by ouabain. The overt toxicity of a high dose of ouabain (1 nmol) was largely prevented by MK-801. Coinjection of the K⁺ channel activator minoxidil (4 nmol) to reduce depolarization attenuated the toxicity of 1 µmol of malonate by ∼60% without affecting malonate-induced ATP depletion. These results indicate that membrane depolarization exacerbates malonate neurotoxicity and that membrane hyperpolarization protects against malonate-induced neuronal damage. We hypothesize that the effects of membrane potential on malonate toxicity are mediated through the NMDA receptor as a result of its combined agonist- and voltage-dependent properties.     

Identification of

The succinate dehydrogenases (SDH: soluble, membrane-extrinsic subunits of succinate:quinone oxidoreductases) from Escherichia coli and beef heart mitochondria each adsorb at a pyrolytic graphite 'edge' electrode and catalyse the interconversion of succinate and fumarate according to the electrochemical potential that is applied. E. coli and beef heart mitochondrial SDH share only ca. 50% homology, yet the steady-state catalytic activities, when measured over a continuous potential range, display very similar catalytic operating potentials and energetic biases (the relative ability to catalyse succinate oxidation vs. fumarate reduction). Importantly, E. coli SDH also exhibits the interesting 'tunnel-diode' behaviour previously reported for the mitochondrial enzyme. Thus as the potential is lowered below ca. -60 mV (pH 7, 38 degrees C) the rate of catalytic fumarate reduction decreases abruptly despite an increase in driving force. Since the homology relates primarily to residues associated with active site regions, the marked similarity in the voltammetry reaffirms our previous conclusions that the tunnel-diode behaviour is a characteristic property of the enzyme active site. Thus, succinate dehydrogenase is an excellent fumarate reductase, but its activity in this direction is limited to a very specific range of potential.     

Prolonged culture of HOS 58 human osteosarcoma cells with 1,25-(OH)2-D3, TGF-beta, and dexamethasone reveals physiological regulation of alkaline phosphatase, dissociated osteocalcin gene expression, and protein synthesis and lack of mineralization

Cultured rodent osteoblastic cells reiterate the phenotypic differentiation and maturation of osteoblasts seen in vivo. As previously shown, the human osteosarcoma cell line HOS 58 represents a differentiated stage of osteoblast development. The potential of HOS 58 for still further in vitro differentiation suggests the line can serve as a model of osteoblast maturation. Using this cell line, we have investigated the influence of 1,25-(OH)2-D3 (D3), TGF-beta and Dexamethasone (Dex) on proliferation and on the protein and mRNA levels of alkaline phosphatase (AP), procollagen 1 (Col 1), and osteocalcin (Oc), as well as mineralization during 28 days in culture. AP mRNA and protein were highly expressed throughout the culture period with further increase of protein AP activity at constant gene expression levels. A differentiation inhibiting effect of either TGF-beta or Dex was seen. Col 1 was investigated without the use of ascorbic acid and showed only minor changes during culture time or stimulation. The gene expression for Oc increased continually whereas protein synthesis peaked at confluence and decreased thereafter. TGF-beta and Dex treatments decreased Oc mRNA and protein levels. Stimulation by D3 was maximal at day 7 with a decrease thereafter. HOS 58 cells showed no mineralization capacity when stimulated with different agents, as measured by energy-dispersive X-ray microanalysis. This was not due to absence of Cbfa1 expression. In conclusion, the HOS 58 osteosarcoma cell line represents a differentiated cell line with highly expressed and physiologically regulated AP expression during further differentiation in culture. We observed a dissociation between osteocalcin gene expression and protein secretion which may contribute to the lack of mineralization in this cell line.     

Succinate dehydrogenase assembly factor 2 is needed for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis

Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T‐DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co‐expressed with a number of genes encoding mitochondrial proteins, including SDH1‐1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin‐adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein‐bound flavin‐adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro‐respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth.     

Delayed mitochondrial dysfunction in apoptotic hair cells in chinchilla cochleae following exposure to impulse noise

Apoptotic death of hair cells (HCs) in the cochlea has been found following exposure to intense noise. The current study was designed to examine the mitochondrial energetic function of HCs during the course of noise-induced apoptosis. Two aspects of the mitochondrial energetic function, succinate dehydrogenase (SDH) activity and mitochondrial membrane potential (MMP), were examined in HCs of chinchilla cochleae following exposure to a series of 75 pairs of impulse noises at 155 dB pSPL. The results showed that nuclear condensation and uptake of propidium iodide or trypan blue appeared at 10 min after the noise exposure, indicating a rapid progression of HC apoptosis. However, SDH activity was preserved at this time point. As the time elapsed (1 hr or 24 hrs) after the noise exposure, all newly-generated apoptotic HCs showed strong SDH activity, indicating the preservation of SDH activity during the course of apoptosis. Examination of MMP with rhodamine 123 staining revealed that MMP was sustained in the apoptotic HCs having mild nuclear condensation, even after the occurrence of cell membrane leakage. MMP was reduced with further progression of nuclear condensation. These results suggest the presence of a delayed mitochondrial dysfunction in apoptotic HCs following exposure to intense noise. Research was supported by the Grant NIDCD 1R03 DC006181-01A1.     

Activity of some mitochondrial enzymes in the progeny of rabbits treated with dichlorvos in the gestation period.

The activities of cytochrome oxidase (CYO) and succinate dehydrogenase (SDH) in brains of progeny of rabbits treated with dichlorvos (DDVP) 6 mg/kg/day during last ten days of pregnancy were investigated. Biochemical estimation of mitochondrial fraction of the brain tissue and the histochemical studies of fresh cryostat slices were carried out in 1, 8 and 16 day old rabbits. In comparison with control animals the decrease in CYO and SDH activity and the disturbances in "enzymatic maturation" rythm were observed in progeny of rabbits submitted to treatment with DDVP. In neuropil the changes were more pronounced than in pericarium of the nerve cells. In the brain structures which are sensitive to the metabolic disturbances (Ammon horn) SDH activities decreased.     

Modification of bovine heart succinate dehydrogenase with ethoxyformic anhydride and rose bengal: evidence for essential histidyl residues protectable by substrates

Purified and membrane-bound succinate dehydrogenase (SDH) from bovine heart mitochondria was inhibited by the histidine-modifying reagents ethoxyformic anhydride (EFA) and Rose Bengal in the presence of light. Succinate and competitive inhibitors protected against inhibition, and decreased the number of histidyl residues modified by EFA. The essential residue modified by EFA was not the essential thiol of SDH, but modification of the essential thiol abolished the protective effect of malonate against inhibition of SDH by EFA. The EFA inhibition was reversed by hydroxylamine nearly completely when the inhibition was less than or equal to 35%, and only partially when the inhibition was more extensive. The uv spectrum of EFA-modified SDH before and after hydroxylamine treatment suggested that extensive inhibition of SDH with EFA may result in ethoxyformylation at both imidazole nitrogens of histidyl residues. Such a modification is not reversed by hydroxylamine. Succinate dehydrogenases and fumarate reductases from several different sources have similar compositions, and the two enzymes from Escherichia coli have considerable homology in the amino acid composition of their respective flavoprotein and iron-sulfur protein subunits. In the former, there is a short stretch containing conserved histidine, cysteine, and arginine residues. These residues, if also conserved in the bovine enzyme, may be the essential active site residues suggested by this work (histidine) and previously (cysteine, arginine).     

Src protein and tyrosine-phosphorylated protein profiles in marrow stroma during osteogenic stimulation

Src protein is essential for the regulation of bone turnover primarily via bone resorption because it is required in osteoclast differentiation and function. We followed temporal changes of Src protein abundance in marrow stromal cells induced to mineralize by dexamethasone (DEX), growth in cold temperature, or both. Given the tyrosine kinase function of Src and its numerous substrates, profiles of phosphotyrosine-containing proteins were followed as well. On day 11 of stimulation, specific alkaline phosphatase (ALP) activity at 30°C decreased under DEX relative to 37°C cultures, in accord with increased cell counts. Mineralization per well under DEX increased by 25% at 37°C, whereas at 30°C it increased by more than threefold regardless of the DEX stimulation. At 30°C, on a per cell basis mineralization increased 2.5 and 3 times with and without DEX, respectively. Cultures at 37°C showed a general drop per cell of many phosphotyrosine-containing proteins on day 3 relative to days 1 and 2 in both DEX-stimulated and nonstimulated cultures; several proteins did recover (recuperate) thereafter. On days 1 and 2, the phosphotyrosine signal was higher in several proteins under DEX stimulation; this trend became inverted after day 3. The changes in abundance per cell of Src protein (pp60src) followed a similar trend, and in addition a truncated Src molecule, p54/52src, was detected as a putative cleavage product presumably representing its carboxy terminus. The pp60src was most abundant, relative to its truncated product, in day 7 nonstimulated cultures, whereas under DEX stimulation the truncated species pp54/52src showed the highest relative abundance on days 7. At 30°C, DEX stimulation accentuated the increase in Src protein on day 3, showed no change on day 7, and returned to increase Src protein on day 10. Potassium ionophorvalinomycin, considered to select against mineralizing osteoprogenitors at 30°C, showed on day 10 in the absence of DEX a relative increase in truncated Src protein compared to both DEX-stimulated and nonstimulated cultures in the absence of valinomycin. On day 7 of DEX stimulation, the presence of valinomycin resulted in low p54/52src. Among phosphotyrosine-containing proteins, a 32–34 kDa band, as yet unidentified, showed the most concordant changes with mineralization induction. P32–34 decreased by DEX on days 2 and 8 and increased by low temperature alone or combined with DEX on day 3. On day 7, p32–34 did not change under DEX, but valinomycin selected cells with less phoshpotyrosine-containing p32–34. Taken together, high Src abundance at the start of osteogenic induction followed by a decrease 1 week later is probably related to energy metabolism-dependent induction of mineralization. This is in temporal accord with the increase in Src truncation and fluctuation in mitochondrial membrane potential (which affects mineralization). The reported binding of amino-terminal Src oligopeptide to p32 ADP/ATP carrier in the mitochondrial inner membrane raises the question of its possible involvement in mitochondria-regulated mineralization. J. Cell. Biochem. 69:316–325, 1998. © 1998 Wiley-Liss, Inc.     

Induction of osteoprogenitor cell differentiation in rat marrow stroma increases mitochondrial retention of rhodamine 123 in stromal cells

Bone marrow stromal cells contain colony forming cells with the potential to differentiate into osteoprogenitor (OPC) cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and β-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3–4 and 10–11. Between days 5 and 10 there was a drop in Rho retention/cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 μg/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 μg/ml Rho for 12 days only slightly increased day 12 ALP activity/cell, had no effect on day-21 mineralization and only the high dose, 10.0 μg/ml, doubled stromal cell proliferation. Under 24 h incubation Rho concentrations of 1.0 μg/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal cells. The results indicate that under both culture conditions stromal cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.     

Salicylic acid and adenine nucleotides regulate the electron transport system and ROS production in plant mitochondria

Although mitochondria have a central role in energy transduction and reactive oxygen species (ROS) production, the regulatory mechanisms and their involvement in plant stress signaling are not fully established. The phytohormone salicylic acid (SA) is an important regulator of mitochondria-mediated ROS production and defense signaling. The role of SA and adenine nucleotides in the regulation of the mitochondrial succinate dehydrogenase (SDH) complex activity and ROS production was analyzed using WT, RNAi SDH1‐1 and disrupted stress response 1 (dsr1) mutants, which show a point mutation in SDH1 subunit and are defective in SA signaling. Our results showed that SA and adenine nucleotides regulate SDH complex activity by distinct patterns, contributing to increased SDH-derived ROS production. As previously demonstrated, SA induces the succinate-quinone reductase activity of SDH complex, acting at or near the ubiquinone binding site. On the other hand, here we demonstrated that adenine nucleotides, such as AMP, ADP and ATP, induce the SDH activity provided by the SDH1 subunit. The regulation of SDH activity by adenine nucleotides is dependent on mitochondrial integrity and is prevented by atractyloside, an inhibitor of adenine nucleotide translocator (ANT), suggesting that the regulatory mechanism occurs on the mitochondrial matrix side of the inner mitochondrial membrane, and not in the intermembrane space, as previously suggested. On the other hand, in the intermembrane space, ADP and ATP limit mitochondrial oxygen consumption by a mechanism that appears to be related to cytochrome bc1 complex inhibition. Altogether, these results indicate that SA signaling and adenine nucleotides regulate the mitochondrial electron transport system and mitochondria-derived ROS production by direct effect in the electron transport system complexes, bringing new insights into mechanisms with direct implications in plant development and responses to different environmental responses, serving as a starting point for future physiological explorations.