Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.     

Intracellular acidosis protects cultured hepatocytes from the toxic consequences of a loss of mitochondrial energization

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.     

Characterization of the plasma and mitochondrial membrane potentials of alveolar type II cells by the use of ionic probes

The lipophilic cation triphenylmethylphosphonium (TPMP+) and the potassium analog Rb+, were used to monitor the membrane potential (delta psi) of freshly isolated rabbit type II alveolar epithelial cells. Type II cells were found to accumulate TPMP+ rapidly at 37 degrees C in Hanks' balanced-salt solution with 5 microM tetraphenyl boron, but this accumulation was partially due to non-membrane potential dependent binding of TPMP+ to the cell. Lysophosphatidylcholine (lysoPC) was found to abolish delta psi and permitted correction for bound TPMP+ or Rb+. TPMP+ remaining in the cell following correction for binding represents the sum of mitochondrial and plasma membrane potential dependent accumulation. The accumulation of Rb+ by the type II cell was found to be independent of the mitochondrial membrane potential and indicated a trans-plasma membrane Rb+ distribution potential of -62.9 +/- 4 mV. A similar value was obtained by estimating the plasma membrane potential dependent accumulation of TPMP+ in type II cells whose mitochondria were depolarized with carbonylcyanide m-chlorophenylhydrazone (CCCP). The release of TPMP+ due to CCCP treatment also permitted an estimation for the trans-mitochondrial membrane potential of -141.8 +/- 10 mV. These techniques of membrane potential measurements were found to be sensitive to changes in delta psi induced by a number of inhibitors and ionophores. The ability to measure the membrane potential of the type II pneumocyte, and the changes caused by various agents, should be useful in characterizing the functional responses of this pulmonary surfactant producing cell.     

Determination of mitochondrial calcium content in hepatocytes by a rapid cellular-fractionation technique. Alpha-adrenergic agonists do not mobilize mitochondrial Ca2+.

A rapid cellular fractionation technique [the preceding paper, Shears & Kirk (1984) Biochem. J., 219, 375-382] was employed to separate a mitochondria-rich fraction from hepatocytes within seconds. Mitochondrial Ca was estimated to be no more than 41% of total cell Ca. At least half of the mitochondrial Ca was present in an energy-dependent pool; 20% of total cell Ca was accessible to EGTA within 10s. The alpha-adrenergic agonist phenylephrine stimulated glycogen phosphorylase activity by 100% within 0.5 min and induced a loss of 20% of total cell Ca after 10 min from the EGTA-inaccessible pool. However, between 0.5 and 10 min after the addition of phenylephrine to hepatocytes there was no significant change in the Ca content of the mitochondria-rich fraction. Hepatocytes that were preloaded with Ca2+ during 90 min incubation at 0-4 degrees C expelled this cation during 20 min incubation at 37 degrees C. After this time, phenylephrine failed to alter the Ca content of a mitochondria-rich fraction. It is concluded that alpha-adrenergic agonists do not mobilize Ca2+ from hepatocyte mitochondria.     

Mitochondrial damage as a mechanism of cell injury in the killing of cultured hepatocytes by tert-butyl hydroperoxide

The killing of cultured hepatocytes by tert-butyl hydroperoxide (TBHP) occurs by different mechanisms depending on the presence or absence of the antioxidant N,N'-diphenylphenylenediamine (DPPD). In either situation there is evidence of mitochondrial damage. The mitochondrial inner membrane potential is lost, a result determined by the release from the cells of the lipophilic cation [3H]triphenylmethylphosphonium (TPMP+). Deenergization of the mitochondria is accompanied by a loss of ATP. Oligomycin reduced ATP stores without release of TPMP+ or without effect on the viability of the hepatocytes over the same time course that TBHP killed the majority of the cells. Monensin, a H+/Na+ ionophore, potentiated the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. By contrast, extracellular acidosis reduced the toxicity of tert-butyl hydroperoxide in the presence or absence of DPPD. Neither monensin nor extracellular acidosis affected the metabolism of tert-butyl hydroperoxide, the release of TPMP+, or the extent of the peroxidation of cellular lipids. These data document the presence of mitochondrial damage in hepatocytes intoxicated with TBHP in both the presence and absence of DPPD. Furthermore, the potentiation by monensin is readily explained by the proposal that mitochondrial deenergization is accompanied by an intracellular acidosis. Such acidosis tends to delay the development of lethal cell injury. The protective effect of extracellular acidosis supports this interpretation.     

Control of mitochondrial content of adenine nucleotides by submicromolar calcium concentrations and its relationship to hormonal effects

Rat liver mitochondria were incubated at 30 degrees C with 4 mM ATP in a medium similar in electrolyte composition to that of hepatic cytosol. Under these conditions, a net increase in mitochondrial adenine nucleotides was observed that was dependent on the concentration of free Ca2+ [( Ca2+]) in the incubation medium. At 0.2 microM [Ca2+] or less, there was no demonstrable uptake of adenine nucleotides; at 0.4 microM [Ca2+], or greater, net uptake occurred. The calcium-dependent accumulation of nucleotides by mitochondria required Mg2+ in the incubation medium and was insensitive to carboxyatractyloside. The uptake of adenine nucleotides was enhanced by the addition of antimycin A or antimycin A together with oligomycin. Accumulation of nucleotides appeared to be associated with a small increase in mean mitochondrial volume, but the membrane potential was not affected. No uptake or loss of NAD-NADH by mitochondria was detected. Ruthenium red failed to inhibit the calcium-dependent uptake of adenine nucleotides by the mitochondria, indicating that stimulation of this process by Ca2+ does not involve transport of the cation into mitochondria by the Ca2+ uniporter. Because glucagon acts to elevate cytosolic [Ca2+] from approximately 0.2 microM to 0.6 microM, the same range affecting nucleotide uptake, it is proposed that the increase in mitochondrial adenine nucleotides that follows treatment with glucagon is mediated by the rise in cytosolic [Ca2+] produced by the hormone. This hypothesis was supported by the observation that epinephrine and A23187, agents that raise cytosolic [Ca2+], increased the content of mitochondrial adenine nucleotides in isolated hepatocytes. Furthermore, cells, incubated under calcium-depleting conditions, had a diminished response to glucagon.     

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: Further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP⁺) has been employed to measure ΔΨ_m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP⁺ uptake allows a reliable measure of ΔΨ_m in intracellular mitochondria, provided that the ratio [TPMP⁺]_i/[TPMP⁺]_e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca²⁺, do not concentrate TPMP⁺. The relationships between ΔΨ_m and two other indicators of cellular energy state, ΔG_Pc and E_h, or between ΔΨ_m and J₀, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between ΔΨ_m and ΔG_Pc, E_h or J₀ over the ΔΨ_m range of 120−160 mV, except in the presence of carboxyatractyloside or oligomycin, where ΔΨ_m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of ΔΨ_m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between ΔΨ_m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.     

Depolarization of plasma membrane of rat brain synaptosomes at extra- and intracellular acidification

To elucidate whether a decrease in pH_o and pH_i has the same effect on presynaptic plasma membrane depolarization, we compared the effects of 1 mM of amiloride which decreased pH_i down to 6.65 and incubation medium acidification down to pH^o 7.0 and 6.0 on DiSC3(5) dye fluorescence. Measurements were performed with and without the mitochondrial toxins: rotenone (10 μM) + oligomycin (5 μg/mL). In the presence of toxins the change in DiSC3(5) fluorescence was specific for plasma membrane potential. In the second case, DiSC3(5) signal represented the total potential of mitochondria and plasma membrane. It was shown that decreasing of pHo works both in the presence of mitochondrial toxins and also in their absence. Amiloride is able to increase DiSC3(5) fluorescence only in the presence of the mixture of rotenone + oligomycin. Therefore, unlike the mitochondrial potential, the plasma membrane potential is sensitive to a decrease in pH_i and pH_o.     

Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells

Mitochondria-targeted molecules comprising the lipophilic TPP (triphenylphosphonium) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However, it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentration in different compartments is uncertain. To address this issue, we compared the uptake into both isolated mitochondria and mitochondria within cells of two hydrophobic TPP derivatives, [3H]MitoQ (mitoquinone) and [3H]DecylTPP, with the more hydrophilic TPP cation [3H]TPMP (methyltriphenylphosphonium). Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was approximately 5-fold greater than that for TPMP, as a result of its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, reaching a steady state within 15 min, compared with approximately 8 h for TPMP. This far faster uptake was the result of the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly located within mitochondria, where it was rapidly reduced to the ubiquinol form, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.     

Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.

Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.     

The effect of hormones on proton compartmentation in hepatocytes

Liver mitochondria isolated from rats treated acutely with glucagon exhibit higher respiration-dependent H+ ion gradients across the mitochondrial inner membrane than mitochondria from control rats. It has been suggested that similar increases in mitochondrial delta pH in situ could stimulate gluconeogenesis, chiefly because the transport of pyruvate into mitochondria would increase in response to the increase in mitochondrial matrix pH. In order to determine whether the increased delta pH observed in vitro in isolated mitochondria also occurs in situ, the effect of glucagon on the pH in the cytosol and mitochondria matrix spaces of isolated hepatocytes was determined. For qualitative results, the spectral responses of intracellularly trapped 6-carboxyfluorescein was used to monitor cytosol pH, while fluorescein-loaded hepatocytes were used to monitor the mitochondrial pH. Hepatocytes were incubated with the diacetate ester derivatives of these dyes. The esters are permeable to the cell membranes, but are rapidly hydrolyzed in the cells. The free unesterified dyes are relatively impermeable to the cell membranes. After being trapped in the cell, 6-carboxyfluorescein remains localized in the cell cytosol, whereas fluorescein is taken up by the mitochondria as a function of the mitochondrial delta pH. In order to quantitate the actual pH in these compartments, the spectral responses (490-465 nm) of 6-carboxyfluorescein-loaded hepatocytes were used to determine the cytosolic pH. Calibration of these responses was obtained within the cell by determination of the dye's differential absorption coefficient (epsilon 490-465 nm) in various high K+ buffers after equilibration of the internal and external pH with valinomycin and the uncoupler 1799. All absorbance values were corrected for dye leakage. Equal hematocrits of unloaded cells were used to correct for absorbance contributions from cellular constituents. The mitochondrial pH was determined by a combination of the indicator dye and [14C]5,5'-demethyloxazolidine-2, 4-dione (DMO) distribution ratio methods. The weak acid DMO freely distributes across the plasma membrane and mitochondrial membrane in whole cells according to the pH gradient across each membrane. Knowledge of the cytoplasmic pH from the 6-carboxyfluorescein data allows the expected distribution of DMO across the plasma membrane to be calculated. The excess accumulation of DMO in intact hepatocytes over that predicted from the plasma membrane pH gradient alone was then used to calculate the pH gradient across the mitochondrial inner membrane. The effects of valinomycin, uncouplers, and hormones on the pH in cytosolic and mitochondrial compartm     

Stimulation of synaptosomal ATP-dependent Ca2+-uptake by N-ethylmaleimide

Treatment of rat brain synaptosomal lysate with N-ethylmaleimide (NEM) was found to stimulate ATP-dependent Ca2+-uptake. This Ca2+-uptake stimulation was blocked by dithioerythritol (DTE), mitochondrial inhibitors oligomycin and sodium azide, but not by vanadate, an inhibitor of plasma membrane Ca2+ pump. Maximal stimulation of Ca2+-uptake was observed at a NEM/protein ratio of 0.1 mumole/0.5-1.0 mg. On fractionation, it was found that NEM did not affect synaptic plasma membrane Ca2+-uptake, but almost doubled it in synaptic mitochondria.     

Dependence of mitochondrial and cytosolic adenine nucleotides on oxygen partial pressure in isolated hepatocytes. Application of a new rapid high pressure filtration technique for fractionation.

By using a new rapid high pressure filtration technique, mitochondrial and cytosolic ATP and ADP contents were determined in isolated hepatocytes at different oxygen partial pressures. At 670 mmHg, subcellular adenine nucleotide contents and ATP/ADP ratios were comparable with values obtained with the digitonin fractionation technique. However at lower oxygen partial pressure ADP appears to be rephosphorylated during digitonin fractionation whereas with high pressure filtration fractionation rephosphorylation of ADP is avoided due to shorter fractionation times. Cytosolic and mitochondrial ATP/ADP ratios decrease if oxygen partial pressure is lowered. However the absolute values of ATP/ADP ratios depend critically on the incubation conditions. Thus incubation of hepatocytes in an oxystat system, where oxygen partial pressure is maintained constant by infusing oxygen-saturated medium and the hepatocyte suspension is continuously stirred, yields much higher subcellular and overall ATP/ADP ratios than incubation in Erlenmeyer flasks gassed with different gas mixtures and shaken in a water bath. This is ascribed to limited diffusion of oxygen from the medium into the cell if the suspension is not mixed thoroughly by stirring. The strong dependence of subcellular ATP/ADP ratios on incubation conditions indicates that oxygen may be one rate-controlling factor for oxidative phosphorylation in the intact cell.     

Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert-butyl hydroperoxide metabolism, prevents the ATP-dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3-hydroxybutyrate in the medium, prevents this hydroperoxide-induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum. Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+-extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.     

The mitochondrial network of human neutrophils: role in chemotaxis,phagocytosis, respiratory burst activation,and commitment to apoptosis

It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC(50) = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 microg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.     

Mechanism of peroxide-induced cellular injury in cultured adult cardiac myocytes

Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse-phase high-pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1-C14]pyruvate. Extracellular glutathione rose 3.3-fold in response to small doses of peroxide (approximately 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50-60% of control values with only a modest (0.95-0.91) reduction in energy charge [ATP + 1/2 ADP)/(ATP + ADP + AMP]. Peroxide-treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose-dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.     

Studies on the transport of acetyl groups from peroxisomes to mitochondria in isolated liver cells oxidizing the polyunsaturated fatty acid 22:4n-6.

The oxidation of the fatty acid [1-(14)C]22:4n-6 was studied in isolated hepatocytes. Labeled acetate was the main acid soluble product identified by HPLC after short incubation periods. At low substrate concentrations and longer incubations [(14)C]acetate was gradually replaced by labeled beta-hydroxybutyrate, acetoacetate and oxaloacetate/malate. Preincubation with 2-tetradecylglycidic acid (TDGA), an inhibitor of mitochondrial fatty acid oxidation, did not reduce the oxidation but acetate was the only product recovered. TDGA also strongly inhibited the metabolism of added [1-(14)C]acetate to mitochondrial oxidation products. During the preparation procedure of hepatocytes the cellular L-carnitine concentration was decreased but it was restored after preincubation with L-carnitine. With low [1-(14)C]22:4n-6, concentrating a low level of [(14)C]acetate and high levels of labeled mitochondrial oxidation products were recovered after preincubation with L-carnitine. A small amount of [(14)C]acetylcarnitine was also detected under this incubation condition. The results suggest that a significant part of labeled acetyl groups from the peroxisomal oxidation of [1-(14)C]22:4n-6 is transported to the mitochondria as free acetate. Moreover, the results also suggest that L-carnitine at physiological concentrations may facilitate the transport of part of the acetyl groups from peroxisomes to mitochondria as acetylcarnitine. However, the possibility that an increased cellular L-carnitine concentration may stimulate oxidation of [1-(14)C]22:4n-6 in mitochondria could not be excluded.     

Modulation of the alpha 1-adrenergic control of hepatocyte calcium redistribution by increases in cyclic AMP

The Ca2+ content of hepatocytes from juvenile male rats (80-110 g) or adult female rats (135-155 g) displayed a biphasic dose-response curve to epinephrine. Low concentrations (less than or equal to 10(-7) M) caused efflux of Ca2+ from the cells, while higher concentrations (10(-6) M and 10(-5) M) induced net Ca2+ uptake which correlated with a large beta 2-adrenergic-mediated increase in cAMP (Morgan, N. G., Blackmore, P. F., and Exton, J. H. (1983) J. Biol. Chem. 258, 5103-5109). Calcium accumulation could be induced in cells from older male rats (180-230 g) by combining a Ca2+-mobilizing hormone with either exogenous cAMP or glucagon (10(-8) M). Readdition of Ca2+ in the presence of glucagon to cells treated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also resulted in enhanced Ca2+ accumulation compared with controls. Addition of vasopressin plus glucagon to the medium perfusing male rat livers also led to cell Ca2+ accumulation, as evidenced by uptake of Ca2+ from the perfusate. Incubation of hepatocytes with antimycin A, oligomycin, and carbonyl cyanide m-chlorophenylhydrazone prevented net Ca2+ accumulation suggesting that mitochondria play a role in the uptake response. This was confirmed by isolation of mitochondria from cells incubated under conditions which promote Ca2+ accumulation. Within 5 min of incubation, the Ca2+ content of these mitochondria was increased 2-fold relative to controls, an effect which was inhibited by oligomycin. These studies demonstrate that a rise in hepatic cAMP can reverse hormonally induced Ca2+ mobilization and point to a major role for the mitochondria in this effect.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.