Protonophoric Uncoupler Research Articles

Extracellular pH Modifies Mitochondrial Control of Capacitative Calcium Entry in Jurkat Cells

It was found that a collapse of the mitochondrial calcium buffering caused by the protonophoric uncoupler CCCP, antimycin A plus oligomycin, or the inhibitor of the mitochondrial Ca2+/Na+ exchanger led to a strong inhibition of thapsigargin-induced capacitative Ca2+ entry (CCE) into Jurkat cells suspended in a medium at pH 7.2. The effect of these inhibitors was markedly less significant at higher extracellular pH. Moreover, dysfunction of the mitochondrial calcium handling greatly decreased CCE sensitivity to extracellular Ca2+ when the pH of extracellular solution was 7.2 (apparent Kd toward extracellular Ca2+ rose from 2.3 +/- 0.6 mm in control cells to 11.0 +/- 1.7 mM in CCCP-treated cells) as compared with pH 7.8 (apparent Kd toward extracellular Ca2+ increased from 1.3 +/- 0.4 mM in control cells to 2.4 +/- 0.4 mM in uncoupler-treated cells). Changes in intracellular pH triggered by methylamine did not influence Ca2+ influx. This suggests that, in Jurkat cells, store-operated calcium channels sense extracellular pH change as a parameter that modifies their sensitivity to intracellular Ca2+. In contrast, in human osteosarcoma cells, changes in extracellular pH as well as mitochondrial uncoupling did not exert any inhibitory effects on CCE.

The Uncoupling of Oxidative Phosphorylationof Mouse-Liver Mitochondria in vivo by Usnic Acid

Usnic acid isolated from the local lichen species Usnea articulata had a median lethal dose of 180 mg/kg to mice (s.c. in- jection). The symptoms of the toxicity exhibited by (+) usnic acid was similar to those of classical uncouplers of oxidative phosphorylation. Mice injected with 200 mg/kg of (+) usnic acid produced significant uncoupling of isolated hepatic mitochondria. The signs of uncoupling included: release of respiratory control, inhibition of respiration (80% in the presence of ADP and 60% in its absence), hindering of ATP bi- osynthesis, and stimulation of Mg +2 -ATPase activity (two-fold in- crease). In this respect, (+) usnic acid, a lipophilic weak acid with two ionizable hydrogens, acts in a similar fashion to many protonophoric uncouplers which dissipate the proton motive force (delta mu H+) across the inner mitochondrial membrane, therefore disrupting the tight coupling mechanism between electron transport and ATP syn- thesis.

Mitochondrial permeability transition as the critical target of N-acetyl perfluorooctane sulfonamide toxicity in vitro.

Perfluorooctanyl compounds with active functional groups have been shown to disrupt mitochondrial bioenergetics by three distinct mechanisms: protonophoric uncoupling of mitochondrial respiration, induction of the mitochondrial permeability transition (MPT), or a nonselective increase in membrane permeability. The purpose of this investigation was to identify the initial target and specific sequence of events associated with the N-acetyl substituted perfluorooctanesulfonamides induced MPT. N-acetyl-perfluorooctanesulfonamide (FOSAA), N-ethyl-N-acetyl-perfluorooctanesulfonamide (N-Et FOSAA), perfluorooctanoic acid (PFOA), perfluorooctanesulfonate (PFOS), and N-ethyl-N-(2-ethoxy)-perfluorooctanesulfonamide (N-Et FOSE) were added individually to liver mitochondria freshly isolated from Sprague-Dawley rats. Mitochondrial swelling and cytochrome c release were recorded spectrophotometrically, oxygen uptake was monitored with a Clark-type oxygen electrode, and reactive oxygen species (ROS) were monitored by dichlorodihydrofluorescein diacetate (H(2)DCFDA) fluorescence. FOSAA (45 microM) and N-Et FOSAA (7.5 microM) induced calcium-dependent mitochondrial swelling, the release of cytochrome c, inhibition of uncoupled mitochondrial respiration, and ROS generation, all of which were inhibited by cyclosporin-A (CsA). PFOA (200 microM) displayed slight CsA sensitive activity, but neither PFOS (10 microM) nor N-Et FOSE (70 microM) induced the MPT. Results of this investigation demonstrate two important findings: (1) MPT induction is specific to the N-acetyl substituted perfluorooctanesulfonamides and, (2) the sequence of events is initiated by induction of the MPT, which causes the release of cytochrome c as well as other cofactors leading to inhibition of respiration and ROS generation. The toxicity of N-acetyl perfluorooctanyl compounds may therefore reflect the mitochondrial dysfunction, which is compounded by the ensuing oxidative injury.

Significant enhancement of photobiological H2 evolution by carbonylcyanide m-chlorophenylhydrazone in the marine green alga Platymonas subcordiformis.

A marine green microalga, Platymonas subcordiformis, photo-synthetically generates H(2) but only transiently at a negligible yield when exposed to light after a period of dark anaerobic incubation. A protonophore uncoupler, carbonyl cyanide m-chlorophenylhrazone (CCCP) significantly increased the yield of H(2) photo-production. CCCP optimally at 15 microM gave 4.9 ml H(2) after 8 h light irradiation in 1 l algal cell culture at 1.8 x 10(6) cells ml(-1). The H(2) yield at 15 microM CCCP was increased by 240-fold when compared to the control. This improvement may be by CCCP disrupting the proton motive force thus facilitating proton transfer across the thylakoidal membrane.

Mitochondrial metabolism underlies hyperoxic cell damage

Exposure of mammals to hyperoxia causes pulmonary and ocular pathology. Hyperoxic damage and cell death may derive from enhanced intracellular formation of reactive oxygen species (ROS), probably of mitochondrial origin. There is, however, controversy on this point. When wild-type and respiration-deficient (ρ°) HeLa cells were cultured in 80% O 2, wild-type cells stopped growing after 5 days and died thereafter whereas ρ° cells survived and grew to confluence. This tolerance of ρ° cells for hyperoxia was not associated with greater resistance to oxidants such as hydrogen peroxide and t-butyl hydroperoxide. Under both 20% and 80% O 2, ρ° cells exhibited substantially decreased ROS production, and, under 80% O 2, ρ° cells showed no suppression of aconitase activity or mitochondrial protein carbonyl formation. Replacement of normal mitochondria in ρ° cells restored ROS production and susceptibility to hyperoxia. Two other approaches that diminished mitochondrial ROS generation also increased tolerance for hyperoxia. HeLa cells constantly exposed to the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone, which enhances respiration but decreases ROS production, showed preferential survival under 80% O 2, as did HeLa cells treated with chloramphenicol, which suppresses both respiration and mitochondrial ROS production. We conclude that interactions between respiring mitochondria and O 2 are primarily responsible for hyperoxic cell damage.

Biochemical characteristics of a mutant of the methanoarchaeon Methanothermobacter thermautotrophicus resistant to the protonophoric uncoupler TCS.

In an attempt to more closely define a protein basis of differences in ATPase and ATP synthase activities in a mutant of the methanoarchaeon Methanothermobacter thermautotrophicus resistant to the protonophoric uncoupler TCS (3,3',4',5-tetrachlorosalicylanilide), the composition of membrane associated proteins from the wild-type and mutant strains has been compared. The uncoupler-resistance in the mutant strain was not accompanied by changes in a protein size or changes in the level of subunits A, B and c (proteolipid) of the A1A0-type ATPase-synthase. On the other hand, we revealed a 670-kDa membrane-associated protein complex that is abundantly present only in the mutant strain; it is composed of at least 5 different subunits of 95, 52, 42, 29 and 22 kDa.

Isolation and characterization of an uncoupler-resistant mutant of Methanothermobacter thermautotrophicus

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the protonophorous uncoupler TCS was isolated. The mutant strain exhibited increased CH 4 formation and elevated level of ATPase activity under non-growing conditions. ATP synthesis driven by methanogenic electron transport as well as by potassium diffusion potential in the presence of either H + or Na + ions was markedly diminished in the mutant strain. An abundant membrane-associated protein complex with molecular mass approximately 670 kDa was detected in the mutant strain after native PAGE. The results indicate that TCS resistance in this mutant has arisen as a consequence of mutation(s) that affects a specific locus coding for an uncoupler binding protein(s) and/or modulate the activity of unidentified ATPase.

Kinetic characteristics of chloroplast glucose transport.

Influx of labelled D-glucose into isolated spinach (Spinacia oleracea L. cv. Melody hybrid) chloroplasts was initially rapid followed by a period of slower influx. The stroma glucose concentration attained equilibrium rapidly with low external glucose concentrations and the two were linearly proportional. The period of slower influx resulted from conversion of glucose to acidic products that remained trapped in the chloroplast. As the external glucose concentration increased, the stroma glucose concentration increased less and less, attaining a maximal concentration of 72 mol m(-3). The maintenance of an equilibrium stroma glucose concentration lower than that in the external medium is evidence that plastid glucose efflux involves secondary active transport. The equilibrium stroma glucose concentration increased in response to light and protonophoric uncouplers. It is proposed that glucose efflux is coupled with a proton and the stroma glucose concentration equilibrates in response to the proton gradient across the membrane. To determine if glucose is a significant product of starch mobilization, chloroplasts were isolated from spinach leaves labelled with 14CO2 during the preceding light period. Chloroplasts degraded starch at the same rate as the intact leaf. Glucose, maltose, and isomaltose were the principal labelled products that appeared in the medium during starch mobilization. The glucose concentration in the chloroplast was 2 mol m(-3), which is similar to the measured Km for zero trans efflux. The data support the role of the glucose translocator as an important component in the pathway for sucrose synthesis at night.

Long-chain fatty acids increase basal metabolism and depolarize mitochondria in cardiac muscle cells.

The effects of long-chain (LC) fatty acids on rate of heat production (heat rate) and mitochondrial membrane potential (DeltaPsi) of intact guinea pig cardiac muscle were investigated at 37 degrees C. Heat rate of ventricular trabeculae was measured with microcalorimetry, and DeltaPsi was monitored in isolated ventricular myocytes with either JC-1 or tetramethylrhodamine ethyl ester (TMRE). Methyl-beta-cyclodextrin was used as fatty acid carrier. Application of 400 microM oleate or linoleate increased resting heat rate by approximately 30% and approximately 25%, respectively. When LC fatty acid was supplied as sole metabolic substrate, resting heat rate was decreased by 3-mercaptopropionic acid. In TMRE-loaded myocytes, neither 40-80 microM oleate nor 40 microM linoleate affected DeltaPsi. At a higher concentration (400 microM) both oleate and linoleate increased TMRE fluorescence by approximately 20% of maximum, obtained using 2,4-dinitrophenol (100 microM), indicating a depolarization of the inner mitochondrial membrane. We conclude that LC fatty acids, at sufficiently high concentration, increase heat rate and decrease DeltaPsi in intact cardiac muscle, consistent with a protonophoric uncoupling action. These effects may contribute to the high metabolic rate after reperfusion of postischemic myocardium.

Structural determinants of fluorochemical-induced mitochondrial dysfunction.

Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) are thought to induce peroxisome proliferation and interfere with mitochondrial metabolic pathways. Direct measurements revealed that PFOA and the unsubstituted sulfonamide of perfluorooctane (FOSA) uncouple mitochondrial respiration by increasing proton conductance. The purpose of this investigation was to characterize structural determinants responsible for the mitochondrial uncoupling effect of several structurally related fluorochemicals. Included in the study were PFOA, PFOS, FOSA, the N-acetate of FOSA (perfluorooctanesulfonamidoacetate, FOSAA), N-ethylperfluorooctanesulfonamide (N-EtFOSA), and the N-ethyl alcohol [2-(N-ethylperfluorooctanesulfonamido)ethyl alcohol, N-EtFOSE] and N-acetic acid (N-ethylperfluorooctanesulfonamidoacetate, N-EtFOSAA) of N-EtFOSA. Each test compound was dissolved in ethanol and added directly to an incubation medium containing substrate-energized rat liver mitochondria. Mitochondrial respiration and membrane potential were measured concurrently using an oxygen electrode and a TPP+ -selective electrode, respectively. All of the compounds tested, at sufficiently high concentrations, had the capacity to interfere with mitochondrial respiration, albeit via different mechanisms and with varying potencies. At sufficiently high concentrations, the free acids PFOA and PFOS caused a slight increase in the intrinsic proton leak of the mitochondrial inner membrane, which resembled a surfactant-like change in membrane fluidity. Similar effects were observed with the sulfonamide N-EtFOSE. Another fully substituted sulfonamide, N-EtFOSAA, at high concentrations caused inhibition of respiration, the release of cytochrome c, and high-amplitude swelling of mitochondria. The swelling was prevented by cyclosporin A or by EGTA, indicating that this compound induced the mitochondrial permeability transition. The unsubstituted and mono-substituted amides FOSA, N-EtFOSA, and FOSAA all exerted a strong uncoupling effect on mitochondria resembling that of protonophoric uncouplers. Among these compounds, FOSA was a very potent uncoupler of oxidative phosphorylation, with an IC50 of approximately 1 microM. These data suggest that the protonated nitrogen atom with a favorable pKa is essential for the uncoupling action of perfluorooctane sulfonamides in mitochondria, which may be critical to the mechanism by which these compounds interfere with mitochondrial metabolism to induce peroxisome proliferation in vivo.

Thyroxine reversibly inhibits the uncoupling action of protonophores on energy production in rat thymus lymphocytes.

Earlier we reported that some thyroid and steroid hormones and also 6-ketocholestanol used in micromolar concentrations modulated the effects of protonophoric uncouplers on isolated mitochondria (Starkov et al. (1997) Biochim. Biophys. Acta, 1318, 173-183). In the present study we investigated the effects of a thyroid hormone, thyroxine, on energy coupling of intact rat thymus lymphocytes and mitochondria isolated from these cells. The resting (oligomycin-inhibited) respiration of the isolated intact lymphocytes was stimulated by the addition of protonophoric uncouplers 2,4-DNP, FCCP, or SF6847. Subsequent addition of micromolar concentrations of thyroxin decreased the rate of uncoupler-stimulated respiration and partially reversed uncoupler-induced decrease of membrane potential (DeltaPsi). In experiments with mitochondria isolated from thymus lymphocytes the re-coupling effect of thyroxine was not observed. In this case thyroxine did not influence mitochondrial respiration stimulated with 2,4-DNP, but did potentiate the stimulation of respiration and DeltaPsi decrease induced with another uncoupler, SF6847. The data are discussed in terms of a hypothesis that aromatic uncouplers are transported into the cell by the thyroxine carrier of the plasma membrane.

Profiling of Drugs for Membrane Activity Using Liposomes as an In Vitro Model System

ABSTRACTThe increasing size of chemical libraries being analyzed by high-throughput screening results in a growing number of active compounds that need to be assessed before moving forward in the drug development process. As a consequence, more rapid and highly sensitive strategies are required to accelerate the process of drug discovery without increasing the cost. Due to the fact that significant numbers of compounds from combinatorial libraries are hydrophobic in nature, approaches are needed to evaluate the potential for these compounds to interfere with the functions of biological membranes. The liposome system was used to detect agents that act as follows: (i) ionophores able to induce specific ion permeability, e.g., valinomycin for K+ and protonophoric uncouplers for H+; (ii) ion antiporters which exchange H+ for other ions, e.g., nigericin; (iii) agents that form low specificity ion channels in the membrane, e.g., gramicidin; and (iv) detergents and other membrane-disrupting agents. We propose using this liposome assay during the drug development process to identify compounds that have membrane activity and, as a consequence, produce a biological effect by altering the physico-chemical properties of the cell membrane rather than interacting with a protein target. Screening of a representative set of biologically-active compounds (198) indicated that the majority of systemic antimicrobial drugs, but not topical drugs, lack membrane activity in this model system.

Ca2+ Efflux in Mitochondria from the YeastEndomyces magnusii

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.

Overexpression of UCP3 in cultured human muscle lowers mitochondrial membrane potential, raises ATP/ADP ratio, and favors fatty acid vs. glucose oxidation.

The skeletal muscle mitochondrial uncoupling protein-3 (UCP3) promotes substrate oxidation, but direct evidence for its metabolic role is lacking. Here, we show that UCP3 overexpression in cultured human muscle cells decreased mitochondrial membrane potential (DYm). Despite this, the ATP content was not significantly decreased compared with control cells, whereas ADP content was reduced and thus the ATP/ADP ratio raised. This finding was contrasts with the effect caused by the chemical protonophoric uncoupler, CCCP, which lowered DYm, ATP, and the ATP/ADP ratio. UCP3-overexpression enhanced oxidation of oleate, regardless of the presence of glucose, whereas etomoxir, which blocks fatty acid entry to mitochondria, suppressed the UCP3 effect. Glucose oxidation was stimulated in UCP3-overexpressing cells, but this effect was inhibited by oleate. UCP3 caused weak increase of both 2-Deoxyglucose uptake and glycolytic rate, which differed from the marked stimulation by CCCP. We concluded that UCP3 promoted nutrient oxidation by lowering DYm and enhanced fatty acid-dependent inhibition of glucose oxidation. Unlike the uncoupler CCCP, however, UCP3 raised the ATP/ADP ratio and modestly increased glucose uptake and glycolysis. We propose that this differential effect provides a biological significance to UCP3, which is up-regulated in metabolic stress situations where it could be involved in nutrient partitioning.

Mitochondrial adaptation to in vivo polyunsaturated fatty acid deficiency: increase in phosphorylation efficiency.

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (delta psi), mitochondrial and cytosolic ATP/ADP-Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP-Pi ratio being maintained despite the lower delta psi. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.

Assessment of mitochondrial energy coupling in vivo by 13C/31P NMR.

The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this report, we demonstrate the use of a noninvasive NMR technique to assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining (13)C NMR to measure rates of mitochondrial substrate oxidation with (31)P NMR to assess unidirectional ATP synthesis flux. These combined (31)P/(13)C NMR measurements were performed in control, 10-day triiodo-l-thyronine (T(3))-treated (model of increased UCP3 expression), and acute 2,4-dinitrophenol (DNP)-treated (protonophore and mitochondrial uncoupler) rats. UCP3 mRNA and protein levels increased 8.1-fold (+/- 1.1) and 2.8-fold (+/- 0.8), respectively, in the T(3)-treated vs. control rat gastrocnemius muscle. (13)C NMR measurements of tricarboxylic acid cycle flux as an index of mitochondrial substrate oxidation were 61 +/- 21, 148 +/- 25, and 310 +/- 48 nmol/g per min in the control, T(3), and DNP groups, respectively. (31)P NMR saturation transfer measurements of unidirectional ATP synthesis flux were 83 +/- 14, 84 +/- 14, and 73 +/- 7 nmol/g per s in the control, T(3), and DNP groups, respectively. Together, these flux measurements, when normalized to the control group, suggest that acute administration of DNP (mitochondrial uncoupler) and chronic administration of T(3) decrease energy coupling by approximately 80% and approximately 60%, respectively, and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach could prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.

Energy-dependent accumulation of calcium antagonists in catecholamine storage vesicles

The calcium antagonists verapamil, nitrendipine, mibefradil, and amlodipine accumulate in chromaffin granule ghosts with apparent equilibrium partition coefficients [(mol/mg membrane lipid)/(mol/mg solvent water)] of 246 ± 105 (N = 8), 2700 ± 600 (N = 4), 7400 ± 2200 (N = 4), and 8100 ± 1100 (N = 5), respectively. In the presence of 1.2 mM MgATP, the partition coefficients were 854 ± 206 (N = 10), 2300 ± 600 (N = 4), 32,700 ± 8,900 (N = 7), and 20,300 ± 5,000 (N = 11) for verapamil, nitrendipine, mibefradil, and amlodipine, respectively. Except for nitrendipine, the apparent partition coefficients in the presence of MgATP were significantly different from the control ( P < 0.001). For amlodipine and verapamil, the vacuolar H +-ATPase inhibitors bafilomycin A 1 (30 nM) and N-ethylmaleimide (2 mM) and the protonophore (uncoupler) carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μM) completely blocked the increase in partition coefficients in response to MgATP. The extra amlodipine, mibefradil, and verapamil that accumulated in response to MgATP were released into the medium by CCCP (10 μM) by 18% (N = 5), 30% (N = 5), and 88% (N = 5) for amlodipine, mibefradil, and verapamil, respectively. Thus, amlodipine, mibefradil, and verapamil, but not nitrendipine, accumulate in catecholamine storage vesicles in response to Δμ H+ generated by the endogenous V-type H +-ATPase, and are partially released by de-energetisation. Hence, these calcium antagonists can reach unexpectedly high concentrations in certain target cells, and give pharmacodynamic properties not shared by nitrendipine.

Sodium Chloride-Induced Volume Changes of Freshwater Cyanobacterium Synechococcus sp. PCC 7942 Cells Can Be Probed by Chlorophyll a Fluorescence

Freshwater species of the cyanobacterial genus Synechococcus import NaCl passively, and export Na+ actively, by means of primary and secondary extrusion mechanisms. As a result of the ion and water fluxes, cell volumes are enlarged. We show in this paper that the NaCl-induced volume enlargement of Synechococcus sp. PCC 7942 cells is attended by a rapid (k = 0.39 s−1) increase in chlorophyll (Chl) a fluorescence. The cell turgor threshold (measured by osmotic titration of Chl a fluorescence) was lower in the absence of NaCl (0.195 Osm kg−1) than in the presence of 0.4 M NaCl (0.248 Osm kg−1) indicating NaCl uptake by the cells. Turgor thresholds of cells suspended in NaCl-containing medium were enlarged further by protonophoric uncouplers, P-type ATPase inhibitors, and light starvation, conditions that are known to interfere with the active extrusion of Na+ ions. Cell swelling exerts probably a regulation on the distribution of phycobilisome (PBS) excitation between photosystem II (fluorescent Chl a) and photosystem I (nonfluorescent Chl a), since it affects PBS-sensitized Chl a fluorescence, but not directly excited Chl a fluorescence. The dependence of the Chl a fluorescence of cyanobacteria on cell volumes allows probing of bioenergetic phenomena that are related to dynamic osmotic volume changes, transmembrane solute and water fluxes, plasma membrane permeabilities, and internal osmotic conditions of cyanobacterial cells. Thus, cyanobacteria may serve as quite convenient models of aquatic microorganisms in experimental studies directed toward the elucidation of perception mechanisms and defense mechanisms of water and solute stresses.

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

The role of external Ca2+ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca2+ in the medium. The observed Ca2+ -mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca2+ in the medium. Thus, the ability of Ca2+ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca2+ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca2+ channel blockers tested, such as verapamil and LaCl3 , inhibited the observed Ca2+ -mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m-chlorophenyl hydrazone, inhibited this Ca2+ -mediated response, whereas monensin, an inhibitor of the Na+ /H+ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca2+ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.

Kinetic Model To Describe the Intrinsic Uncoupling Activity of Substituted Phenols in Energy Transducing Membranes

A new approach to understand the increased toxicity of uncouplers as compared to baseline toxicity (narcosis) is presented here. The overall uncoupling activity is quantitatively separated into the contribution of membrane concentration and speciation and intrinsic activity. This approach is a further step toward the development of improved Quantitative Structure−Activity Relationships (QSAR) and of toxicokinetic models used in risk assessment. The protonophoric uncoupling activity of seven nitro- and chlorophenols has been investigated as a function of pH and concentration using time-resolved spectroscopy on photosynthetic membranes. The experimental data are described by a kinetic model that includes a monomeric and a dimeric protonophoric shuttle mechanisms. Input parameters of the model are the experimental data for relaxation of the membrane potential, the biomembrane-water distribution constants of the phenol and phenoxide species, and the acidity constant of the phenol. Adjustable parameters are the translocation rate constants of all phenolic species and the heterodimer formation constant. These parameters constitute the intrinsic uncoupling activity. Hydrophobicity and acidity govern the partitioning of phenols into the membrane but appear not to be the sole determining factors for the intrinsic uncoupling activity of phenolic compounds. Additional factors include steric effects and charge distribution within the molecule.