Glucagon effects on the membrane potential and calcium uptake rate of rat liver mitochondria

It has been widely reported that the in vivo administration of glucagon to rats results in the stimulation of calcium influx in subsequently isolated liver mitochondria. The mechanism of this effect is investigated through simultaneous measurements of calcium uptake rate and mitochondrial membrane potential. This allows the measurement of the calcium uniporter conductance independent of hormonal effects on electron transport or respiration. Two experimental approaches are used. The first involves measuring the uptake of 40-50 nmol of Ca2+/mg of mitochondrial protein with the calcium dye antipyrylazo III; the second uses 45Ca2+ to follow uptake in the presence of 0.5 to 1.5 microM free calcium, buffered with HEDTA. In both cases a tetraphenyl phosphonium electrode is used to follow membrane potential, and membrane potential is varied using either malonate or butylmalonate in the presence of rotenone. The relative merits of these two approaches are discussed. The conductance of the calcium uniporter is found not to be stimulated by glucagon pretreatment. Also, the relative glucagon stimulation of both calcium influx and membrane potential is found to increase with increasing malonate concentration. These results imply that there is no direct stimulation of calcium uptake into liver mitochondria following glucagon treatment. The results are consistent with a glucagon stimulation of substrate transport, substrate oxidation, or a stimulation of electron transport resulting in an increased membrane potential and secondary stimulation of calcium uptake.     

Phosphate transport,membrane potential,and movements of calcium in rat liver mitochondria

The membrane potential and calcium accumulation of mitochondria were followed by ion-specific electrodes in the presence of the proton-donor anions phosphate, acetate, glutamate, and beta-hydroxybutyrate. Phosphate was the only anion which allowed rapid and complete restoration of both the membrane potential and the steady-state extramitochondrial calcium concentration after the uptake of 100–200 nmol calcium per mg protein. If there was no influx of any proton-donor anion, the extent of calcium uptake depended on the intramitochondrial phosphate content. Both the fall of the membrane potential and the increase of the external calcium concentration brought about by a given amount of uncoupler were counteracted by phosphate transported into the mitochondria.     

Effect of thyroxine in vitro on the transmembrane potential of rat liver mitochondria

It has been shown in in vitro experiments that a certain latent period after the addition of thyroxine (T4) and triiodothyronine (T3) was necessary for the manifestation of their effects on transmembrane potential (TMP) of the rat liver mitochondria. The duration of the lag-period decreased upon an increase in the concentrations of these hormones, and T4 at a dose of 2.10(-4) M produced a fall in TMP immediately after its addition. The rate of TMP fall was in proportion with the concentrations of thyroid hormones introduced into the cell, with T3 30-40% more effective than T4. It was established that the action of I2 resembled that of thyroid hormones, namely, a fall in TMP, mitochondrial swelling, activation of transhydrogenase Kl was ineffective. It is suggested that the appearance of the lag-period upon the action of thyroid hormones might be explained by the period of time necessary for the formation of the active iodine forms, as well as by the formation of fatty acids (donators of H+) by mitochondrial phospholipases. All these factors lead to TMP fall resulting in decreased formation of sufficient ATP quantities in mitochondria.     

Calcium release from the rat liver mitochondria during collapse of the membrane potential

Ca(2+)-release from rat liver mitochondria after protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP)-induced membrane depolarisation is studied. It is shown that the release of calcium is accompanied by an increase of the inner mitochondrial membrane permeability as the result of the opening of permeability transition pore (PTP). Calcium is released from mitochondria through the uniporter working in reverse mode and also by PTP mechanism which accounts for ruthenium red (RR)-insensitive component of total. Ca(2+)-release. Unlike Ca2+, the strontium release from the mitochondria is completely sensitive to RR, specific uniporter blocker, which shows the absence of rapid Sr(2+)-efflux mechanisms other than uniporter of bivalent cations. The data obtained also give an evidence that the lifetime of the open state of the pore is limited, and barrier properties of the mitochondrial membrane are restored after the closure of the pore.     

Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.     

Bulk phase proton fluxes during the generation of membrane potential in rat liver mitochondria.

The addition of oxygen to anaerobic rat liver mitochondria incubated at 15 degrees C in the absence of permeant cations produced negligible rapid H+ ejection, monitored spectroscopically with phenol red, which corresponded kinetically to the rise in delta psi, as monitored by merocyanine 540. Slow H+ translocation was observed under these conditions during the aerobic phase, the extent of which was proportional to the amount of oxygen added and the rate dependent on the rate of counter-ion movement. Measurement of H+ disappearance in the mitochondrial matrix, as monitored by neutral red, likewise showed little or no rapid H+ change in the absence of counter-ion movements. In the presence of permeant cations, the H+ disappearance in the matrix was readily measured. This observation argues against the importance of the mitochondrial outer membrane and intermembrane space in masking H+ movements. The H+ translocation required in the generation of maximal or static head delta psi was determined by following the spectral response of merocyanine to increasing oxygen additions. The amount of oxygen giving maximal Delta psi corresponds to an extrusion of 2-3 ng ions of H+ . mg of protein-1. The absence of H+ movement of near this magnitude during the development of the Delta psi argues against the Delta psi-driven backflow of H+ ions as the sole explanation of these observations.     

Effect of Mg2+ on membrane fluidity and K+ transport in rat liver mitochondria

1. The effects of Mg2+ on the fluidity and on the transport properties of mitochondrial inner membrane were compared in parallel experiments. The fluidity was measured by intercalated fatty acid spin probes. Valinomycin-induced K+ uptake was followed using an ion-selective electrode. 2. The rotational diffusion rate of lipids was very slightly affected by Mg2+, whereas the ordering of the probed region of the inner membrane increased considerably above 30 degrees C in the presence of Mg2+. Mg2+ strongly inhibited K+ transport, particularly above 30 degrees C. 3. In the presence of different concentration of MgCl2 (0--30 mM) the order parameter showed no significant variation, whereas the rotational correlation time had essentially biphasic character with a minimum (i.e., faster diffusion rate) at 10 mM MgCl2. 4. We conclude that Mg2+ induces structural changes in the mitochondrial inner membrane and concomitant changes in its functional properties. The term 'fluidity' is inadequate for the interpretation of the data, since changes in the order parameter and in the characteristic correlation time of the inner membrane upon addition of Mg2+ did not show parallel tendencies.     

Response of the electrochromic dye,merocyanine 540, to membrane potential in rat liver mitochondria

Summary We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca^2–] _a -dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca^2–-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca²⁺-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K^–] _a , nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba²⁺] _a . All these data are consistent with the concept that PTX may act on Ca^2– channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca²⁺ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca²⁺ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca²⁺ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca²⁺ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca²⁺-channel conductance (6 nS/cell at 2.6mm [Ca²⁺] _a ). PTX evoked the activation of a new class of Ca²⁺-selective channels (5 pS in 25mm [Ca²⁺]_pipet), which are rather insensitive to membrane potential. We have termed theseG-type calcium channels. These data suggest that treatment with PTX not only increases the probability of L-type Ca²⁺-channel activation at more negative potentials, but also increases the probability of opening of an entirely new, voltage-independent, Ca²⁺ channel. These actions of PTX should promote Ca²⁺ entry and might explain the stimulation by the toxin of CA secretion from medullary chromaffin cells in culture.     

Effect of bovine serum albumin on membrane potential in plant mitochondria

The membrane potential in highly coupled potato ( Solanum tuberosum L.) mitochondria, as measured by changes in safranine absorbance, was significantly increased by addition of bovine serum albumin. Purification of potato mitochondria on Percoll, in removing 50% of free unsaturated fatty acids, decreased the BSA-de-pendent membrane potential. The effect of added linoleic acid and of the natural accumulation of fatty acids during aging was studied. The response of membrane potential to addition of bovine serum albumin appeared to be directly correlated to the amount of free unsaturated fatty acids. Aging in vitro, in releasing free fatty acids, decreased respiratory control and ADP:O ratios and collapsed the membrane potential. During 2–3 h of incubation, addition of BSA completely restored membrane potential and oxidative phosphorylation.
It is concluded that both in fresh and in aged potato mitochondria the effect of bovine serum albumin on oxidative phosphorylation can be ascribed to an effect on membrane permeability to ions. BSA, in binding free unsaturated fatty acids, restored maximal membrane potential. The bovine serum albumin-dependent membrane potential appears to be a sensitive criterion of the functional integrity of the inner mitochondrial membrane.     

Proton translocation in cytochromec oxidase: Redox linkage through proximal ligand exchange on cytochromea 3

An analysis of resonance Raman scattering data from CO-bound cytochromec oxidase and from the photodissociated enzyme indicates that histidine may not be coordinated to the iron atom of cytochromea ₃ in the CO-bound form of the enzyme. Instead, the data suggest that either a water molecule or a different amino acid residue occupies the proximal ligand position. From these data, it is postulated that ligand exchange on cytochromea ₃ can occur under physiological conditions. Studies of mutant hemoglobins have demonstrated that tyrosinate binds preferentially to histidine in the ferric forms of the proteins. In cytochromec oxidase tyrosine residues are located near the histidine residues recently implicated in coordination to cytochromea ₃ (Shapleighet al., 1992; Hosleret al., this volume). Expanding on these concepts, we propose a model for proton translocation at the O₂-binding site based on proximal ligand exchange between tyrosine and histidine on cytochromea ₃. The pumping steps take place at the level of the peroxy intermediate and at the level of the ferryl intermediate in the catalytic cycle and are thereby consistent with the recent results of Wilkstrom (1989) who found that proton pumping occurs only at these two steps. It is shown that the model may be readily extended to account for the pumping of two protons at each of the steps.     

The role of phospholipase A2 in lipid peroxidation-induced fall of membrane potential of rat liver mitochondria

Cumene hydroperoxide (230 microM)-induced fall of the membrane potential takes place only in Ca2(+)-loaded mitochondria. Inhibitor of phospholipase A2 p-bromphenacyl bromide prevents uncoupling of mitochondria, having no effect on the accumulation of lipid peroxidation products.     

Effect of inflammatory mediators on respiration in rat liver mitochondria

Some inflammatory mediators have been studied for their influence on the energy reactions of the liver mitochondria. Mediators were injected intraperitoneally to rats 15 min before decapitation in the following doses (per 100 g of the body) weight: histamine--0.5 mg, serotonin--0.5 mg, bradykinin--0.2 mg, andekalin--0.5 units. Histamine action in the body is connected with modification of the respiratory mitochondria chain and, like the oligomycin action, is directed to attended oxidation and phosphorylation points. Serotonin increases the mitochondria sensitivity to separating agents in succinate oxidation. It is supposed that serotonin-induced inhibition of oxidation of NAD-dependent substances is connected with NADH2 dehydrogenase inhibition or transhydrogenase reaction activation. Bradykinin has activated NAD-dependent substance oxidation and increased respiratory chain sensitivity on the SoQ link to 2,4-dinitrophenol action. Andekalin exerts an analogous effect intensifying ADP-, DNP- and Ca-stimulated respiration of mitochondria during succinate oxidation. Mechanism of the inflammatory mediators influence on the energy metabolism is discussed.