Jörg W. Stucki

Stimulation of respiration by methylene blue in rat liver mitochondria

The effect of methylene blue on isolated rat liver mitochondria in the presence and absence of chloroacetaldehyde was investigated. Fatty acid oxidation was inhibited by chloroacetaldehyde and subsequently stimulated by methylene blue. Assessment of tightly coupled mitochondria revealed decreasing respiratory control ratios induced by increasing concentrations of methylene blue and methylene blue provoked mitochondrial swelling. In uncoupled mitochondria, methylene blue promoted a concentration‐dependent stimulation of respiration. These findings provide evidence that methylene blue, the redox dye currently used as an antidote for encephalopathy associated with alkylating chemotherapy, uncouples oxidative phosphorylation and acts as an electron transfer mediator to stimulate mitochondrial respiration.

Regulation of the degree of coupling of oxidative phosphorylation in intact rat liver.

The degree of coupling of oxidative phopshorylation q was determined in isolated perfused livers and in livers in vivo from fed and fasted rats. This determination of q was based on a simple nonequilibrium-thermodynamic representation of the major reactions of cytosolic adenine nucleotides, and made use of the measured cytosolic concentrations of adenine nucleotides, phosphate, and lactate/pyruvate ratios in extracted livers. The deviations of the measured values from the theoretically predicted ones at different mass action ratios of the adenylate kinase reaction showed that the basic assumptions of the model, including linearity between flows and thermodynamic forces, were fulfilled in intact liver within the experimental error. The degree of coupling was higher in livers from fed rats than in livers from fasted rats. In particular, the determined values of q were close to the theoretical degrees of coupling qecp and qecf which allow maximization of output power and output flow of oxidative phosphorylation for fed and fasted states, respectively, at optimal efficiency and minimal energy costs. This finding indicates that conductance matching between the load and phosphorylation is fulfilled in vivo. Moreover, it was found that fatty acids lower the degree of coupling in a concentration-dependent manner. This suggested that in livers in the fasted state q is decreased due to elevated fatty-acid levels. Thus fatty acids could act as metabolic regulators of the degree of coupling, enabling the cell to optimize efficiency of oxidative phosphorylation under different metabolic regimes.

Simultaneous measurements of Ca2+ in the intracellular stores and the cytosol of hepatocytes during hormone‐induced Ca2+ oscillations

Simultaneous Ca2+ measurements in the cytosol and intracellular stores (IS) of rat hepatocytes were performed using two Ca2+‐sensitive probes (Fluo‐3 and Mag‐fura‐2), and combined whole‐cell patch clamp and fluorescence microscopy. A steady‐state Ca2+ concentration of ∼630 μM was estimated in the IS. α1‐Adrenergic stimulation induced periodic elevations of cytosolic Ca2+ and parallel synchronized transient declines in the IS. Subsequent application of the intracellular Ca2+‐pump inhibitor thapsigargin resulted in a release of Ca2+ from the IS to reach a level of Ca2+ depletion much lower than the lowest transient decline observed during the oscillations.

Efficiency of energy conversion in model biological pumps: Optimization by linear nonequilibrium thermodynamic relations

Experimental investigations showed linear relations between flows and forces in some biological energy converters operating far from equilibrium. This observation cannot be understood on the basis of conventional nonequilibrium thermodynamics. Therefore, the efficiencies of a linear and a nonlinear mode of operation of an energy converter (a hypothetical redox-driven H+ pump) were compared. This comparison revealed that at physiological values of the forces and degrees of coupling (1) the force ratio permitting optimal efficiency was much higher in the linear than in the nonlinear mode and (2) the linear mode of operation was at least 10(6)-times more efficient that the nonlinear one. These observations suggest that the experimentally observed linear relations between flows and forces, particularly in the case of oxidative phosphorylation, may be due to a feedback regulation maintaining linear thermodynamic relations far from equilibrium. This regulation may have come about as the consequence of an evolutionary drive towards higher efficiency.

A critical evaluation of in situ measurement of mitochondrial electrical potentials in single hepatocytes.

The range of applicability and the critical parameters involved in the assessment of mitochondrial electrical potential (delta psi mit) using epifluorescence microscopy were evaluated based on both theoretical and experimental analysis. Rat hepatocytes loaded with the potential-dependent fluorescent dye rhodamine 123 exhibited the expected heterogeneity of fluorescence distribution with dark regions corresponding to the nucleus and bright regions corresponding to the mitochondria-rich cytosol. Calibration of the signal was performed by permeabilizing the cell membrane for monovalent cations using nystatin and gramicidin, and equilibrating the cell with a K(+)-free bath solution. A voltage-clamp at defined delta psi mit was then achieved after addition of valinomycin in the presence of different K+ concentrations in the bath. Theoretical analysis indicated that the ratio of fluorescence intensity measured in mitochondria-rich and mitochondria-poor regions of cell was related with delta psi mit and yielded quantitative estimates of electrical potential with an accuracy of 10-20 mV. The ratio tended to plateau at potentials more negative than-140 mV, showing a limitation of the technique. Manoeuvres such as imposing a heavy ATP demand or interfering with the mitochondrial respiration depolarized mitochondria, while delta psi mit was not altered in a measurable manner during Ca2+ oscillations consecutive to alpha 1-agonist stimulation.

The adenylate kinase reaction acts as a frequency filter towards fluctuations of ATP utilization in the cell.

The buffering ability of the adenylate kinase reaction with respect to the phosphate potential and efficiency of oxidative phosphorylation in the presence of a fluctuating load conductance were studied by computer simulations. Fluctuations of the load conductance, i.e., of the irreversible ATP-utilizing reactions in the cell, were generated by integrating an Ornstein-Uhlenbeck diffusion process. This real or colored noise was then injected into the set of differential equations describing the rate laws for the changes of the adenine nucleotide concentrations based on a simple nonequilibrium thermodynamic model of oxidative phosphorylation. Numerical integration of this system of stochastic differential equations allowed us to investigate the influence of different parameters on the performance of this energy converter. Probability density estimates revealed that the variance of the efficiency about its optimal value was significantly reduced by the adenylate kinase reaction. It was found that the buffering ability of this enzyme is restricted to a specific frequency domain of the fluctuations of the load conductance. This frequency filtering was confirmed by substituting the random fluctuations of the load conductance by simple sinusoidal perturbations. All these studies revealed that for each domain of frequencies of the load perturbations there exists an optimal activity of the adenylate kinase which minimizes deviations from optimal efficiency of oxidative phosphorylation.

Hormone-induced intracellular calcium oscillations and mitochondrial energy supply in single hepatocytes

Abstract Mitochondria in liver cells behave as a regulated stable power supply — optimized with respect to efficiency and stability against fluctuations of ATP-utilizing reactions in the case of starvation — even in the presence of intracellular Ca 2+ oscillations.

Transient kinetics of thermodynamic buffering

The transient response of mitochondrial ATP production towards perturbations was studied by analyzing the trajectories leading from arbitrary initial conditions of the adenine nucleotide pool to the final steady state. These trajectories were calculated from differential equations based on linear relations between flows and thermodynamic forces of the adenylate kinase system including oxidative phosphorylation. The motion of the system along the trajectories consists of two phases: (1) a rapid phase leading from initial states to a common relaxation curve; and (2) a slow phase leading along the relaxation curve to the final steady state. The first phase corresponds to a motion close to the loci of constant adenylic energy charge. In line with this observation is the finding that the energy charge is a constant of motion of the adenylate kinase reaction. The second phase corresponds to a motion along a relaxation curve characterized by minimal Lyapunov exponents in the concentration space of the adenine nucleotides. Thus, both phases of the transient kinetics can be approximated in terms of thermodynamic functions to a high degree of precision. Incubations with isolated rat liver mitochondria were in excellent agreement with the theoretical predictions. In summary, these studies show that the adenylate kinase system not only optimizes the efficiency of oxidative phosphorylation through thermodynamic buffering but, in addition, also deeply influences the transient response of the whole system.

Perturbation of myo-Inositol-1,4,5-Trisphosphate Levels during Agonist-Induced Ca2+ Oscillations

Agonist-induced Ca2+ oscillations in rat hepatocytes involve the production of myo-inositol-1,4,5-trisphosphate (IP3), which stimulates the release of Ca2+ from intracellular stores. The oscillatory frequency is conditioned by the agonist concentration. This study investigated the role of IP3 concentration in the modulation of oscillatory frequency by using microinjected photolabile IP3 analogs. Photorelease of IP3 during hormone-induced oscillations evoked a Ca2+ spike, after which oscillations resumed with a delay corresponding to the period set by the agonists. IP3 photorelease had no influence on the frequency of oscillations. After photorelease of 1-(alpha-glycerophosphoryl)-D-myo-inositol-4,5-diphosphate (GPIP2), a slowly metabolized IP3 analog, the frequency of oscillations initially increased by 34% and declined to its original level within approximately 6 min. Both IP3 and GPIP2 effects can be explained by their rate of degradation: the half-life of IP3, which is a few seconds, can account for the lack of influence of IP3 photorelease on the frequency, whereas the slower metabolism of GPIP2 allowed a transient acceleration of the oscillations. The phase shift introduced by IP3 is likely the result of the brief elevation of Ca2+ during spiking that resets the IP3 receptor to a state of maximum inactivation. A mathematical model of Ca2+ oscillations is in satisfactory agreement with the observed results.

Non-equilibrium thermodynamic sensitivity of oxidative phosphorylation

A new method was developed to analyse the dynamic properties of oxidative phosphorylation, in particular the sensitivity of the phosphate potential with respect to fluctuating cellular ATP utilization. This treatment is based on the eigenvalue sensitivity analysis of an experimentally supported non-equilibrium thermodynamic model of oxidative phosphorylation. Such an analysis allows direct access to the kinetic information, while circumventing the awkward conventional numerical integration of a set of nonlinear differential equations. This procedure revealed, for the parameters characteristic for liver of starved rats in vivo, that the sensitivity of oxidative phosphorylation to a fluctuating ATP utilization is minimal at a degree of coupling q = 0.95. This means that the phosphate potential is highly buffered with respect to fluctuating energy demands at that degree of coupling. This value of q agrees well with the degree of coupling qecf, at which net ATP production of oxidative phosphorylation – at optimal efficiency – occurs in the most economic way. This simultaneous maximization of kinetic stability and economic thermodynamic efficiency at the same degree of coupling appears to be a coincidence.