Electroorientation effect of isolated mitochondria in different functional states

For the first time, the electroorientation effect of isolated rat liver mitochondria has been investigated in a nonuniform alternating electric field at frequencies from 50 to 10(7) Hz. Two relaxation regions of mitochondria electroorientation have been revealed; they are due to the presence of low-frequency alpha - and high-frequency beta-dispersion. It has been shown that incubation of mitochondria in a medium with a low electroconductivity as well as in the presence of ionophores (valinomycin, 2,4-dinitrophenol) results in a decreased electroorientation value in the region of the high-frequency structural polarization. This is explained by a marked lowering of the effective electroconductivity of mitochondria due to changes in the permeability of the inner membrane of organelles. Upon the addition of ionophores, the decrease in the electroorientation effect value becomes more pronounced. Inhibition of respiration of mitochondria by antimycin A resulted in a slight diminution of the effect, followed by time-dependent stabilization, probably due to a lowered electroconductivity of the mitochondrial matrix, induced by the reduction of pH in the intramitochondrial space, change in the ionization of inner enzyme structures, and a partial limitation of the inner ion mobility.     

Chemical modification of mitochondria. Uncoupler binding by mitochondria in different metabolic states

At low uncoupler concentrations the binding of carbonyl-cyanide-m-chlorophenyl-hydrazone to mitochondria was found to depend sensitively on the metabolic state of mitochondria. The binding data are consistent with the assumption that at low concentrations and pH 7.4 the uncoupler is bound mainly in anionic form to the inner mitochondrial membrane and that upon energization the inner membrane undergoes conformation change, exposes buried ionizable groups and hence acquires a negative net membrane charge. Deenergization of the inner membrane by a small amount of uncoupler removes the negative net membrane charge and consequently increases the apparent binding constants. Based upon the present results on uncoupler binding and previous observations on the physiological properties of alkylating uncouplers, a possible molecular mechanism involving electron carriers and coupling factors is suggested for coupling electron transport to phosphorylation.     

Freeze-fracture analysis of isolated liver mitochondria in different metabolic states

Isolated liver mitochondria were crosslinked with glutaraldehyde during different respiratory states and then analyzed either by freeze-fracturing, or in thin sections prepared according to a low denaturation embedding technique. In freeze-fractured material, large intracristal spaces were found in 86% of the mitochondria freshly isolated in a sucrose medium. These results were in good agreement with mitochondria analyzed in thin sections, in which case 88% of the mitochondria contained intracristal spaces. In freeze-fractured mitochondria, only 24% of the mitochondria crosslinked after 3 min in respiratory state 4 contained intracristal spaces, while 36% of the mitochondria contained intracristal spaces during state 3. These values compared favorably with those obtained in embedded material in which case 21 and 41% of the mitochondria contained intracristal spaces during states 3 and 4, respectively. The agreement between the observations made with the two methods demonstrated that the disappearance of intracristal spaces is independent of the embedding technique, and is not a special effect caused by dehydration in ethylene glycol.     

Evaluation of structuro-functional heterogeneity of isolated mitochondria from the normal and the ischemic myocardium

The structural and functional heterogeneity of mitochondria isolated from intact and ischemic (after 60 min exposure at 37 degrees C) rabbit myocardium was evaluated. In the presence of cytochrome c. a relatively high (260 +/- 26 ng at O/min . mg of protein) rate of rotenone-sensitive NADH oxidation was observed, which was increased in ischemia. Cytochrome c stimulated the increase of NADH oxidation in mitochondria of normal and ischemic myocardium by the factors of 3.5 and 3.4, respectively. Succinate oxidation in the presence of bromthymol blue in normal and ischemic myocardium mitochondria was activated by cytochrome c 3.3- and 2.9-fold, respectively. The percentage of mitochondria with both structurally damaged membranes was 15% and 25% in normal and ischemic myocardium preparations, respectively. In the absence of ADP, cytochrome c contributed to the increase of the succinate oxidase activity in ischemic mitochondria; that in the 3rd state was inhibited in ischemia and normalized by cytochrome c. A principle was proposed for estimating the percentage of mitochondria with damaged outer membranes, the indices being equal to 34% in control and to 56% in ischemic myocardium. Evidence was obtained suggesting that this mitochondrial fraction was characterized by lowered coupling and absence of rotenone-sensitive NADH: oxidase activity. The percentage of intact mitochondria, in which succinate oxidation is inhibited by bromthymol blue and does not need exogenous cytochrome c, is 51% in control and 19% in ischemic myocardium mitochondria.     

Biochemical and stereological analysis of rat liver mitochondria in different thyroid states

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.     

Cyclic AMP activation of respiration of the liver mitochondria in different metabolic states]

cAMP 10(-6) activates the liver mitochondria respiration in all the metabolic states and failed to change or increased the phosphorylation rate in the oxidation of saturating concentration of succinate and isocitrate. Preincubation of mitochondria or homogenate of the liver with cAMP is obligatory for this effect. The fraction V of serum albumin and EDTA did not prevent the effect. Noradrenaline enhanced the mitochondrial respiration only in incubation with the homogenate. The effect of noradrenaline and cAMP was not summed up. Probably the noradrenaline effect was mediated through cAMP. The data obtained are against the decisive role of the respiration and phosphorylation uncoupling or the oxidation substrate accumulation and lead to the assumption on the mitochondria enzymes activation.     

Localized states qualitatively change the response of ecosystems to varying conditions and local disturbances

The response of dynamical systems to varying conditions and disturbances is a fundamental aspect of their analysis. In spatially extended systems, particularly in pattern-forming systems, there are many possible responses, including critical transitions, gradual transitions and locally confined responses. Here, we use the context of vegetation dynamics in drylands in order to study the response of pattern-forming ecosystems to oscillating precipitation and local disturbances. We focus on two precipitation ranges, a bistability range of bare soil with a patterned vegetation state, and a bistability range of uniform vegetation with a patterned vegetation state. In these ranges, there are many different stable states, which allow for both abrupt and gradual transitions between the system states to occur. We find that large amplitude oscillations of the precipitation rate can lead to a collapse of the vegetation in one range, while in the other range, they result in the convergence to a patterned state with a preferred wavelength. In addition, we show that a series of local disturbances results in the collapse of the vegetation in one range, while it drives the system toward fluctuations around a finite average biomass in the other range. Moreover, it is shown that under certain conditions, local disturbances can actually increase the overall vegetation density. These significant differences in the system response are attributed to the existence of localized states in one of the bistability ranges.     

Identification of two discrete states of energized mitochondria: Experiments on single mitochondria

Dynamic phase microscopy was used to measure the refractivity of a single mitochondrion. Our previous studies showed that application of an electric potential to artificial and natural mitochondrial membranes sharply increases their refractivity. Under the conditions of proton pump activity, the refractivity of a single mitochondrion is 2 to 4 times higher than an average refractivity of deenergized mitochondria. This study demonstrates that the membrane potential of energized mitochondria varies depending on environmental conditions and is controlled by the mitochondrial osmoregulation system. The refractivity of energized mitochondria, i.e., the difference between the refraction indexes of a single mitochondrion and the medium, is 0.02 ± 0.01, i.e., several times lower than that of the energized mitochondria whose membranes bear an electric charge. Earlier it was shown that refractivity of model multilayer systems formed from purified natural lecithin depends linearly on the electric field strength. These data point to a relationship between the refractivity of a single mitochondrion and the membrane potential generated during operation of the proton pump. Under normal conditions (250 mOsm), the mitochondrion behaves as a dynamic system oscillating on a minute scale between two functional states with different values of the refractivity index and different membrane potentials. The transition time is 10–30 s; the lifetime of both states is several minutes. The histograms reflecting the distribution of refractivities of single energized mitochondria within a population (n = 20–30) revealed the presence of two independent peaks (fractions II and III) with average refractivity values of 0.05 ± 0.01 and 0.09 ± 0.01, respectively; these fractions correspond to two long-lived states of mitochondria. However, under hypotonic conditions (120 mOsm), only one (“static”) state was identified, in which oscillations were absent and the refractivity of the overall mitochondrial population does not exceed 0.05 ± 0.01 (fraction II). Studies on mitoplast showed that values of refractivity are related to the inner mitochondrial membrane. It is inferred from these data that there exist two discrete states of mitochondria. Analysis of low-amplitude fluctuations of the refractivity of single mitochondria revealed the presence of frequency components at 1–3 Hz, presumably generated in response to non-uniform functioning of mitochondrial proton pumps. It is suggested that frequency components at 1.8-2.6 Hz are more characteristic of the ATPase pump, while the 1–1.3 Hz frequencies predominate during the functioning of respiratory proton pumps.     

Two different conformational states of the KirBac3.1 potassium channel revealed by electron crystallography

Potassium channels allow the selective flow of K(+) ions across membranes. In response to external gating signals, the potassium channel can move reversibly through a series of structural conformations from a closed to an open state. 2D crystals of the inwardly rectifying K(+) channel KirBac3.1 from Magnetospirillum magnetotacticum have been captured in two distinct conformations, providing "snap shots" of the gating process. Analysis by electron cryomicroscopy of these KirBac3.1 crystals has resulted in reconstructed images in projection at 9 A resolution. Kir channels are tetramers of four subunits arranged as dimers of dimers. Each subunit has two transmembrane helices (inner and outer). In one crystal form, the pore is blocked; in the other crystal form, the pore appears open. Modeling based on the KirBac1.1 (closed) crystal structure shows that opening of the ion conduction pathway could be achieved by bending of the inner helices and significant movements of the outer helices.     

Reinforcer satiation and resistance to change of responding maintained by qualitatively different reinforcers

In previous research on resistance to change, differential disruption of operant behavior by satiation has been used to assess the relative strength of responding maintained by different rates or magnitudes of the same reinforcer in different stimulus contexts. The present experiment examined resistance to disruption by satiation of one reinforcer type when qualitatively different reinforcers were arranged in different contexts. Rats earned either food pellets or a 15% sucrose solution on variable-interval 60-s schedules of reinforcement in the two components of a multiple schedule. Resistance to satiation was assessed by providing free access either to food pellets or the sucrose solution prior to or during sessions. Responding systematically decreased more relative to baseline in the component associated with the satiated reinforcer. These findings suggest that when qualitatively different reinforcers maintain responding, relative resistance to change depends upon the relations between reinforcers and disrupter types.     

Altered release of carnitine palmitoyltransferase activity by digitonin from liver mitochondria of rats in different physiological states.

The release of carnitine palmitoyltransferase (CPT) activity from rat liver mitochondria by increasing concentrations of digitonin was studied for mitochondrial preparations from fed, 48 h-starved and diabetic animals. A bimodal release of activity was observed only for mitochondria isolated from starved and, to a lesser degree, from diabetic rats, and it appeared to result primarily from the enhanced release of approx. 40% and 60%, respectively, of the total CPT activity. This change in the pattern of release was specific to CPT among the marker enzymes studied. For all three types of mitochondria there was no substantial release of CPT concurrently with that of the marker enzyme for the soluble intermembrane space, adenylate kinase. These results illustrate that the bimodal pattern of release of CPT reported previously for mitochondria from starved rats [Bergstrom & Reitz (1980) Arch. Biochem. Biophys. 204, 71-79] is not an immutable consequence of the localization of CPT activity on either side of the mitochondrial inner membrane. Sequential loss of CPT I (i.e. the overt form) from the mitochondrial inner membrane did not affect the concentration of malonyl-CoA required to effect fractional inhibition of the CPT I that remained associated with the mitochondria. The results are discussed in relation to the possibility that altered enzyme-membrane interactions may account for some of the altered regulatory properties of CPT I in liver mitochondria of animals in different physiological states.     

Axonal transport blockade and denervation have qualitatively different effects upon skeletal muscle metabolism

The activity and isoenzyme pattern of muscle lactic dehydrogenase (LDH) was measured at different times after axonal transport blockade by colchicine or after denervation. After denervation, total LDH activity decreased and the isoenzyme pattern was altered, LDH-1 being the most affected form. In contrast, after axonal transport blockade there was a decrease in LDH activity but the isoenzyme pattern was not modified. Denervation abolishes both nerve-evoked muscle activity and the release of neuro trophic substances from the nerve whereas colchicine blocks axonal transport without affecting the nerve capacity to conduct action potentials or neuromuscular transmission. It is then concluded that nerve-evoked muscle activity is the most important factor in the regulation of muscle LDH isoenzyme distribution. On the other hand, muscle metabolism can also be regulated by axonally transported molecules. The results presented here show that there is a qualitative difference between the effects of denervation and those of axonal transport blockade upon the muscle, since only denervation altered the isoenzyme pattern of muscle LDH.     

Oscillatory states of mitochondria. Studies on the oscillatory mechanism of liver and heart mitochondria

Oscillatory movements of ions across the inner membrane of liver and heart mitochondria in vitro have been investigated. Our results indicate that the inverse of the square of oscillatory period is linear with respect to (a) the permeant cation concentration; (b) the inverse of the permeant weak acid anion concentration, and (c) the rate of energyproduction. It has been shown that various factors contribute to damping of the oscillations. These factors include: substrate utilization, mitochondrial deterioration, imperfect mitochondrial synchronization, and, possibly, an oscillatory mechanism which dictates damping. An increased period length and extensive damping of oscillations occurs at a critical mitochondrial protein concentration (less than 0.6 mg protein/ml). Such inhibition can be reversed by the addition of cytochrome c.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states

It has been shown that K_M values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The K_i values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 μM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adenienucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

Dermorphin: synthesis of analogs and structuro-functional relations

The review analyzes structure-activity relations among dermorphin analogues. Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is one of natural opioid peptides having a unique structure and exerting a very potent and prolonged antinociceptive effect. Methods of dermorphin synthesis are summarized together with data on more than 300 dermorphin-like peptides: the physico-chemical characteristics and data on opioid tests in vitro and in vivo are discussed. Based on these studies, conclusions have been drawn on the functional role of each amino acid residue in the dermorphin molecule and on modifications leading to analogues with high and differential opioid activity.     

The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states

The intensity of light scattered at 90° to the incident beam and the effective hydrodynamic radii of mitochondria incubated under a variety of conditions have been measured. Addition of high concentrations of uncouplers to respiring mitochondria resulted in a decrease in scatter which was not due to swelling. Addition of valinomycin to mitochondria depleted of substrate in K⁺-free medium produced an increase in scatter that was not due to shrinking. It is concluded that changes in the intensity of scattered light are not reliable indices of changes of volume of mitochondria, and that changes in conformation with changes in metabolic state dominate changes in light scatter. A molecular mechanism for the effect of metabolic state upon the scattered intensity is suggested.     

Endocrine function of the heart: structuro-functional aspects

Modern notions, accumulated during the period of investigation of before poorly studied endocrine++ function of the heart are presented. History of the problem, structural and functional aspects on argumentation of the cardiac endocrine function, place and role of physiologically active cardiac peptides in regulation of liquor and electrolytic homeostasis are followed, as well as interactions of the cardiac endocrine apparatus with the hormonal system of the organism. The authors' experience on the investigation connected with the problem is also demonstrated.