Influence of the redox-state potential of biophase on electrically stimulated skeletal muscles (myographic and voltage-clamp analysis).

Redox modulation of cholinergic and adrenergic mechanisms of excitatory tissues have been analyzed rather satisfactorily until recently. The aim of the present work is to give some initial guiding information about the redox modulation of electrogenic excitatory processes in skeletal muscles. It was observed, that on increasing the tissue redox-state potential (E0'), the amplitudes of muscle contractions are higher by 18.5 per cent than controls, but decreasing E0', the amplitudes of muscle contractions are lower by 10.5 per cents than controls. It means, that muscle contractions elicited by electric stimulation are also under redox control. One of the mechanisms responsible for this phenomenon is that following increased E0' values both peak current (Ip) and steady-state current (ISS) increases, but after decreased E0' values ISS decreases. The role of other site of actions and mechanisms, i.e. ion channels, active transport, excitation-contraction coupling, and Na(+)-Ca2+ exchange diffusion processes, are also discussed.

Correlations between the actual redox-state potential (E0') of biophase and heart activity in vivo.

In CFY rats the tissue redox-state potential (E0') in heart, m. vastus medialis and in the liver, and the heart frequency and QRS amplitudes were measured parallel. It was observed that following compensatory redosis caused by konakion both the autorhythmic heart frequency and QRS amplitudes increased, while after compensatory oxidosis induced by urea occurred the opposite. Following compensatory redosis caused by konakion acetylcholine decreased, but adrenaline increased the heart frequency and QRS amplitudes more intensively than at normal E0' values. After compensatory oxidosis caused by urea, acetylcholine decreased the heart frequency and QRS amplitudes significantly less. Adrenaline decreased the heart frequency in such milieu. On the basis of these data the following conclusions are proposed; For the realization of autorhythmic activity, for negative type acetylcholine and for positive type adrenaline effects in heart, a relatively low primordial tissue E0' value is an essential back-ground element. Among the mechanisms controlling autorhythmicity and sympathetic/parasympathetic effects the actual and permanently changing tissue E0' value is also an important modifying, or through biochemical redox feed-back mechanisms even a regulatory factor of heart activity.

Analysis of the influence of aeroions on ECG and correlation between this action and tissue redox-state potential.

It was observed in rats, that following negative aeroionization heart frequency and the altitude of P-waves increased. After positive ionization these interrelationships took place inversely. As between these effects and the tendency of tissue redox-state potential changes correlation was seen, the results were grouped also on redox basis, independently on whether the increase or decrease of redox-state potential was caused by negative or positive aeroions. The results of this grouping showed, that following an elevation of tissue redox-state potential (+delta E'0) heart frequency dropped, and the altitude of T-waves increased. After a decrease of tissue redox-state potential (-delta E'0) these interrelationships were realized inversely. After -delta E'0 the positive chronotropic influence of noradrenaline increased, but consequent to +delta E'0 the classical positive chronotropic effect of this catecholamine was reversed. These results corroborate our earlier notion, that aeroions exert their action on heart through changing tissue redox-state potential.

Influence of 3-methylcholanthrene on the redox-state of liver and red muscles in vivo.

It was observed, that following an injection of 3-methylcholanthrene (MC), the tissue redox-state potential is modified expressively both in liver and in red muscles. In the liver in the first day an oxidosis develops, which is followed by redosis, but in the muscle a redosis can be observed already in the first day. It is a meaningful fact, that MC influences biochemical processes in the early phase of its effect not only in the liver but also in the red muscle. By reason of this data the possibility of a prevention of the MC influence by adequate redox agents might also be arised.

Model of redox regulation of hyper- or hypothyreoidism.

Since the triiodothyronine (T3) shifts the tissue metabolism to oxidative direction, one should await that in hyperthyreoidism caused by T3 an oxidosis will be formed, whilst in hypothyreoidism called forth by subtotal thyreoidectomy, redosis will be emerged. However, according to our experiments these interrelationships proved to be inverse. These "paradoxal" changes of redox-state are the consequences of the flowing redox compensations elicited by the tissue redox-buffer capacity (RBC). The pathomechanism can be modelled with differential equation (computer analysis). The redox-state changes are characteristics in each tissues. Between the RBC of the tissue and the shift of redox-state potential (E'0) there is a negative correlation. As an autoregulatory mechanims, the redosis formed in hyperthyreoidism will increase the hormone synthesis in the thyroid gland, while the oxidosis after hypothyreoidism will decrease the synthesis. In other words, these processes strengthen each other. Changes of the heart frequency show correlation with the E'0, and can be described by differential equation. Our theoretical model for the redox regulation might answer also the question of the reversibility-irreversibility range of the autoregulation in the pathomechanism.

Correlations between the tissue redox-state and K(+)-contractures.

Analyzing the mechanisms of redox-modulation of the excitatory-contractory process, recently the amplitude of K(+)-contractures, tissue redox-state potential and electrical burst activity were simultaneously measured in the rectus abdominis muscle of the frog (Rana esculenta) following oxidant (thionine) and reductant (ascorbate) treatments. Pretreatment with oxidant in parallel with the increment of redox-state potential increased, while pretreatment with reductant, parallel with the decrement of redox-state potential decreased significantly both the amplitudes of K(+)-contractures and the electrical burst activity. The main mechanisms of action of this phenomenon, at least of the phasic portion, in all probability is the increase of intracellular quotient of the ionized/bound calcium after oxidizing, but a decrease of this quotient following reducing shifts. In the case of tonic portion an increase of Ca2(+)-influx through the Na(+)-Ca2(+)-exchange diffusion mechanisms seems feasible. Other mechanisms are also discussed. Hence, the mechanism of K(+)-contractures is under the control of tissue redox-state potential as well.

Redox agents modulate a(K+)0 changes evoked by acetylcholine and adrenaline in frog heart.

It was observed earlier, that in the presence of oxidizing agents the acetylcholine exerted a positive ino- and chronotropic effect, while the positive ino- and chronotropic action of adrenaline was decreased. In the presence of reducing agents both the negative inotropic effect of acetylcholine and the positive inotropic action of adrenaline was increased. Analyzing the ionic mechanism background of these correlations, the changes of extracellular K(+)-activity (a(K+)0) were followed and it was established that; In relation to slow transient changes (in min time ranges) an oxidant decreased the a(K+)0 following acetylcholine, while it increased the a(K+)0 after adrenaline application. A reductant increased the a(K+)0 with acetylcholine, but decreased a(K+)0 in the presence of adrenaline. Because of the inverse character of redox modulation on a(K+)0 levels, a reverse change in a(K+)0 should be (at least one of) the site of action of the opposite effects of oxidants or reductants exerted on ino- and chronotropism of acetylcholine or adrenaline.

Opposite effects of methylene blue and ascorbate on lipid peroxidation in muscles. Correlation with the redox state. I. Experiments on satisfied frogs.

Homogenates of heart, stomach and rectus abdominis muscles of the frog have shown different degrees of malondialdehyde (MDA) formation. MDA content was highest in heart, and lowest in stomach musculature. The resultant tissue redox-state potential (RSP) and redox potential (E'0) in homogenates determined potentiometrically also showed differences with opposite signs in relation to MDA levels. An electron acceptor, methylene blue (MB), decreased but an electron donor, ascorbate (Asc), increased the MDA level in each of the muscles. These effects were dependent upon the concentration of MB and Asc and proportional to the control MDA content in each muscle. Thus an inverse interdependence between MDA level and redox state existed even when a positive change in redox potentials was induced by MB, and also when a negative change was induced by Asc. Since there was a close negative correlation between the changes of MDA concentration and redox potential in the homogenates, it is strongly suggested that the changes of redox state in muscle are implicated in the processes leading to lipid peroxidation (LP).

Correlations between positive and negative aeroions, tissue redox-state potential and heart frequency in rats.

It was observed in rats that following positive aeroionization the redox-state potential (E'0) in skeletal muscles and liver was decreased, and the heart frequency increased. After negative ionization these interrelationships took place inversely. It was also established that upon adding an oxidant (menadione) i.v., the E'0 was decreased (compensatorily) in the organs mentioned above, parallel with the increment of heart frequency. Following injection of reductants (cysteine, thiamine) a reverse image was observed. Applying simultaneously positive ionization and reducing agents, the E'0 change and the heart frequency alteration failed to appear. The phenomenon was the same after simultaneous application of negative ionization and oxidant (menadione) injection. Because the heart effect of positive and negative ionization could readily be prevented by a respective redox agent, it seemed that actions of aeroions are exerted through shifts in tissue E'0. The most probable site of action of E'0, is the pacemaker mechanism, but an action on serotonin liberation may also be assumed.

Changes in autorhythmic heart frequency elicited by redox agents.

In isolated frog heart it was established that methylene-blue (MB, an oxidizing agent) decreased, while ascorbate (ASC, a reducing agent) increased the frequency of autorhythmic heart contractions. After MB treatment, in parallel with this phenomenon, the extracellular K+ concentration [K+]o showed a slow increase, but following ASC application a slow decrease occurred. Since these correlations are in good accordance with the idea that the pacemaking ability of heart, among other properties, depends on the voltage and time-dependent decrease in potassium conductance following the spike, changes in [K+]o might be one mechanism by which oxidizing and reducing agents modulate heart frequencies. On the basis of the effect of insulin (INS) and K-strophantoside (STR) on these modulatory influences, it is presumed that the changes in slow delta [K+]o transients might result, at least partly, from the effect of redox agents on the active transport system. In light of the increase in passive K+ fluxes after oxidant treatment and the decrease in this parameter following reductant treatment an effect of redox agents on the characteristics of the K+-channel is also postulated.

Correlations between redox-state potential changes in different tissues and the heart frequency in vivo.

Redosis evoked in different tissues by methylene-blue or menadione (oxidants), resulted in an increase in heart frequency, while oxidosis evoked by thiamine or cysteine (reductants) diminished the frequency. In isolated organ tissues where compensatory redox feed back overshoots are rarely to develop, owing to the low redox buffer capacity and lack of the influence of nervous and humoral factors, the heart frequency decreased in response to direct oxidosis induced by the application of oxidants, and increased following reductant application; this suggested an environmental type redox regulatory influence of the agents rather than specific action of the agents. This environmental type effect can result from direct action on isolated organs, or from direct and indirect actions in vivo. An increased redox-state potential resulted in decreased heart frequency and inversely. In a pathological situation provoked by complete strangulation of aortae, a significant oxidosis developed in parallel with a decrease in heart frequency. On increasing the redox buffer capacity by application of methylene-blue (oxidant), or thiamine (reductant) both the redox and the resulting heart frequency changes could readily be counteracted. When cigarette smoke was pumped through an intratracheal tube, a significant redosis developed in the heart ventricle in parallel with an increased heart frequency. These data show that regardless of the origin of redox-state potential changes in tissues, a shift to oxidosis decreases and a shift to redosis increases the heart frequency.

Inverse modulation of extracellular Na+- and K+-activities by ascorbate or methylene blue.

On analyzing the mechanisms of the internal environment type redox regulation of physiological processes it was observed on frog rectus muscles that during acetylcholine contractures methylene blue pretreatment inhibited, but ascorbate pretreatment enhanced the slow transient changes of extracellular Na+-activity. At the same time, these modifications were inverse for K+-transients. Because k-strophantoside was capable of influencing these effects radically it seems highly plausible to assume that the principal site of action of these modulations is the inhibitory impact of methylene blue, while the enhancing effect of ascorbate on (Na+ + K+)-ATPase may likely be explained on redox basis.

Effect of redox agents on the non-electrolyte isotonic concentrations and on the equivalent pore radius of skeletal muscles of the frog.

Oxidizing and/or reducing agents inversely influence the alterations in the speed of mass changes (dw/dt) due to osmotic perturbations: an oxidizing agent increases while a reducing agent decreases it. The values for isotonic concentration (Ciso) increased for all of the tested non-electrolytes in the presence of an oxidizing agent, while decreased in the presence of a reducing one. The Staverman's reflection coefficient values (sigma) showed changes opposite in direction, so that the direct correlation between the size of test molecules and sigma values remained unchanged. An oxidizing agent increases and a reducing agent decreases the equivalent pore radius (EPR).

In vivo redox changes in some organs in response to Gramoxone.

The effects of Gramoxone on the redox state potential (E'0) of the liver, heart, skeletal muscle and lung of guinea-pig were examined at different times. The E'0 values were compared with control measurements. Gramoxone was found to cause mainly a significant E'0 increase with time in lung tissues. This lends support to the general experience that the primary target organ for paraquat (PQ) toxicity is the lung. It is suggested as explanation of the observation that the lung is most actively connected with molecular oxygen.

Might the cell damaging effect of alcohol be prevented by redox agents?

It was found that alcohol caused pathological and irreversible decrements of the redox-state potential (redosis) in the pancreas and the liver. On giving reducing agents, a compensatory oxidosis developed, and this could prevent the cell damaging effect of alcohol. This effect was most marked in the reversible phase, while it could be effective also in the irreversible phase. By measuring the redox buffer capacity in the tissues it is possible to determine the measure of reversibility of the injury caused by alcohol.

Lipid peroxidation in the membrane damaging effect of silica-containing dust on rat lungs.

Rats were injected intratracheally with a single dose of 50 mg silica (DQ-12) suspended in 0.5 ml saline. The animals were sacrificed at 3, 6, 12 and 21 days following exposure, and the rate of lipid peroxidation as well as the activities of superoxide dismutase and catalase were measured in the lung tissue. Parallelly the redox state potential of the lung tissues was also determined during this period. It was found that under the effect of silica, lipid peroxidation gradually increased in the lung. This effect was accompanied by enhancement of superoxide dismutase and catalase activity. The redox state potentials were shifted to oxidation. It is suggested that silica, though indirectly, induces free radical production associated with oxidosis, which, through lipid peroxidation, increases to membrane injury.

Redox regulation of autorhythmic heart contractions and the effect of acetylcholine failed to manifest itself by decreasing [Ca2+]0.

Earlier it was described by us, that the amplitude and frequency of cyclic autorhythmic contraction of frog heart was decreased by oxidants but increased by reductants. Increasing the redox state potential, acetylcholine effects a positive and not a negative inotropic action. Decreasing the redox state potential, the negative inotropic effect is increased. In search of the site of action of these regulatory influences, the role of [Ca2+]0 was investigated recently and it was established, that: 1. Reducing the [Ca2+]0 by 50% and oxidation diminished the amplitude and frequency of cyclic autorhythmic contraction more expressively, than in normal saline. 2. The increase in cyclic rhythmicity caused by reductant failed to manifest itself in Ca2+ deficient Ringer. 3. The reversing action of oxidation on ACh effects was practically abolished following a decline in [Ca2+]0. 4. The effect of reductant to intensify the negative inotropic actions of acetylcholine suffered an inversion. Because both the redox regulation of quantitative and/or qualitative parameters of cyclic autorhythmic contractions and the ACh effect altered significantly and in a non-additive manner, following a decrement in [Ca2+]0, it seems to be clear, that as a site of action of redox regulation in normal saline, a change in the involvement of extracellular Ca2+ (presumably by altering its influx or absorbtive parameters) has to be taken into consideration.

Redox state potential influences (+/-) delta [Na+]o activity values during acetylcholine contractures of frog skeletal muscles.

1. The slow alterations in the activity of extracellular Na+ concentrations were measured by using ion-selective electrodes. 2. In the first min there is an increase in the changes of the activity of extracellular Na+ following acetylcholine evoked contractures. 3. This increment was significantly inhibited by k-Strophantoside application, suggesting an active transport origin of this first peak. 4. In the second min a strong decrease in the activity of extracellular Na+ concentration followed. 5. Reducing agent (ascorbate) increased, while oxidant (methylene-blue) decreased both the first increment and the sequential decrement in the activity of the extracellular Na+ concentrations. 6. On the basis of these experiments a site of action of the redox regulation of excitatory processes could be the modification of Na+ fluxes.

Relationship between the tissue redox state potential and dak/dt changes of [K+]0 activity during k-strophantoside or acetylcholine induced contractures.

It was established that oxidosis evoked by methylene blue during k-strophantoside and acetylcholine contractures in skeletal muscle was accompanied by an increase in the changes of [K+]0 activity, while redosis evoked by ascorbate under the same conditions was accompanied by a decrease in the changes of [K+]0 activity. These changes in [K+]0 activity parallelled the alterations of contractures caused by oxidosis or redosis suggesting that changes in the [K+]0 transient play an important role in these phenomena.