Effect of bivalent metal ions on the fluctuations of ion currents in rat liver mitochondria

Ions of bivalent metals are shown to arrange in the Sr2+ greater than Ca2+ greater than Ba2+ greater than Mn2+ series as to their ability to induce ion flow vibration in the rat liver mitochondria. Application of Sr2+ results in the most stable prolonged vibrations of ion flows in mitochondria. Ca2+, Ba2+ and Mn2+ induce slightly pronounced and intensively damped vibrations. The studied Mg2+, Co2+, Ni2+, Pb2+ Fe2+ cations have effect on valinomycin-induced K+ transport in mitochondria and do not induce vibrations. It is established that the ability of bivalent cations to induce vibrations is associated with the possibility of their transfer through the mitochondrion membrane and accumulation in the matrix. Inhibitors of the electrogenic Ca2+ transport in mitochondria produce the similar effect on vibrations induced by Sr2+, Ca2+, Ba2+ and Mn2+.     

Influence of oxidative processes in mitochondria on contractility of the frog Rana temporaria heart muscle. Effects of cadmium

The inotropic Cd2+ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd2+ at concentrations of 1, 10, and 20 microM is established to decrease dosedependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Ca2(+)-channels of the L-type (Ca 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd2+ at concentrations of 15 and 25 microM produces swelling of non-energized and energized mitochondria in isotonic (with KNO2 and NH4NO3) and hypoosmotic (with 25 mM CH3COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 microM Cd2+ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd2+ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP-uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd2+ is due not only to its direct blocking action on Ca2(+)-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K+ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

The respiration of rat brain mitochondria purified by phase partition. Effects of K+, Mg2+ and Ca2+

Highly purified brain mitochondria have been prepared by Na+, NH4+ or K+-containing two-phase systems. K+ stimulated the basal rate of respiration in the three mitochondrial preparations. However, K+ only stimulated the maximal oxidation rate (state 3 respiration rates) in those mitochondria prepared by K+-free (Na+ or NH4+-containing) two-phase systems. The increase in the basal rates of respiration induced by exogenous K+ correlates with the mitochondrial swelling rates. The stimulatory effect of K+ on maximal oxidation rates seems to reflect the K+ depletion of brain mitochondria when prepared by K+-free procedures.     

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.     

Inhibition of Ca-induced calcium release from mitochondria by antiischemic phenol-based antioxidants

Effects of different inhibitors of lipid peroxidation (LP), such as sulphur-containing oligoquinone hypoxen, natural flavonoid dihydroquercetin (DHQ), and β-ionol, on Ca²⁺-induced calcium release from rat liver mitochondria (RLM) were investigated during oxidation of various substrates. The hypothesis about interrelation between antioxidant properties and influence of selected substances on spontaneous calcium release from mitochondria was verified. Degree of antioxidant activity of the selected substances was estimated by the inhibition of LP induced by Fe²⁺/ATP complex in phospholipid emulsion or in rat liver mitochondria (RLM). According to the inhibition efficacy the investigated substances were ordered as follows: β-ionol ≫ hypoxen > DHQ. 50% inhibition of oxygen consumption during LP of phospholipid emulsion was reached in presence of 3.2 ± 0.6 μM of β-ionol, 15.0 ± 1.1 μM of hypoxen, or 19.8 ± 1.7 μM of DHQ. Among the investigated antioxidants hypoxen only decreased spontaneous release of calcium from RLM after calcium accumulation by RLM. The impact of the antioxidants onto calcium current depended on the oxidized substrate. Hypoxen effect was most expressed during the oxidation of NAD-dependent substrate. The direct relationship between the antioxidant activity of the selected antioxidants and their influence on calcium transport in RLM was not revealed. The results indicate that the choice of antiischemic preparations should not only rely on their antioxidant activities.     

Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.     

Effects of hirsutine and dihydrocorynantheine on the action potentials of sino-atrial node, atrium and ventricle

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla MIQ. JACKSON with antihypertensive, negative chronotropic and antiarrhythmic activity, and its C3 structural epimer, dihydrocorynantheine, on membrane potentials of rabbit sino-atrial node and guinea-pig right ventricle and left atrium were studied with microelectrode techniques. In sino-atrial node preparations, hirsutine and dihydrocorynantheine (0.1 microM to 10 microM) concentration-dependently increased cycle length, decreased slope of the pacemaker depolarization (phase 4 depolarization), decreased maximum rate of rise and prolonged action potential duration. In atrial and ventricular preparations, both compounds (0.1 microM to 30 microM) concentration-dependently decreased maximum rate of rise and prolonged action potential duration. These results indicate that hirsutine and dihydrocorynantheine have direct effects on the action potential of cardiac muscle through inhibition of multiple ion channels, which may explain their negative chronotropic and antiarrhythmic activity.     

Inhibition of mitochondrial respiration by neutral, monocationic, and dicationic bis-pyridines related to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium cation (MPP+)

The cytotoxic effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) is believed to be associated with a compromise in cellular energy arising as a consequence of its persistent inhibition of mitochondrial respiration. MPP+ is a rather weak inhibitor of electron transport, but it undergoes passive accumulation inside actively respiring mitochondria in response to the transmembrane electrochemical potential gradient. In order to test the prediction that dicationic analogs of MPP+ might be concentrated to a much greater extent and thereby exert especially potent inhibition of respiration on the intact organelle, we synthesized four differently spaced bis-pyridines, each in neutral, monocationic, and dicationic forms, and evaluated their inhibitory activities in intact mitochondria and in electron transport particles (ETP). Compared to the neutrals, the monocations and especially the dications exhibit reduced inhibition in ETP, but the inhibition in mitochondria is enhanced selectively for the cationic inhibitors presumably on account of their accumulation in the mitochondrial matrix. This enhancement is limited by the relatively poor ability of the cationic bis-pyridines to enter mitochondria, as judged from experiments which evaluated the rate of onset of inhibition (without preincubation), in the absence and presence of tetraphenylborate (TPB-). The dications appear to be transported less well than the monocations, and only the most lipophilic dication exhibited a substantially greater accumulation-dependent enhancement of inhibitory activity on mitochondria than did the corresponding monocation. The compounds studied here constitute a novel class of respiratory chain probes which may be useful for a variety of studies on mitochondria.     

Carnitine acyltransferase activities in rat liver and heart measured with palmitoyl-CoA and octanoyl-CoA. Latency, effects of K+, bivalent metal ions and malonyl-CoA.

1. Liver carnitine acyltransferase activities with palmitoyl-CoA and octanoyl-CoA as substrates and heart carnitine palmitoyltransferase were measured as overt activities in whole mitochondria or in mitochondria disrupted by sonication or detergent treatment. All measurements were made in sucrose/KCl-based media of 300 mosmol/litre. 2. In liver mitochondria, acyltransferase measured with octanoyl-CoA, like carnitine palmitoyltransferase, was found to have latent and overt activities. 3. Liver acyltransferase activities measured with octanoyl-CoA and palmitoyl-CoA differed in their response to changes in [K+], Triton X-100 treatment and, in particular, in their response to Mg2+. Mg2+ stimulated activity with octanoyl-CoA, but inhibited carnitine palmitoyltransferase. 4. The effects of K+ and Mg2+ on liver overt carnitine palmitoyltransferase activity were abolished by Triton X-100 treatment. 5. Heart overt carnitine palmitoyltransferase activity differed from the corresponding activity in liver in that it was more sensitive to changes in [K+] and was stimulated by Mg2+. Heart had less latent carnitine palmitoyltransferase activity than did liver. 6. Overt carnitine palmitoyltransferase in heart mitochondria was extremely sensitive to inhibition by malonyl-CoA. Triton X-100 abolished the effect of low concentrations of malonyl-CoA on this activity. 7. The inhibitory effect of malonyl-CoA on heart carnitine palmitoyltransferase could be overcome by increasing the concentration of palmitoyl-CoA.     

Effect of freezing-thawing rates on the functional state and ionic permeability of rat liver mitochondria

The effects of various rats of freezing-thawing reactions on the functional state and ionic permeability of rat liver mitochondria were studied. The degree of mitochondrial damage during the freezing -- thawing process depended on the rate of thawing rather than on that of freezing. The mitochondria which were slowly or rapidly frozen down to --196 degrees and subsequently slowly thawed revealed a higher membrane permeability for K+ Na+ and H+ and a more than 2-fold increase of the ATPase activity and the maximal rate of NADH oxidation via the antimycin-insensitive pathway in the presence of cytochrome c. This was concomitant with a complete inhibition of the ATP-synthetase activity and a marked inhibition of the respiratory chain function due to the efflux of cytochrome c from the inner mitochondrial membrane. After freezing and rapid thawing the functional activity of mitochondria changed insignificantly. A comparison of different cryoeffects demonstrated that the minimal damaging effects were exerted by rapid freezing -- rapid thawing, when the mitochondria partly restored their ability for oxidative phosphorylation.     

Energy-driven uptake of N-methyl-4-phenylpyridine by brain mitochondria mediates the neurotoxicity of MPTP

The oxidation of NAD+-linked substrates by rat brain mitochondria is completely inhibited by pre-incubation with 0.5 mM N-methyl-4-phenylpyridine (MPP+). The effect is dependent on the integrity of the mitochondria because far higher concentrations of MPP+ are required to inhibit NADH oxidation in inverted mitochondria or isolated inner membrane preparations. The reason for this difference in behavior has been traced to a novel system for the uptake of MPP+ into mitochondria against a concentration gradient. The uptake system is energized by the transmembrane potential, as shown by the fact that valinomycin plus K+, which collapses this gradient, abolishes MPP+ uptake, while agents which collapse the proton gradient have no effect on the process. If an uncoupler is added to mitochondria preloaded with MPP+, efflux of the latter occurs with the concentration gradient. The uptake system has been studied in liver, whole brain, cortex, and midbrain preparations from rats. It may be readily distinguished from the synaptic dopamine reuptake system, since the former is blocked by uncouplers and respiratory inhibitors, but not by dopamine or mazindol, whereas the synaptic system is blocked by mazindol and competitively inhibited by dopamine but is not affected by respiratory inhibitors or uncouplers. Energy-driven uptake of MPP+ by brain mitochondria may be a crucial step in the complex sequence of events leading to the neurotoxic actions of its precursor, MPTP.     

The influence of ATP-dependent K(+)-channel diazoxide opener on the opening of mitochondrial permeability transition pore in rat liver mitochondria

The influence of mitochondrial ATP-dependent K(+)-channel (K+(ATP)-channel) opener, diazoxide (DZ) on the mitochondrial permeability transition pore (MPTP) opening in rat liver mitochondria is studied. In the absence of DZ the MPTP opening leads to the increase in the rate of K(+)- and Ca(2+)-cycling supported by the simultaneous functioning of K(+)-channels and K+/H(+)-antiporter, and also Ca(2+)-uniporter together with MPTP as the cations influx and efflux pathways. Independent of MPTP opening, the activation of both constitutes of K(+)-cycle, K(+)-uptake as well as K+/H(+)-exchange, by DZ is observed. It is shown that the activation of transmembrane exchange of K+, combined with MPTP opening, results in partial inhibition of the latter. A simple methodical approach for the estimation of DZ influence on the open state of mitochondrial pore is proposed. It is shown that MPTP closure followed by Ca2+ reentry to the matrix is accompanied by the K+/H(+)-exchange inhibition which takes place in the same timeframes as the increase in matrix Ca2+ content. Relevant to physiological conditions, an important physiological function of MPTP is revealed, that is the maintenance of relatively low matrix level of Ca2+ accompanied by the acceleration of transmembrane ion exchange (K+ and Ca2+) which could strongly influence the energy state and energy-dependent processes in mitochondria.     

Effect of long-chain fatty acids and acyl-CoA on mitochondrial permeability,transport, and energy-coupling processes

The following effects of fatty acids and acyl-CoA thioesters on energy metabolism of mitochondria can now be assumed: (1) Inhibition of adenine nucleotide translocation. This effect may increase the energy state of mitochondria respiring under state 3 conditions and decrease phosphorylation potential in the surrounding medium (the cytoplasm). (2) Increased permeability to monovalent cations. This may lead to a partial energy dissipation due to a futile recycling of K+ (or another cation), namely and energy-dependent uptake and a passive outflow. (3) True uncoupling due to increased permeability to protons. This effect probably occurs at high concentrations of fatty acids only. (4) Substrate effect. Fatty acids in the form of acyl-CoA are excellent respiratory substrates for mitochondria of most tissues. Their oxidation is coupled to the generation of high energy state of the mitochondrial membrane and, consequently, to ATP synthesis.     

Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel

The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mitochondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K+(ATP)-channel) opener, diazoxide (Dz). Activation of the K+(ATP)-channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.     

Inhibition of succinate oxidation and K+ transport in mitochondria during hibernation

Respiration of liver mitochondria of ground squirrels changes with physiological state. The inhibition of respiration at the level of dehydrogenases occurs during hibernation which is spontaneously removed during arousal. The main mechanism causing a decrease in respiration during hibernation seems to be the inhibition of succinate oxidation, induced by oxaloacetic acid. This is evidenced by the removal of the inhibition by glutamic and isocitric acids. A close correlation between the changes of K+ transport in mitochondria and of the physiological state of hibernator is observed. During hibernation the K+ transport rate decreases 3 times and during arousal it increases 1.5-fold in comparison with the active animals. The K+ content in mitochondria of hibernating and active ground squirrels is the same, whereas during arousal it increases 2-fold.     

Effect of oxidative processes in mitochondria on contractility of heart muscle of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>. Actions of Cd<Superscript>2+</Superscript>

The inotropic Cd²⁺ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd²⁺ at concentrations of 1, 10, and 20 mM is established to decrease dose dependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Na²⁺-channels of the L-type (Ca_v 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd²⁺ at concentrations of 15 and 25 mM produces swelling of non-energized and energized mitochondria in isotonic (with KNO₂ and NH₂NO₃) and hypoosmotic (with 25 mM CH₃COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 mM Cd²⁺ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd²⁺ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP—uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd²⁺ is due not only to its direct blocking action on Ca²⁺-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K⁺ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

Ischemic preconditioning inhibits mitochondrial respiration,increases H2O2 release,and enhances K+ transport

Ischemic preconditioning, or the protective effect of short ischemic episodes on a longer, potentially injurious, ischemic period, is prevented by antagonists of mitochondrial ATP-sensitive K+ channels (mitoKATP) and involves changes in mitochondrial energy metabolism and reactive oxygen release after ischemia. However, the effects of ischemic preconditioning itself on mitochondria are still poorly understood. We determined the effects of ischemic preconditioning on isolated heart mitochondria and found that two brief (5 min) ischemic episodes are sufficient to induce a small but significant decrease ( approximately 25%) in mitochondrial NADH-supported respiration. Preconditioning also increased mitochondrial H2O2 release, an effect related to respiratory inhibition, because it is not observed in the presence of succinate plus rotenone and can be mimicked by chemically inhibiting complex I in the presence of NADH-linked substrates. In addition, preconditioned mitochondria presented more substantial ATP-sensitive K+ transport, indicative of higher mitoKATP activity. Thus we directly demonstrate that preconditioning leads to mitochondrial respiratory inhibition in the presence of NADH-linked substrates, increased reactive oxygen release, and activation of mitoKATP.     

The effect of alkanols on Ca2+ transport in brain mitochondria.

Ethanol stimulates the Na(+)-dependent Ca2+ efflux in brain mitochondria and inhibits the Na(+)-independent Ca(2+)-efflux. Here, we studied the effects of n-alkanols on the various Ca2+ transport processes in brain mitochondria. Only short-chain alcohols (i.e. methanol, ethanol and propanol) stimulated Na+/Ca2+ exchange. The inhibition of H+/Ca2+ exchange was significant only with ethanol. Short-chain alcohols inhibit while long-chain alcohols activate the cyclosporin-sensitive Ca(2+)-efflux. These data suggest that the mechanism of the alkanols' effects on Na+/Ca2+ exchange, H+/Ca2+ exchange and the cyclosporin sensitive pore are entirely different. Alkanols have no effect on the electrogenic Ca2+ uniporter. Ethanol did not affect the apparent K0.5 for Na+ (7.5 mM) of the Na+/Ca2+ exchange. Similarly, the magnitude of the effect of ethanol did not depend on matrix Ca2+ concentration, suggesting that short-chain alkanols do not stimulate the rate of Na+/Ca2+ exchange by increasing the affinity of the carrier to Ca2+in or Na+out. High concentrations of K+, Mg2+ and Ca2+ enhanced the ethanol effect. It is possible that high surface potential attenuates the effect of ethanol. It is suggested that ethanol stimulation of Na+/Ca2+ exchange depends on the modulation of the surface dielectric constant.     

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.     

Endobain E, a brain Na+, K+ -ATPase inhibitor, decreases norepinephrine uptake in rat hypothalamus

The ability of an endogenous brain Na+, K+ -ATPase inhibitor, termed endobain E, to increase [3H]norepinephrine release in rat hypothalamus was previously reported. Endobain E effect on neurotransmitter uptake was studied by assaying [3H]norepinephrine uptake in rat hypothalamus preparations, to observe uptake inhibition, which reached 60% with endobain E equivalent to 100 mg fresh cerebral cortex, an effect achieved with 40 or 400 microM ouabain. Results support the proposal that endobain E behaves as an ouabain-like substance. Taken jointly results obtained on neurotransmitter release and uptake, the suggestion that endobain E may enhance norepinephrine availability in the synaptic gap and thus lead to an increase in noradrenergic activity is advanced.