Effects of pelargonidine and a benzocaine analogue p-diethylaminoethyl benzoate on mitochondrial K(ATP) channel.

ATP-dependent K+ (K(ATP)) channel was purified from the inner mitochondrial membrane and reconstituted into the bilayer lipid membrane. The K(ATP) activity was inhibited by high concentrations of ATP and ADP, but was activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethyl benzoate (DEB) acted as a K(ATP) opener: at micromolar concentrations, it reversed the inhibitory effect of ATP and ADP, and also prevented K(ATP) rundown. Pelargonidine extracted from Pelargonium flowers reduced the spontaneous activity of K(ATP) channels and attenuated DEB-induced channel activation. The opposite action of DEB and pelargonidine on K(ATP) corresponded to the opposite redox properties of the two agents in reactions with free radicals: DEB behaved as an electron donor, while pelargonidine acted as an electron acceptor. We suggest that the thiol groups in mitochondrial K(ATP) are targets for redox-active ligands.

Redox properties of local anesthetics: A structural determinant of closed channel blockers in BTX-modified Na+ channels.

Single channel analyses and macroscopic current measurements have shown that benzocaine is a predominantly closed channel blocker in BTX-modified Na+ channels; cocaine is an open channel blocker; and tetracaine, a dual channel blocker (Wang & Wang, 1994; Wang et al., 1994). The reason for such a selective state-dependent block by local anesthetics in BTX-modified Na+ channels is not clear. We assessed the redox properties of tetracaine, benzocaine, cocaine, and various derivatives by their ability to donate electrons to radical intermediates of eosin dye excited by visible light. Electron-donor properties of the drugs were previously proposed to be involved in Na+ channel blockade (Marinov, 1991). Our results provide evidence that redox properties of tetracaine, benzocaine, and their homologs correlate with their ability to enhance Na+ channel inactivation in BTX-modified Na+ channels. This correlation may be explained in terms of the previously proposed redox model of ion channels.

Dose-dependent potentiation and inhibition of single Ca2+-activated K+ channels by flufenamic acid.

Using the patch-clamp technique in an inside-out configuration, we studied the action of an antiinflammatory drug, flufenamic acid (FFA), on single large-conductance Ca2+-activated K+ channels in cultured Vero kidney cells. Depending on its concentration, FFA caused either potentiation or inhibition of K(Ca) channel activity of the same channel. Within the concentration ranges of about 5 to 10 microM and of 50 to 500 microM, FFA increased the channel activity; and within the intermediate range of about 10 to 50 microM, FFA inhibited the channels. The effects were only partially reversible. The activating phases were accompanied by an increase in the channel open time and decreases in the channel closed time and slope factor of the Ca2+ concentration-response curve. An apparent dissociation constant of Ca2+ interaction with the channel changed slightly. Possible mechanisms of the FFA effects are discussed.

Reconstitution of the mitochondrial ATP-dependent potassium channel into bilayer lipid membrane.

Electrical properties and regulation of the mitochondrial ATP-dependent potassium channel were studied. The channel protein was solubilized from the mitochondrial membrane using an ethanol/water mixture. Reconstituted into a bilayer lipid membrane BLM), the protein formed a slightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl. Often, several channels worked simultaneously (clusters) when many channels were incorporated into the BLM. The elementary channel and the clusters were both highly potassium selective. At concentrations of 1 to 10 microM, ATP favors channel opening, while channels become closed at 1-3 mM ATP. GDP (0.5 mM) reactivated the ATP-closed channels without affecting the untreated channels. The sulfhydryl-reducing agent ditiothreitol increased the open probability at concentrations of 1 to 3 mM, but damaged the selectivity of the channel.

Regulation of mitochondrial KATP channel by redox agents.

The ATP-dependent K+ channel (KATP) was purified from the inner mitochondrial membrane and reconstituted into lipid bilayer membranes. KATP activity was inhibited by high concentrations of ATP and ADP, but activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethylbenzoate (DEB) acted as a KATP opener: at micromolar concentrations, it reversed inhibition by ATP and ADP and it also prevented KATP rundown. Pelargonidine, extracted from flowers of Pelargonium, reduced spontaneous activity of KATP channels and diminished their potentiation by DEB. Their opposite action on KATP corresponded with their opposite redox properties in reactions with free radicals: DEB behaved as an electron donor, whereas pelargonidine acted as an electron acceptor. We hypothesize that thiol groups on mitoKATP are targets for redox-active ligans.

A possible role of the redox interactions in the dual, activatory and inhibitory, action of DPI 201-106 on the potential-dependent Na+ channels.

Dual, activatory and inhibitory, effects of a cardiotonic drug DPI 201-106 on Na+ channels were compared with the redox properties of the DPI itself and of the constituents of its molecule, indole and piperazine. The indole component accepted electrons from radical intermediates of the light excited dye; the piperazine part of DPI acted as an electron donor in the same radical reactions. These data extend the previously obtained results which characterized organic blockers of Na+ and Ca2+ channels as electron donors, whereas activators of these channels were shown to be electron acceptors in test radical reactions. The whole DPI 201-106 molecule revealed both the electron-donor and electron-acceptor activity. The described electrophysiological effects of this compound (G. Wang et al., 1989) are discussed within the framework of the Na+ channel redox model (B. Marinov, 1991).

[Redox properties of peptides potentially regulating animal hibernation]. / Okislitel'no-vosstanovitel'nye svoistva peptidov, potentsial'nykh reguliatorov gibernatsii zhivotnykh.

Weak redox properties of peptides isolated from the brain of hibernating animals were determined in reactions with free radicals. The peptides Thr-Ser-Lys-Tyr-Arg (NKT) and Tyr-Arg diminished concentration of anion-radicals of dye excited by light, thus demonstrating electron-acceptor properties toward radicals. In contrast, the peptides Thr-Ser-Lys-Tyr (RZ-5) and Asp-Tyr acted as electron donors in the same radical reactions. The possible role of redox properties of these peptides in their regulation of Ca currents in excitable tissues is discussed.

[The effect of catecholamines on the luminol-dependent chemiluminescence of rat polymorphonuclear leukocytes]. / Vliianie katekholaminov na liuminolzavisimuiu khemiliuminestsentsiiu polimorfnoiadernykh leikotsitov krysy.

The effects of catecholamines on reactive oxygen species (ROS) generation by polymorphonuclear leucocytes were studied using chemiluminescence (CL). It has been found that catecholamines in 10(-9)-10(-10) M concentration range are able to increase reactive oxygen species generation. Within the micromolar concentration range (10(-6)-10(-5) M) cetacholamines (CA) strongly suppress CL intensity and ROS production. The efficiency of inhibitory action of CA: noradrenaline > adrenaline > dopamine > L-DOPA correspond to their redox properties. A conclusion is made that peroxidase is one of the main targets of CA effect.

Redox properties of benzocaine and its homologs.

Redox properties of the local anesthetic benzocaine and its homologs were studied by their reactions with free radical intermediates of dyes excited by the visible light. Homologs with more hydrophobic substituents at the benzene ring appeared to be better electron donors and facilitated photobleaching of the dye Methylene Blue or photoreduction of hemin. The electron-donor properties decreased in the same order as their blocking activities (C. Quan, W. M. Mok, and G. K. Wang, Biophys. J. 70:194-201 (1996)). This correlation is considered as additional evidence in favour of the ion channel redox model.

Estimation of redox properties of chemical compounds by their reactions with free radicals.

The electron-donor and electron-acceptor properties of biologically active compounds were estimated on the basis of their reactions with free radical intermediates of the excited dye eosin. After dye excitation by a short flash of light, the kinetics of dye anion radicals in the pure dye solution were compared with those in the presence of the natural reducing agent NADH or a number of substances which are known as local anesthetics (LA). LA individually slowed the decay of dye anion radicals and enhanced their concentration to a lesser extent than NADH, thus acting as electron donors toward dye radicals. The electron-donor properties of LA studied correlate with their ability to block sodium channels. In contrast, the sodium channel agonist veratridine decreased the concentration of dye anion radicals, thus showing electron-acceptor properties. The opposite redox features of the channel antagonist and the agonist were revealed also by a modified method suitable for steady-light spectroscopy. Promoted by NADH, eosin photobleaching was slowed by the electron acceptor ferricyanide as well as by veratridine and some phenols. The electron-acceptor properties of the latter compounds correlated with their ability to inhibit ATP synthesis by submitochondrial particles. Thus, these methods may be used to evaluate the redox properties of compounds not characterized by electrochemical methods and to predict their biological activity.

[Inhibition of mitochondrial ATP-synthetase by redox-agents]. / Ingibirovanie ATF-sintetazy mitokhondrii redoks-agentami.

Effects of phenol and phenothiazine on ATP synthesis and electron transport in submitochondrial particles were studied. Nitrophenols and phenothiazines inhibited ATP synthesis without notable effect on electron transport. On the contrary chlorphenols equally decreased the velocities of electron transport and ATP synthesis. The inhibitors studied showed the properties of electron acceptors in relation to the radicals, their acceptor properties corresponding to their ability to inhibit ATP synthesis.

Ion channel redox model.

Na(+)- and Ca2(+)-channel blockers behave as electron donors in reactions with excited dye radicals, while agonists of these channels behave as electron acceptors in the same reactions. The opposite redox characteristics of channel blockers and agonists may reflect their opposite action on the channels. The observed regularities of channel-modulator reactions with free radicals, as well as, the ability of many proteins to influence long-range electron transfer, are the basis for a model of channel function and regulation by various agents in which labile electrons in the channel-forming protein acts as electric field sensors.

[Opposite control of O2 affinity for hemoglobin by donors and acceptors]. / Protivopolozhno napravlennaia donorami i aktseptorami reguliatsiia srodstva kisloroda k hemoglobinu.

The effects of electron donors and acceptors on O2 binding by hemoglobin were studied. 2,4-Dinitrophenol, levomycetin and pelargonidine-3,5-diglycoside which act as electron acceptors in free radical reactions, enhance this process. In contrast, N-propylajmalin which is known to be an electron donor in the above reactions, suppresses the O2 binding. Diphosphoglycerate and inositol hexaphosphate, the natural inhibitors of O2 binding, exhibit, similar to N-propylajamlin, the properties of electron donors, the latter being a more potent electron donor than the former.

[The mechanism of superoxide dismutase functioning: a multicenter model]. / Mekhanizm funktsionirovaniia superoksiddismutazy: mnogotsentrovaia model'.

A new model of superoxide dismutase (SOD) functioning has been proposed on the basis of recent data. This model takes into account both experimental data and results of quantum mechanics calculations. One substrate molecule (superoxide radical) binds copper ion in active center of SOD, and the second superoxide radical interacts with a peripheral region of the enzyme. Uncoupled electron from the active center is transferred to the peripheral superoxide anion. This results in formation of oxygen molecule in the active center, and of hydrogen peroxide molecule in the peripheral region.

[Changes in superoxide dismutase activity in the presence of electron donors and acceptors]. / Izmenenie aktivnosti superoksiddismutazy pod deistviem donorov i aktseptorov élektronov.

The activity of superoxide dismutase (SOD) from bovine erythrocytes was measured by the inhibition of nitrotetrazolium blue reduction rate in superoxide anion radical generation systems--xanthine/xanthine oxidase of NADH/phenazine methasulfate. The enzyme activity increases in the presence of compounds acting as electron donors in radical-involving reactions and decreased in the presence of compounds possessing the properties of electron acceptors. Activation of SOD by electron donors and its inhibition by electron acceptors was dependent on the concentration of the above compounds. In the absence of SOD electron donors and acceptors did not change the rate of tetrazolium blue reduction by superoxide anion radicals. The role of the new type of SOD regulation for the enzyme functioning in the cell is discussed.

Modulation of superoxide dismutase by electron donors and acceptors.

The competition between superoxide dismutase (SOD) and nitroblue tetrazolium (NBT) for O2- radicals in the presence of a number of physiologically active compounds was studied. The Na+ channel blockers, ajmaline, tetracaine, bipuvacaine, lidocaine and etmozine produced an increase in the amount of O2- reacting with SOD. Nitroprusside, ferricyanide, BAY K8644, levomycetin, cGMP, cAMP and GMP acted in the opposite way. All the SOD activtors had in common the property of being electron donors in the reactions with the light-induced free radicals of eosin whereas the SOD inhibitors behaved as electron acceptors. The electron activity of SOD modulators correlated qualitatively with their regulating efficacy.

Norepinephrine with its precursors and their antagonists haloperidol and phentolamine interact with dye free radicals in opposite ways.

In the reactions with dye free radicals, catecholamines exhibited reversible electron donor and acceptor properties with the effectiveness increasing from tyrosine to norepinephrine. The physiological antagonists haloperidol and phentolamine showed opposite patterns of behaviour in the same reactions, changing their properties as acceptors to electron donors. The regularity observed is similar to that demonstrated earlier by a variety of Na+ and Ca2+ channel modulators.

Dihydropyridine Ca2+ agonists and channel blockers interact in the opposite manner with photogenerated unpaired electrons.

Interaction of Ca2+-channel antagonists (felodipine, ryocidil, verapamil, diltiazem) and agonists (dihydropyridine derivatives Bay K 8644 and CGP 28392) was studied by the methods of absorption spectroscopy. Ca2+-channel antagonists were found to act as electron donors, the agonists being electron acceptors in the interaction with dye free radicals in solution. Redox transitions in channel-forming protein were proposed as a possible mechanism of the modulation of channel activity by the compounds tested.