Warren L. Erdahl

Ca2+ transport properties of ionophores A23187, ionomycin, and 4-BrA23187 in a well defined model system.

Models for the electroneutral transport of Ca2+ by ionophores A23187, ionomycin, and 4-BrA23187 have been tested in a defined system comprised of 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine vesicles prepared by freeze-thaw extrusion. Quin-2-loaded and CaCl2-loaded vesicles were employed to allow the investigation of transport in both directions. Simultaneous or parallel measurements of H+ transport and membrane potential, respectively, indicate that for any of these ionophores, electrogenic transport events do not exceed 1 in 10,000 when there is no preexisting transmembrane potential. When a potential of approximately 150 mV is imposed across the membrane, transport catalyzed by A23187 remains electroneutral; however, for ionomycin and 4-BrA23187, approximately 10% of transport events may be electrogenic. The defined vesicle system has also been utilized to determine how the rate of Ca2+ transport varies as a function of ionophore and Ca2+ concentration and with the direction of transport. Some aspects of the results are unexpected and should be considered by investigators using ionophores in biological systems. These include the apparent failure of these compounds to fully equilibrate Ca2+ with a high affinity Ca2+ indicator when these species are separated by a membrane, rates of transport that vary markedly with the direction of transport, and extents of transport that are a function of ionophore concentration. At least some of these unexpected behaviors can be explained by a strong influence of delta pH on forward and reverse transport kinetics. In the case of A23187, the data also give some initial insights into the relationship between formation of the transporting species and the entry of this species into the membrane hydrophobic region.

Ischemic Preconditioning Decreases Mitochondrial Proton Leak and Reactive Oxygen Species Production in the Postischemic Heart

BACKGROUND Proton leak (H(+) leak) dissipates mitochondrial membrane potential (mΔΨ) through the re-entry of protons into the mitochondrial matrix independent of ATP synthase. Changes in H(+) leak may affect reactive oxygen species (ROS) production. We measured H(+) leak and ROS production during ischemia-reperfusion and ischemic preconditioning (IPC) and examined how changing mitochondrial respiration affected mΔΨ and ROS production. MATERIALS AND METHODS Isolated rat hearts (n = 6/group) were subjected to either control-IR or IPC. Rate pressure product (RPP) was measured. Mitochondria were isolated at end reperfusion. Respiration was measured by polarography and titrated with increasing concentrations of malonate (0.5-2 mM). mΔΨ was measured using a tetraphenylphosphonium electrode. H(+) leak is the respiratory rate required to maintain membrane potential at -150 mV in the presence of oligomycin-A. Mitochondrial complex III ROS production was measured by fluorometry using Amplex-red. RESULTS IPC improved recovery of RPP at end reperfusion (63% ± 4% versus 21% ± 2% in control-IR, P < 0.05). Ischemia-reperfusion caused increased H(+) leak (94 ± 12 versus 31 ± 1 nmol O/mg protein/min in non-ischemic control, P < 0.05). IPC attenuates these increases (55 ± 9 nmol O/mg protein/min, P < 0.05 versus control-IR). IPC reduced mitochondrial ROS production compared with control-IR (31 ± 2 versus 40 ± 3 nmol/mg protein/min, P < 0.05). As mitochondrial respiration decreased, mΔΨ and mitochondrial ROS production also decreased. ROS production remained lower in IPC than in control-IR for all mΔΨ and respiration rates. CONCLUSIONS Increasing H(+) leak is not associated with decreased ROS production. IPC decreases both the magnitude of H(+) leak and ROS production after ischemia-reperfusion.

Monensin Improves the Effectiveness of meso-Dimercaptosuccinate when Used to Treat Lead Intoxication in Rats

Among divalent cations, the ionophore monensin shows high activity and selectivity for the transport of lead ions (Pb2+) across phospholipid membranes. When coadministered to rats that were receiving meso-dimercaptosuccinate for treatment of Pb intoxication, monensin significantly increased the amount of Pb removed from femur, brain, and heart. It showed a tendency to increase Pb removal from liver and kidney but had no effect of this type in skeletal muscle. Tissue levels of several physiologic (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, zinc) and nonphysiologic (arsenic, cadmium, chromium, nickel, strontium) elements were also determined after the application of these compounds. Among the physiologic elements, a number of significant changes were seen, including both rising and falling values. The size of these changes was typically around 20% compared with control values, with the largest examples seen in femur. These changes often tended to reverse those of similar size that had occurred during Pb administration. Among the nonphysiologic elements, which were present in trace amounts, the changes were smaller in number but larger in size. None of these changes appears likely to be significant in terms of toxicity, and there were no signs of overt toxicity under any of the conditions employed. Monensin may act by cotransporting Pb2+ and OH– ions out of cells, in exchange for external sodium ions. The net effect would be to shuttle intracellular Pb2+ to extracellular dimercaptosuccinic acid thereby enhancing its effectiveness. Thus, monensin may be useful for the treatment of Pb intoxication when applied in combination with hydrophilic Pb2+ chelators.

Ischemic preconditioning preserves mitochondrial membrane potential and limits reactive oxygen species production

BACKGROUND Mitochondrial superoxide radical (O(2)(•¯)) production increases after cardiac ischemia/reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O(2)(•¯) production, but the mechanism is unknown. Mitochondrial membrane potential (mΔΨ) is known to affect O(2)(•¯) production; mitochondrial depolarization decreases O(2)(•¯) formation. We examined the relationship between O(2)(•¯) production and mΔΨ during IR and IPC. MATERIALS/METHODS Rat hearts were subjected to Control or IPC. Mitochondria were isolated at end equilibration (End EQ), end ischemia (End I), and end reperfusion (End RP). mΔΨ was measured using a tetraphenylphosphonium electrode. Mitochondrial O(2)(•¯) production was measured by electron paramagnetic resonance using DMPO spin trap. Cytochrome c levels were measured using high-pressure liquid chromatography. RESULTS IPC preserved mΔΨ at End I (-156 ± 5 versus -131 ± 6 mV, P < 0.001) and End RP (-168 ± 2 versus -155 ± 2 mV, P < 0.05). At End RP, IPC attenuated O(2)(•¯) production (2527 ± 221 versus 3523 ± 250 AU/mg protein, P < 0.05). IPC preserved cytochrome c levels (351 ± 14 versus 269 ± 16 picomoles/mg protein, P < 0.05) at End RP, and decreased mitochondrial cristae disruption (10% ± 4% versus 33% ± 7%, P < 0.05) and amorphous density formation (18% ± 4% versus 28% ± 1%, P < 0.05). CONCLUSION We conclude that IPC preserves mΔΨ, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O(2)(•¯) production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in mΔΨ decrease O(2)(•¯) production, our results indicate that preservation of mΔΨ is associated with decreased O(2)(•¯) and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve mΔΨ depolarization, but rather preservation of mitochondrial electrochemical potential.

Mitochondrial uncoupling does not decrease reactive oxygen species production after ischemia-reperfusion.

Cardiac ischemia-reperfusion (IR) leads to myocardial dysfunction by increasing production of reactive oxygen species (ROS). Mitochondrial H(+) leak decreases ROS formation; it has been postulated that increasing H(+) leak may be a mechanism of decreasing ROS production after IR. Ischemic preconditioning (IPC) decreases ROS formation after IR, but the mechanism is unknown. We hypothesize that pharmacologically increasing mitochondrial H(+) leak would decrease ROS production after IR. We further hypothesize that IPC would be associated with an increase in the rate of H(+) leak. Isolated male Sprague-Dawley rat hearts were subjected to either control or IPC. Mitochondria were isolated at end equilibration, end ischemia, and end reperfusion. Mitochondrial membrane potential (mΔΨ) was measured using a tetraphenylphosphonium electrode. Mitochondrial uncoupling was achieved by adding increasing concentrations of FCCP. Mitochondrial ROS production was measured by fluorometry using Amplex-Red. Pyridine dinucleotide levels were measured using HPLC. Before IR, increasing H(+) leak decreased mitochondrial ROS production. After IR, ROS production was not affected by increasing H(+) leak. H(+) leak increased at end ischemia in control mitochondria. IPC mitochondria showed no change in the rate of H(+) leak throughout IR. NADPH levels decreased after IR in both IPC and control mitochondria while NADH increased. Pharmacologically, increasing H(+) leak is not a method of decreasing ROS production after IR. Replenishing the NADPH pool may be a means of scavenging the excess ROS thereby attenuating oxidative damage after IR.

Transport Properties of the Calcium Ionophore ETH-129

The transport mechanism and specificities of ionophore ETH-29 have been investigated in a highly defined phospholipid vesicle system, with the goal of facilitating the application of this compound to biological problems. ETH-129 transports Ca(2+) via an electrogenic mechanism, in contrast to A23187 and ionomycin, which function in a charge neutral manner. The rate of transport is a function of membrane potential, increasing by 3.9-fold per 59 mV over a broad range of that parameter. Rate is independent of the transmembrane pH gradient and strongly stimulated by the uncoupler carbonyl cyanide m-chlorophenylhydrazone when no external potential has been applied. The effect of uncoupler reflects the collapse of an opposing potential arising during Ca(2+) transport, but also reflects the formation of a mixed complex between the uncoupler, ETH-129, and Ca(2+) that readily permeates the vesicle membrane. Oleate does not substitute for the uncoupler in either regard. ETH-129 transports polyvalent cations according to the selectivity sequence La(3+) > Ca(2+) > Zn(2+) approximately equal to Sr(2+) > Co(2+) approximately equal to Ni(2+) approximately equal to Mn(2+), with the magnitude of the selectivity coefficients reflecting the cation concentration range considered. There is little or no activity for the transport of Na(+), K(+), and Mg(2+). These properties suggest that ETH-129 will be useful for investigating the consequences of a mitochondrial Ca(2+) overload in mammalian cells, which is difficult to pursue through the application of electroneutral ionophores.