A.J. Merola

The interaction of nonionic detergents with lipid bilayer membranes

Abstract Sublytic concentrations of polyoxyethylene ether detergents are able to reduce the direct current (dc) resistance of lipid bilayer membranes. The order of cation effectiveness in lowering bilayer resistance is: K + > Rb + > Cs + > Na + = Li + . The decrease in bilayer resistance is transient when the detergent is present on one side of the film. The time required for maximum decrease in resistance is dependent on the concentration of detergent added. Multimolecular aggregates of Triton are involved in the resistance-lowering process. The data indicate that these aggregates interact with lipid in the bilayer. A mechanism is proposed by which lipid-Triton complexes enhance the permeability of bilayer membranes to cations, particularly to K + . The studies summarized in this communication and the accompanying manuscript suggest that detergents with a polyoxyethylene ether structure act as ionophores which are rather specific for K + .

Ion transport by heart mitochondria. XXII. Spontaneous, energy-linked accumulation of acetate and phosphate salts of monovalent cations.

Abstract The accumulation of monovalent cations by isolated beef heart mitochondria has been studied by evaluating the efficiency of energy-dependent osmotic swelling. Extensive osmotic swelling occurs spontaneously when isolated heart mitochondria are suspended in 0.1 m acetate or phosphate salts. The swelling and ion uptake depend on either respiration or the presence of exogenous ATP, and the initial rate of swelling is proportional to the initial rate of respiration or ATP hydrolysis, respectively. The efficiency of the reaction varies somewhat from preparation to preparation but approaches a limit of about 2 cations accumulated per pair of electrons traversing a phosphorylation site. All monovalent cations tested support the reaction, but the most efficient energy-dependent swelling occurs with K + . Weak acid anions are required for the ion accumulation and swelling and the reaction appears to depend on the amount of free acid available in the suspension. Permeant strong acid anions, such as NO 3 − , fail to support the swelling reaction in the presence of energy. Valinomycin increases both the amount and the efficiency of ion uptake under these conditions. Mg 2+ decreases both of these values whereas p -chloromercuriphenyl sulfonate increases both. These responses are discussed in terms of current models of mitochondrial ion transport.

Ion transport by heart mitochondria. Retention and loss of energy coupling in aged heart mitochondria.

Abstract The retention and loss of energy-coupling reactions in isolated beef heart mitochondria have been examined under anaerobic conditions using suspending media chosen to mimic the intracellular milieu. In long-term incubations at 37 °C, a loose coupling develops which can be controlled by adding serum albumin. This lesion closely resembles that produced by addition of free fatty acids which has been described in previous studies. Shorter incubation times produce an increased susceptibility to hydrogen peroxide which is characterized by elevated ATPase activity, increased permeability to monovalent cations, and increased proton ejection on transition from the anaerobic to the aerobic state. This peroxide sensitivity is prevented by chelators such as EGTA and appears to involve a time-dependent release of metal ions. Of the metabolites which are known to increase in concentration in the ischemic heart cell, Na + , P 1 , lactate, and H + all promote swelling of isolated heart mitochondria and contribute to a decline in energy coupling. The relationship of these results to the pathological deterioration of mitochondria in ischemic heart tissue is discussed.

Ion transport by heart mitochondria: XXV. Activation of energy-linked K+ uptake by non-ionic detergents

Abstract The energy-dependent uptake of K + salts by isolated heart mitochondria is markedly activated by Triton X-100 and several other nonionic detergents. The detergent-mediated reaction closely resembles that seen in the presence of low concentrations of the ionophore valinomycin. Triton increases the efficiency of energylinked K + uptake, induces passive permeability to K + , and affects respiration, phosphorylation, O 2 pulses, and ATPase activity in a manner similar to valinomycin. Studies of equilibrium extraction of cations into organic solvents indicate that detergents with the poly(ethyleneoxy) ethanol structure can function as ionophores with properties roughly analogous to the cyclic polyethers. The effects of detergents with the ability to act as ionophores are compared with those of other classes of surface-active reagents.

The permeability of heart mitochondria to creatine.

Abstract Isolated beef heart mitochondria contain substantial amounts of creatine (over 20 nmol/mg of protein) which appears to be sequestered in the matrix compartment. In contrast, rat heart mitochondria contain very little creatine (less than 2 nmol/mg). The inner membrane of both rat and beef heart mitochondria is relatively impermeable to creatine in the cold but shows a measurable permeability at 37 °C. Entrance of creatine into the matrix does not appear to be carrier-mediated or to depend on the metabolic state of the mitochondrion. The evidence suggests that the concentration of creatine in the intermembrane space increases in the ischemic period following the death of the animal and, since ATP from oxidative phosphorylation is not available for the normal conversion to creatine phosphate in this compartment, the creatine diffuses into the matrix, a compartment in which it has no metabolic role. Once the preparation of mitocnondria has begun, the low temperature of the isolation medium prevents further movement of creatine across the inner membrane in either direction. The presence of substantial amounts of creatine in an otherwise functional mitochondrion is therefore an indication that the organelle has survived a period of ischemia sufficient to produce an extensive alteration of cellular metabolism.

Inhibition of mitochondrial oxidation and uncoupling of phosphorylation by antispermatogenic bis-dichloroacetamides

Abstract Antispermatogenic bis-dichloroacetamides affect both electron transport and the energy conservation system in heart mitochondria and submitochondrial particles. Oxidation of NADH or pyridine nucleotide-linked substrates is inhibited at a site prior to coenzyme Q but after the primary dehydrogenase, similar to rotenone and amytal inhibition and as with these latter inhibitors the oxidation of succinate is essentially unaffected at similar concentrations of drug. In this respect the dichloroacetamides also resemble antabuse (tetraethylthiuram disulfide), but unlike the inhibition due to antabuse, the effects of the dichloroacetamides are not prevented by mercaptoethanol. Structure-activity studies suggest the importance of a substantial degree of lipid solubility and an unhindered dichloroacetamide group for activity. Activity in intact mitochondria appears in both succinate and pyridine nucleotidelinked substrate supported systems. N , N ′-bis(dichloroacetyl)-1,12-dodecanediamine was synthesized and is the most active member of this group of compounds found thus far. This compound results in a marked decrease in phosphorylation coupled to oxidation, as well as a release from ADP-limited respiration and an increase in the Mg 2+ -stimulated ATPase activity. Dinitrophenol-stimulated ATPase activity is inhibited at low concentrations of drug, but rises somewhat at higher concentrations. The most dramatic changes in each of these parameters occur at quite similar concentrations of N , N ′bis(dichloroacetyl)-1,12-dodecanediamine, i.e. about 5 μM. The implications of these results and their relationship with other pharmacological properties of these drugs are discussed.

The inhibition of drug metabolism by antispermatogenic N,N′-bis(dichloroacetyl) diamines

2 dichloroacetylamines NN-bis(dichloroacetyl-1-8)octamethylenediamine (WIN 18446) NN-14-xylene-bis(N-ethyldichloroacetamide) (N-ethyldichloroacetamide) (WIN 13099) seem to inhibit drug metabolism. They are firmly bound to subcellular membrane systems and possess potent antispermatogenic activi ty. WIN 13146 is a structural analog of WIN 13099 which lacks antispe rmatogenic activity. The antispermatogenic compounds produce a two-to t hreefold increase in sleeping time in rats when tested with sodium hexob arbital (80 mg/kg ip). WIN 13146 resulted in little or no increase in hexobarbital sleeping time. The inhibition of hexobarbital metabolism s eems to be independent of chain length of these compounds. WIN 13146 h as little effect on N-demethylation of 4-dimethylaminoantipyrine or O-de methylation of p-acetanisidide in rat liver homogenates. The antisperma togenic compounds lowered N and O-demethylation from 100 in controls to as low as 49 average activity. These compounds showed little effect on S-demethylation of 2-(methylthio)benzothiazole.

A simple large-volume cell suitable for multiparameter measurements in the aminco-chance spectrophotometer☆☆☆

In order to better understand cellular and subcellular phenomena it is desirable to observe multiple parameters such as pH, various ion movements, optical density at various wavelengths, and kinetics of oxygen consumption. Instrumentation for the measurement of these kinds of parameters has been carefully described by Pressman (11, who used a rectangular cuvet with a plastic covrr fitted with an oxygen electrode, a I(+ electrode and a pH electrode. Fluorescence and light scattering measurements are made with the appropriate light sources and photomultipliers situated at the sides of the cuvet and after proper amplification the signals from these sensors are recorded simultaneously. In this communication we would like to describe a simple cuvet suitable for multiparameter measurements adapted for use with the spectrophotometer designed by Chance (2) and commercially available from the American Instrument Co. as a dual-wavelength split-beam spectrophotometer. Figure 1 shows the essential features of the cell, including the measurements which are critical for the positioning of the cell in the spectrophotometer. Since the cell is patterned after the Bl-65103 cuvet carrier, it fits without modification into the spect’rophotometer just as one would position the cell carriers which come as standard equipment with the instrument. This also ensures that t’he light beam will strike the cuvet properly. The entire cell was made from cast methyl methacrylate except the optical surfaces, viz., t’he two sides of the stirring chamber and one end which is used for fluorescence or light scattering measurements.

Structural requirements in the uncoupling of oxidative phosphorylation by N,N'-bis(dichloroacetyl) diamines.

Abstract The nature and extent of the disruption of mitochondrial function by bisdichloroacetamides depends on their chemical nature and lipid solubility (partition coefficient). Secondary and tertiary amides are active in the inhibition of electron transport as are mono-, di- and trichloroacetamides. Uncoupling of oxidative phosphorylation is very much more dependent on chemical structure and it is shown that only lipid soluble secondary dichloroacetamides are active in affecting energy-linked mitochondrial function. Uncoupling phosphorylation from the oxidation of TMPD-ascorbate at the third site of phosphorylation appears to be less extensive than that supported by succinate or pyruvate plus malate. An apparant alkalization of the medium occurs when mitochondria are treated with the dichloroacetamide but not when treated with inactive acetamides. These data are discussed in light of the known respiratory effects of the dichloroacetamides on rodent testes.

Halidohydrolytic conversion of N, N′-BIS-(dichloroacetyl)-N,N′-diethyl-1,4-xylylenediamine in rat liver

Abstract N , N ′-bis-(dichloroacetyl)- N , N ′-diethyl-1,4-xylylenediamine (WIN 13099) was synthesized with tritium on the xylylene carbon which did not exchange with body water. This compound was metabolized by 140,000 g supernatant liquid and the mitochondrial fraction of rat liver. Deproteinized 140,000 g supernatant liquid was required for the mitochondrial activity. Although WIN 13099 effectively inhibits drug metabolism by rat liver microsomes in vitro and in vivo , it is not metabolized by rat liver microsomes in vitro . The major metabolite in vitro of WIN 13099, N -dichloroacetyl- N ′-hydroxyacetyl- N , N ′-diethyl-1,4-xylylenediamine, was isolated and characterized and found not as effective as the parent compound in inhibiting electron transport by sub-mitochondrial particles or drug metabolism by rat liver microsomes.