Douglas R. Pfeiffer

Cyclosporin A-sensitive and insensitive mechanisms produce the permeability transition in mitochondria.

Cyclosporin A is a potent inhibitor of the mitochondrial permeability transition, possibly by blocking an inner membrane pore through which solute movements occur [Broekemeier et al. (1989) J. Biol. Chem. 264, 7826-7830]. The inhibitory effect of cyclosporin, however, is transient. Trifluoperazine, at concentrations which inhibit the mitochondrial phospholipase A2, also produces a transient inhibition. When both inhibitors are used together, the inhibitory effect is long lasting. These findings suggest that the transition can be caused by two overlapping and/or interactive mechanisms, one dependent on an inner membrane pore and the other on phospholipase A2.

Cyclosporin A protects hepatocytes subjected to high Ca2+ and oxidative stress

Hepatocytes incubated with 0.8 mM t‐butylhydroperoxide are protected by cyclosporin A when the medium Ca2+ concentration is 10 mM, but not when it is 2.5 mM. The highest Ca2+ level is associated with an inhibition of t‐butylhydroperoxide‐dependent malondialdehyde accumulation and with mitochondrial Ca2+ loading within the cells. These findings are new evidence that t‐butylhydroperoxide can kill cells by peroxidation‐dependent and ‐independent mechanisms, and suggest that the mitochondrial permeability transition and the resultant de‐energization are components of the peroxidation‐independent mechanism. Cyclosporin A may have considerable utility for the protection of cells subjected to oxidative stress.

Release of mitochondrial matrix proteins through a Ca2+-requiring, cyclosporin-sensitive pathway

Induction of the inner membrane permeability transition, normally associated with the release of small molecules and ions from the mitochondrial matrix, also causes the release of matrix proteins. The release is linear with time and slow when compared to the time course of mitochondrial swelling. Transient induction of the high permeability state is reflected in transient release of proteins. Cyclosporin A (0.5 nmol/mg protein) or chelation of free Ca2+, which reverses the permeability transition, also block the subsequent release of protein even when added after extended preincubation. Possible mechanisms of protein release are discussed.

Lipids and lipolytic enzyme activities of rat heart mitochondria

Abstract The lipid composition and lipolytic enzyme activities in rat cardiac mitochondria were examined. Subsarcolemmal mitochondria were prepared by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by exposure of the remaining low-speed pellet to the protease, nagarse. These procedures are known to yield two functionally different populations of mitochondria. However, their phospholipid contents and compositions were identical, as were the positional distributions of the constituent fatty acids. Of the ethanolamine phospholipids, 20% were plasmalogens, and about 2% of the choline phospholipids consisted of this alkenylacyl species. Both subsarcolemmal and interfibrillar mitochondria contained a Ca2+-activated phospholipase A2, as evidenced by the Ca2+-dependent release of unsaturated fatty acids and lysophosphatidylethanolamine from endogenous lipids. Ruthenium red prevented the activation of this enzyme by Ca2+, indicating that the activity is located in the matrix space or associated with the inner surface of the inner membrane. Both mitochondrial fractions produced free fatty acids and lysophosphatidylethanolamine in the absence of free Ca2+ apparently due to an outer membrane phospholipase A1. The activity of this enzyme decreased with time, particularly in interfibrillar mitochondria, providing that Ca2+ was absent. Nagarse treatment of subsarcolemmal mitochondria resulted in a preparation with the same phospholipase A1 properties as interfibrillar mitochondria. The possibility that differences in phospholipase A1 properties account for some of the functional variations between the two mitochondrial types is discussed.

Factors affecting solute entrapment in phospholipid vesicles prepared by the freeze-thaw extrusion method: a possible general method for improving the efficiency of entrapment

It is often assumed that the internal solute concentrations of phospholipid vesicles are equal to those in the medium in which they were prepared, particularly when freeze-thaw cycles are employed during the procedure. Conditions are reported here which when used to prepare vesicles by the polycarbonate filter extrusion method, produce approximately 12- and approximately 7-fold higher internal concentrations of Ca2+ and sucrose, respectively, than exist in the external medium. Formation of these large gradients is dependent upon the use of freeze-thaw cycles during preparation, on the presence of tetraethylammonium perchlorate in the medium, and is independent of media pH across the region of pH 5-9. Gradient formation is antagonized by high concentrations of an impermeant solute (NaCl). It is proposed that gradients form because solutes are concentrated by exclusion from ice during freezing but that they are normally dissipated by osmotic lysis during thawing. The presence of a permeant solute such as tetraethylammonium perchlorate provides an alternative mechanism to balance osmotic pressure, thereby preserving the gradients of impermeable species.

Lipid composition and (Na+ + K+)-ATPase activity in rat lens during triparanol-induced cataract formation

The development of triparanol cataracts in rats is accompanied by the loss of lens (Na+ + K+)-ATPase activity and by alteration in the lens content and composition of phospholipids, sterols and phospholipid acyl groups. The lipid changes occur along the same time course as the loss of (NA+ + K+)-ATPase activity. Triparanol feeding produces a decrease in lens phospholipid content. The percentage contents of phosphatidylcholine and phosphatidyl-serine decrease while the content of sphingomyelin substantially increases. The amounts of oleic acid in lens phospholipids decrease while stearic and palmitic acids increase; however, these changes are relatively small. Sterol content is also decreased while the percentage content of desmosterol increases markedly. Feeding of the cataractogenic agents galactose and diazacholesterol also alters the lens lipid compositions and (Na+ + K+)-ATPase activity. A loss of phosphatidylserine is the only change in lipid properties which always accompanies a loss of the enzyme activity. The possible relationships between the lens content of phosphatidylserine, (Na+ + K+)-ATPase activity and the mechanism of triparanol-induced cataract formation are discussed.

A COMPARISON OF PHOSPHOLIPID DEGRADATION BY OXIDATION AND HYDROLYSIS DURING THE MITOCHONDRIAL PERMEABILITY TRANSITION

The peroxidation and hydrolysis of mitochondrial phospholipids has been examined under conditions which are referable to induction of the permeability transition by t-butylhydroperoxide. Over a 30-min time course, the peroxide causes formation of 0.3 nmol/mg protein of malondialdehyde. This value is little effected by Ca2+, Sr2+, or Mn2+ but is increased approximately fivefold by Fe2+. The latter cation, but not the others, results in malondialdehyde formation in the absence of added peroxide. Partially oxidized phosphatidylethanolamine is present in normal mitochondria and is increased by approximately 50% following t-butylhydroperoxide treatment; however, the amounts observed are in the range of 0.4-0.6 mol% of total phosphatidylethanolamine. The minor degradation by peroxidation is in contrast to approximately 2.5 mol% degradation which occurs by hydrolysis. This degree of hydrolysis is accompanied by mitochondrial swelling and Mg2+ release, while a comparable level of peroxidation (malondialdehyde formation) is not. It is concluded that induction of the permeability transition by t-butylhydroperoxide does not represent damage to the membrane lipid phase caused by peroxidation. It is possible, however, that peroxidation accelerates the accumulation of phospholipid hydrolysis products and is thereby a factor which favors the transition.

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens

The partial purification of (Na+ + K+)-ATPase from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in rho-nitrophenylphosphatase activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa alpha subunit of (Na+ + K+)-ATPase which can be phosphorylated by reaction with [gamma-32P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51000 and thus appears to be the beta subunit of the enzyme. The enzyme is sensitive to ouabain with the I50 for (Na+ + K+)-ATPase and rho-nitrophenylphosphatase inhibition being 1.2 and 1.3 microM, respectively. Several agents which inhibit (Na+ + K+)-ATPase from other tissues such as oligomycin, Ca2+, vanadate, N-ethylmaleimide, rho-chloromercuribenzenesulfonic acid (PCMBS) and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K+ are partially effective in activating the (Na+ + K+)-ATPase and rho-nitrophenylphosphatase activities. The K+ congeners were relatively more effective in supporting (Na+ + K+)-ATPase compared to rho-nitrophenylphosphatase activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of (Na+ + K+)-ATPase activity, rho-nitrophenylphosphatase activity and fluorescence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.

General features in the stoichiometry and stability of ionophore A23187-cation complexes in homogeneous solution

Existing literature describing the stoichiometry and stability of complexes between A23187 and divalent cations in solution has been extended to include additional transition series cations, the heavy-metal cations Cd2+ and Pb2+, plus seven lanthanide series trivalent cations. Stability constants of 1:1 complexes between the ionophore and the divalent cations vary by 6.2 orders of magnitude between Cu2+ and Ba2+ which are the strongest and weakest complexes, respectively. Considering alkaline-earth and first-series transition cations together, the pattern of stability constants obeys the extended Irving-Williams series as is seen with many nonionophorous liganding agents. Cd2+ and Pb2+ are bound with an affinity similar to those of Mn2+ and Zn2+, whereas the lanthanides are bound with little selectivity and slightly higher stability. Titration of the ionophore in the 10(-5) M concentration range with di- and trivalent cations gives rise first to complexes of stoichiometry MA2 and subsequently to MA as the metal concentration is increased. The second stepwise stability constants for formation of the MA2 species exceeds the first constant by approximately 10-fold. With lanthanides, heavy metals, and transition-metal cations, OH-, at near physiological concentrations, competes significantly with free ionophore for binding to the 1:1 complexes. This competition is not apparent when Ca2+ or Mg2+ are the central cations. Possible implications of the 1:1 complex selectivity pattern, the ionophore-hydroxide competitive binding equilibria, and potential ternary complexes involving 1:1 ionophore:cation complexes and other anions present in biological systems are discussed with respect to the ionophores transport selectivity and biological actions.

Effects of solute concentration on the entrapment of solutes in phospholipid vesicles prepared by freeze-thaw extrusion

Phospholipid vesicles prepared by the freeze-thaw extrusion method contain internal solute concentrations which are much higher than the external values (entrapment ratios much greater than 1). This concentrating effect is a complex function of the total impermeant solute concentration in the medium used to prepare vesicles, the presence or absence of permeant solutes in the medium and the apparent competitive binding interactions between solutes and phospholipid. Increases in water phase solute concentration during freezing are thought to underlie the concentrating phenomenon, while osmotic pressure driven lysis of vesicles during thawing appears to limit its magnitude. By judicious selection of solute concentration and physical properties, further increases in the entrapment ratio should be obtainable, improving the usefulness of these vesicles as drug delivery vesicles and experimental systems.