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M Crompton

Publications and source records attributed to M Crompton.

At least 19 recordsLinked to original sources
PubMed1995

Evidence for the involvement of a membrane-associated cyclosporin-A-binding protein in the Ca(2+)-activated inner membrane pore of heart mitochondria.

Heart and liver mitochondria contain a pore in the inner membrane that is activated by Ca2+ and oxidative stress and that has been implicated in cell injury. Pore opening is blocked by cyclosporin A (CSA). Following previous indications that the interaction of CSA with the pore is inhibited by Ca2+ and promoted by ADP, we have investigated how covalent labelling of heart mitochondria by a photoactive CSA derivative is influenced by these agents. In situ photolabelling of an 11-22-kDa (approximately) membrane fraction was selectively increased in the presence of ADP and decreased in the presence of Ca2+. This fraction also accounted for all the high affinity [3H]CSA-binding capacity and contained peptidylprolyl cis-trans isomerase activity (PPIase). The membrane PPIase was extracted using Chaps as detergent, and was purified to a 22-kDa protein (SDS/PAGE). The enzyme was inhibited by CSA (Ki 5 nM). The major component of the 11-22-kDa fraction, photolabelled in an ADP/Ca(2+)-sensitive manner, also migrated at 22 kDa on SDS/PAGE; a minor 11-kDa component was also detected. On the basis of these criteria, it is suggested that the membrane PPIase may be the target for CSA when it blocks the pore. The presence of a similar PPIase in the membrane fraction of liver mitochondria was also demonstrated. The implications of these findings are discussed.

Adenosine Diphosphate↗
PubMed1994

On the interactions of Ca2+ and cyclosporin A with a mitochondrial inner membrane pore: a study using cobaltammine complex inhibitors of the Ca2+ uniporter.

The mitochondrial inner membrane contains a Ca(2+)-activated pore of possible relevance to the pathogenesis of ischaemia/reperfusion injury which is inhibited by the immunosuppressant cyclosporin A (CSA). The present study employs a number of novel cobaltammine complex inhibitors of the Ca2+ uniporter (mediating Ca2+ uptake) to examine whether intramitochondrial Ca2+ influences the capacity of CSA to block the pore. Using dissipation of the inner membrane potential as a means of monitoring the state of the pore, it is shown that CSA blockade is facilitated as Ca2+ uptake is restricted. Ca2+ also depresses and reverses the binding of [3H]CSA to mitochondria, but Ca2+ is ineffective when its uptake is prevented. It is concluded that a high intramitochondrial Ca2+ concentration antagonizes pore inhibition by CSA. The significance of this is discussed.

Animals↗
PubMed1994

An ADP-sensitive cyclosporin-A-binding protein in rat liver mitochondria.

Mitochondria contain a structure which forms a large aqueous pore in the inner membrane after Ca2+ overload in the presence of Pi. In the present study, pore activation in liver mitochondria was monitored using the collapse of the inner membrane potential (delta psi). Ca(2+)-induced pore opening (delta psi collapse) was prevented by the immunosuppressant cyclosporin A, but cyclosporin A did not reverse pore opening (i.e. allow delta psi regeneration) unless ADP was also added. At concentrations that produced substantial pore blockade, [3H]cyclosporin partitioned more or less equally between membrane and soluble fractions, but the distribution was shifted slightly to the membranes in the presence of ADP. ADP also increased the binding of [3H]cyclosporin A to membranes washed free of soluble components. The indication that cyclosporin A inhibition of the pore is mediated by an ADP-sensitive membrane component was examined using a tritiated photoactivable derivative of cyclosporin A. ADP selectively increased covalent binding of this derivative to a membrane component. This component eluted from molecular-sizing columns as a 13-17-kDa-protein in the presence of 0.5% Chaps as detergent and migrated as a 10-kDa (approximately) protein in SDS/PAGE. These findings provide the first evidence that a protein of approximately 10 kDa may be part of the cyclosporin-A receptor of the Ca(2+)-activated pore. The possible implications of these findings are discussed.

Adenosine Diphosphate↗
PubMed1993

On the involvement of a cyclosporin A sensitive mitochondrial pore in myocardial reperfusion injury.

Mammalian cardiomyocytes may withstand prolonged periods of ischaemia, only to die on reperfusion. We review data that implicate mitochondrial dysfunction as a basis for reperfusion induced cell injury, and present some new evidence that suggests that such a mechanism operates in intact cardiomyocytes. The mitochondrial dysfunction is the consequence of the opening of high conductance pores in the inner mitochondrial membrane, which uncouple mitochondrial oxidative phosphorylation, promoting ATP hydrolysis. The conditions required to open the pores correlate closely to conditions that prevail upon reperfusion of the ischaemic heart: a high [Ca2+]i and Pi, low [ATP], and oxidative stress. Pore opening is suppressed by physiological concentrations of ATP. Pore opening may be prevented by cyclosporin A. Studies in isolated myocytes show that mitochondria become uncoupled after reoxygenation, and that this is associated with the hypercontracture that signals cell death. Cyclosporin A reduces the proportion of hypercontracted myocytes in populations of cells rendered anoxic.

Adenosine Triphosphate↗
PubMed1992

The involvement of cyclosporin A binding proteins in regulating and uncoupling mitochondrial energy transduction.

The uncoupling of mitochondrial energy transduction by excess Ca2+ may be a factor in the pathogenesis of tissue injury brought about by energy deprivation, for example, in ischaemia. In isolated mitochondria the lesion appears as a large, 20 A, pore in the inner membrane. The pore is blocked potently by the immunosuppressant cyclosporin A. Cyclosporin A also markedly retards collapse of the mitochondrial inner membrane potential in energy-deprived (respiration-inhibited) cardiomyocytes as judged by changes in rhodamine 123 fluorescence, and prolongs cell viability. A potential mitochondrial target for cyclosporin A is the matrix protein cyclophilin. Purified cyclophilin activates the respiratory chain of submitochondrial particles. This might reflect not only a physiological function of this protein, but also a component involved in the generation of the 20 A pore under pathological conditions.

Animals↗
PubMed1991

Cytochrome oxidase content of rat brain during development.

The cytochrome oxidase concentration and content of rat brain during development was measured using a simple new assay for cytochrome a. The cytochrome oxidase concentration increased from 1.2 nmol/g wet wt. of brain at birth to about 5.5 nmol/g in the adult, most of the change occurring between 5 and 25 days after birth.

Animals↗
PubMed1991

Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A.

Cyclosporin A is a potent immunosuppressant used to prevent graft rejection. The cellular target of cyclosporin A in T lymphocytes is believed to be cyclophilin, a ubiquitous protein with peptidyl prolyl cis trans isomerase activity located in both the cytosol and mitochondria. Recently, cyclosporin A-inhibition of mitochondrial cyclophilin has been implicated in the prevention of mitochondrial dysfunction induced in vitro by Ca2+ overload and other factors potentially relevant to ischaemic cell injury. This study investigates the effect of cyclosporin A on injury to cardiomyocytes induced by substrate-free anoxia. It is shown that cyclosporin A retards progression of the injury, most probably at a late step in the injury process.

Animals↗
PubMed1990

The presence of two classes of high-affinity cyclosporin A binding sites in mitochondria. Evidence that the minor component is involved in the opening of an inner-membrane Ca(2+)-dependent pore.

The inner membrane of rat liver mitochondria contains a reversible Ca(2+)-dependent pore, opening of which is largely blocked by cyclosporin A. Analyses of [3H]cyclosporin binding to rat liver mitochondria demonstrate two classes of high-affinity binding site with capacities of less than 5 pmol and approximately 60 pmol cyclosporin.mg mitochondrial protein-1 in addition to partitioning into membrane phospholipids (0.03 pmol.mg mitochondrial protein.nM-1). Direct measurement [14C]sucrose entry into the matrix space indicates that cyclosporin A inhibits pore opening by interacting with the low-capacity sites. The same low-capacity sites (Kd cyclosporin, 8 nM) are possibly attributable to peptidylprolyl cis-trans-isomerase, although investigation of pore state interconversion from the rapid kinetics of [14C]sucrose entrapment in the matrix space does not indicate that cyclosporin-sensitive prolyl isomerization occurs at the actual step of pore opening/closure. It is suggested that the low-capacity cyclosporin-binding component may stabilize the open pore state; this is supported by the observations that Ca2+ decreases cyclosporin binding to this component and that cyclosporin brings about closure of the pre-opened pore. The implications for the possible number of functional pores in mitochondria are discussed.

Amino Acid Isomerases↗
PubMed1990

A heart mitochondrial Ca2(+)-dependent pore of possible relevance to re-perfusion-induced injury. Evidence that ADP facilitates pore interconversion between the closed and open states.

The permeability properties of a putative Ca2(+)-activated pore in heart mitochondria, of possible relevance to re-perfusion-induced injury, have been investigated by a pulsed-flow solute-entrapment technique. The relative permeabilities of [14C]mannitol, [14C]sucrose and arsenazo III are consistent with permeation via a pore of about 2.3 nm diameter. Ca2+ removal with EGTA induced pore closure, and the mitochondria became 'resealed'. The permeability of the unresealed mitochondria during resealing was markedly stimulated by 200 microM-ADP, and the relative permeabilities to solutes of different size were stimulated equally, indicating an increase in open-pore number, rather than an increase in pore dimensions. This is paradoxical, since ADP also stimulated the rate of resealing. The rate of EGTA-induced resealing was also stimulated by the Ca2+ ionophore A23187, which indicates that the rate of removal of matrix free Ca2+ is limiting for pore closure. An explanation for the paradox is suggested in which ADP facilitates pore interconversion between the closed and open states in permeabilized mitochondria, and pore closure in Ca2(+)-free mitochondria occurs much faster than previously thought.

Adenosine Diphosphate↗
PubMed1988

Kinetic evidence for a heart mitochondrial pore activated by Ca2+, inorganic phosphate and oxidative stress. A potential mechanism for mitochondrial dysfunction during cellular Ca2+ overload.

Evidence that the Ca2+-induced permeabilization of mitochondria is attributable to a reversible Ca2+-activated pore [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29] has been further investigated. Permeabilization is induced in a wholly synergistic manner by either Ca2+ plus phosphate or Ca2+ plus tert-butyl hydroperoxide. When permeabilization is complete, extramitochondrial [14C]sucrose equilibrates with the matrix space with a half-time of about 800 ms; [14C]mannitol equilibrates at least threefold faster. Permeabilization is essentially fully reversed on Ca2+ chelation with EGTA, when the half time for [14C]sucrose equilibration is increased 600-1400-fold (to 550-1150 s). A pulsed-flow [14C]solute-entrapment technique has been developed to measure the kinetics of EGTA-induced resealing. The technique incorporates a suitable choice of [14C]solute and an appropriate model for data analysis, and is competent to measure permeation state changes occurring in 100 ms. The data obtained are consistent with exponential resealing of mitochondria in which pores of any single mitochondria close with a high degree of synchrony. The rate of resealing is increased about eight-fold by ADP (half-time approximately 1 s; Km approximately 30 microM). CoA, Mg2+, AMP and also ATP, when account is taken of ADP arising by hydrolysis, are essentially ineffective. It is concluded that heart mitochondria do contain a pore whose permeation state is controlled over an approximate 1000-fold range by Ca2+ and other factors including phosphate, oxidative stress and ADP. The possible involvement of the pore in reoxygenation-induced injury in heart is discussed.

Adenosine Diphosphate↗
PubMed1988

Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.

The capacity of cyclosporin A to inhibit opening of a Ca2+-dependent pore in the inner membrane of heart mitochondria was investigated. Whereas in the presence of 25 nmol of Ca2+/mg of mitochondrial protein and 5 mM-Pi mitochondria were unable to maintain accumulated Ca2+, inner-membrane potential and sucrose impermeability, all three parameters were preserved when cyclosporin was included. Pore opening was assayed directly by [14C]sucrose entry and entrapment in the matrix space. [14C]Sucrose entry induced by both Ca2+ plus Pi and Ca2+ plus t-butyl hydroperoxide was almost completely inhibited by 60 pmol of cyclosporin/mg of mitochondrial protein. It is concluded that cyclosporin A is a potent inhibitor of the pore.

Animals↗
PubMed1987

Evidence for the presence of a reversible Ca2+-dependent pore activated by oxidative stress in heart mitochondria.

Rat heart mitochondria became permeabilized to sucrose when incubated with 100 nmol of Ca2+/mg of protein in the presence of Pi. Ca2+ chelation with EGTA restored impermeability to sucrose, which became entrapped in the matrix space. t-Butylhydroperoxide markedly promoted permeabilization in the presence of Ca2+ but not in its absence, and Ca2+-plus-t-butylhydroperoxide-induced permeabilization was reversed by EGTA. The data suggest that Ca2+ and oxidative stress synergistically promote the reversible opening of an inner membrane pore.

Animals↗
PubMed1987

The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria.

The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria ('mitosomes') were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.

Adenine Nucleotides↗
PubMed1987

Effects of adrenergic agonists and mitochondrial energy state on the Ca2+ transport systems of mitochondria.

This study investigates the effects of adrenergic agonists and mitochondrial energy state on the activities of the Ca2+ transport systems of female rat liver mitochondria. Tissue perfusion with the alpha-adrenergic agonist phenylephrine and with adrenaline, but not with the beta-adrenergic agonist isoprenaline, induced significant activation of the uniporter and the respiratory chain. Uniporter activation was evident under two sets of experimental conditions that excluded influences of delta psi, i.e., at high delta psi, where uniporter activity was delta psi independent, and at low delta psi, where uniporter conductance was measured. Preincubation of mitochondria with extracts from phenylephrine-perfused tissue quantitatively reproduced uniporter activation when comparison was made with mitochondria treated similarly with extracts from tissue perfused without agonist. Similar, but more extensive, data were obtained with heart mitochondria pretreated with extracts from hearts perfused with the alpha-adrenergic agonist methoxamine. Phenylephrine did not affect Ca2+ efflux mediated by the Na+-Ca2+ carrier or the Na+-independent system. In contrast, the liver mitochondrial Na+-Ca2+ carrier was activated by tissue perfusion with isoprenaline; the Na+-independent system was unaffected. Na+-Ca2+ carrier activation was not associated with any change in a number of basic bioenergetic parameters. It is concluded that the Ca2+ transport systems of liver mitochondria may be controlled in an opposing manner by alpha-adrenergic agonists (promotion of Ca2+ influx) and beta-adrenergic agonists (promotion of Ca2+ efflux). At delta psi values greater than 110 mV, the Na+-independent system was activated by increase in delta psi; the uniporter and Na+-Ca2+ carrier activities were insensitive to delta psi changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

The reversible Ca2+-induced permeabilization of rat liver mitochondria.

Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed.

Animals↗
PubMed1986

The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria.

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.

Animals↗