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M Crompton

Publications and source records attributed to M Crompton.

At least 19 recordsLinked to original sources
PubMed1997

Selective cell ablation in transgenic mice expression E. coli nitroreductase.

The gene encoding E. coli nitroreductase (NTR) was expressed in the luminal cells of the mammary gland of transgenic mice using the ovine beta-lactoglobulin promoter. Treatment of NTR expressing animals with the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) resulted in a rapid and selective killing of this population of cells whereas the closely associated myoepithelial cells were unaffected. NTR-mediated inducible cell ablation offers a number of advantages over the use of HSV1-tk for the selective killing of cells in vivo.

Animals↗
PubMed1996

The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.

1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN-_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re-establishment of millimolar concentrations of ATP.

Adenosine Triphosphate↗
PubMed1996

Involvement of cyclophilin D in the activation of a mitochondrial pore by Ca2+ and oxidant stress.

Heart and liver mitochondria contain a structure that is able to form a large non-selective pore in the inner membrane under conditions of high matrix Ca2+ and oxidant stress. The pore is blocked by cyclosporin A (CSA). In this study, rat liver mitochondria were covalently labelled with a photoactive CSA derivative in the presence and absence of the pore ligands Ca2+ and ADP. Photolabelling of a 21-kDa protein was selectively depressed by Ca2+ in a manner reversed by ADP. The protein exhibited peptidyl-prolyl cis-trans isomerase (PPIase) activity and was inhibited by CSA (Ki, 8 nM). The PPIase was associated with the outside of sonicated submitochondrial particles but dissociated in 0.5 M NaCl. When mitochondria were treated with increasing concentrations of digitonin, the 21-kDa PPIase fractionated with the matrix marker enzyme, malate dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane-space marker, adenylate kinase. Photolabelling of the 18-kDa PPIase was unaffected by Ca2+ or ADP. The 21-kDa PPIase was digested with endoproteinase Asp-N and 11 of the peptides were N-terminally sequenced. The sequences were most similar to those of human cyclophilin-D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findings are discussed.

Adenosine Diphosphate↗
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↗