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A P Halestrap

Publications and source records attributed to A P Halestrap.

At least 73 records · Page 4Linked to original sources
PubMed1993

The mechanisms by which mild respiratory chain inhibitors inhibit hepatic gluconeogenesis.

(1) Liver cells from starved rats were incubated with 10 mM L-lactate, 1 mM pyruvate and 0.3 microM glucagon in the presence and absence of the mild respiratory inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at 0.5 mM. (2) The whole cell concentrations of phosphoenolpyruvate, 2-phosphoglycerate and 3-phosphoglycerate increased about 2-fold, whilst the triose and hexose phosphate concentrations all decreased significantly. Similar results were obtained with 0.15 microM oligomycin and 10 microM atractyloside. (3) These data can be explained by a substantial decrease in the cytosolic free concentration ratio of ATP/ADP acting on the equilibrium of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. (4) The increase in cytosolic phosphoenolpyruvate concentration can account for the observed increase in pyruvate kinase flux that occurs under these conditions (Pryor et al. (1987) Biochem. J. 247, 449-457). (5) An inhibition of pyruvate carboxylase was also implied by a decrease in calculated tissue oxaloacetate concentrations, confirming a role for both enzymes in the inhibition of gluconeogenesis. (6) Whole cell concentrations of effectors of pyruvate carboxylase activity were measured; only the ATP/ADP ratio decreased significantly. (7) Subcellular fractionation studies showed a good correlation between the measured mitochondrial ATP/ADP ratio and rates of gluconeogenesis both in the presence and absence of oleate. (8) A similar correlation could be observed between rates of pyruvate carboxylation and the measured matrix ATP/ADP ratio in isolated liver mitochondria from starved rats. (9) Data are also presented suggesting an additional effect of DCMU on the rate pyruvate carboxylation in situ under some circumstances, mediated by decreases in mitochondrial acetyl-CoA and cytosolic pyruvate concentrations. (10) It is noted that the effects of phenylethylbiguanide (phenformin) on the rate of gluconeogenesis and metabolite profiles in the perfused liver (Cooke et al. (1973) J. Biol. Chem. 248, 5272-5277) are similar to those caused by DCMU, supporting a mitochondrial locus of action for this hypoglycaemic agent.

Adenosine Diphosphate↗
PubMed1993

Pyrophosphate metabolism in the perfused heart and isolated heart mitochondria and its role in regulation of mitochondrial function by calcium.

1. Langendorff-perfused rat hearts were treated, either alone or in combination, with acetate, adrenaline or supraphysiological [Ca2+] to mimic increased workload. No significant increases in the PPi content of the freeze-clamped tissue were observed. 2. In contrast with liver mitochondria, isolated rat heart mitochondria incubated at a free [Mg2+] of 1.5 mM showed little or no increase in matrix PPi content or volume in the presence of 0.75 microM Ca2+; neither did 5 mM acetate cause heart mitochondrial PPi to increase. 3. At 0.1 mM Mg2+ some increase in matrix PPi content of heart mitochondria was observed, and increases in liver mitochondria were significantly greater. 4. Substantial increases in heart mitochondrial-matrix PPi content were observed at 1.5 mM Mg2+ when [Ca2+] > 1 microM, especially in the presence of acetate, or at 0.75 microM Ca2+ in the presence of bongkrekic acid to inhibit the adenine nucleotide translocase. 5. Total pyrophosphatase activity was similar in heart and liver mitochondrial matrix extract and toluene-permeabilized mitochondria. 6. These data suggest that under physiological conditions Ca2+ does not regulate heart mitochondrial [PPi], because higher matrix [Mg2+] overcomes the inhibition of pyrophosphatase by Ca2+, and also significant loss of PPi from the matrix on the adenine nucleotide translocase may occur. 7. It is concluded that, in contrast with the situation in liver [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382], Ca(2+)-induced PPi-mediated increases in mitochondrial volume are unlikely to play a physiological role in the heart. 8. The substantial swelling of liver and kidney mitochondria caused by addition of supraphysiological [Ca2+] (20 nmol/mg of protein) was associated with significant increases in PPi content, but was not observed in heart mitochondria. 9. This substantiates the role that we have proposed for PPi in the opening of a non-specific pore by Ca2+ overload of mitochondria [Halestrap and Davidson (1990) Biochem. J. 268, 153-160].

Animals↗
PubMed1993

Characterization of the inhibition by stilbene disulphonates and phloretin of lactate and pyruvate transport into rat and guinea-pig cardiac myocytes suggests the presence of two kinetically distinct carriers in heart cells.

1. The kinetics of transport of pyruvate (Km 0.20 mM), L-lactate (Km 2.2 mM) and D-lactate (Ki 10.2 mM) into rat cardiac myocytes were studied and compared with those for guinea-pig heart cells [Poole, Halestrap, Price and Levi (1989) Biochem. J. 264, 409-418] whose equivalent values were 0.07, 2.3 and 6.6 mM respectively. Maximal rates of transport were about 5-fold higher in the rat heart cells. 2. 4,4'-Dibenzamidostilbene-2,2'-disulphonate (DBDS), a powerful inhibitor of monocarboxylate transport into erythrocytes [Poole & Halestrap (1991) Biochem. J. 275, 307-312], was found to be a potent but apparently partial inhibitor of lactate and pyruvate transport, with an apparent Ki value at 0.5 mM L-lactate of about 16 microM in both species. Maximal inhibition was 50% and 80% in rat and guinea-pig cells respectively. 3. The maximal extent of inhibition and apparent Ki values were dependent on both the substrate transported and its concentration. Maximum inhibition was less and the Ki was greater at higher substrate concentrations. 4. A variety of other stilbene disulphonates were studied which showed different Ki values and maximal extents of inhibition. 5. Phloretin was a significantly less potent inhibitor of transport into both rat (Ki 25 microM) and guinea-pig (Ki 16 microM) heart cells than into rat erythrocytes (Ki 1.4 microM). In the rat but not the guinea-pig heart cells, inhibition appeared partial (maximal inhibition 84%). 6. We demonstrate that our results can be explained by the presence of two monocarboxylate carriers in heart cells, both with Km values for L-lactate of about 2 mM and inhibited by alpha-cyano-4-hydroxycinnamate, but with different affinities for other substrates and inhibitors. One carrier is sensitive to inhibition by stilbene disulphonates and has lower Km values for pyruvate (0.05-0.10 mM) and D-lactate (5 mM), whereas the other has higher Km values for pyruvate (0.30 mM) and D-lactate (25 mM), and is relatively insensitive to stilbene disulphonates. Rat heart cells possess more of the latter carrier and guinea-pig heart cells more of the former. 7. The significance of these results for the study of lactate transport in the perfused heart is discussed.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1993

Effects of tyrosine kinase inhibitors on protein kinase-independent systems.

Tyrosine kinase inhibitors have been widely used to probe the role of tyrosine phosphorylation in cellular signalling. These inhibitors exhibit an apparent specificity for tyrosine kinases over the serine/threonine kinases but little is known about their effects on other enzymes or biological systems. We demonstrate that genistein, erbstatin and alpha-cyanocinnamamides (tyrphostins) have inhibitory effects on fatty acid synthesis, lactate transport, mitochondrial oxidative phosphorylation and aldehyde dehydrogenase. We propose, therefore, that results obtained using tyrosine kinase inhibitors should be interpreted with caution, particularly if used at concentrations sufficient to inhibit these non-protein kinase-dependent events.

Aldehyde Dehydrogenase↗
PubMed1993

Protection by Cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts.

Reperfusion following a period of ischemia can salvage the myocardium only if the ischemic episode has not exceeded a certain time limit; beyond this point damage becomes irreversible. A key feature of the transition from reversible to irreversible injury is mitochondrial dysfunction which may involve the opening of a non-specific pore in the mitochondrial inner membrane. Pore opening can be induced in vitro by exposure of isolated mitochondria to high [Ca2+] and Pi. Such pore formation is sensitized by adenine nucleotide depletion and oxidative stress and can be blocked by the immunosuppressant cyclosporin A. Here we show that in isolated perfused rat hearts subjected to 30 min ischemia and 15 min reperfusion, 0.2 microM cyclosporin A restored the ATP/ADP ratio and AMP content (decreased and increased respectively during ischemia) to pre-ischemic values. In separate experiments functional recovery was assessed by monitoring the restoration of left ventricular developed pressure (LVP) during reperfusion after 30, 40 or 45 min ischemia. LVP was substantially improved in the presence of 0.2 microM cyclosporin A but did not return to pre-ischemic levels. The cyclosporin analogues G and H were less effective than cyclosporin A in protecting the heart during reperfusion. This is consistent with their reduced ability to protect isolated mitochondria from damage caused by Ca2+ overload. Surprisingly, reperfusion of hearts with 1 microM cyclosporin A reversed the protective effect seen at 0.2 microM.

Adenine Nucleotides↗
PubMed1993

Lactate-proton co-transport and its contribution to interstitial acidification during hypoxia in isolated rat spinal roots.

Exposure of nervous tissue to hypoxia results in interstitial acidification. There is evidence for concomitant decrease in extracellular pH to the increase in tissue lactate. In the present study, we used double-barrelled pH-sensitive microelectrodes to investigate the link between lactate transport and acid-base homeostasis in isolated rat spinal roots. Addition of different organic anions to the bathing solution at constant bath pH caused transient alkaline shifts in extracellular pH; withdrawal of these compounds resulted in transient acid shifts in extracellular pH. With high anion concentrations (30 mM), the largest changes in extracellular pH were observed with propionate > L-lactate approximately pyruvate > 2-hydroxy-2-methylpropionate. Changes in extracellular pH induced by 10 mM L- and D-lactate were of similar size. Lactate transport inhibitors alpha-cyano-4-hydroxycinnamic acid and 4,4'-dibenzamidostilbene-2,2'-disulphonic acid significantly reduced L-lactate-induced extracellular pH shifts without affecting propionate-induced changes in extracellular pH. Hypoxia produced an extracellular acidification that was strongly reduced in the presence of alpha-cyano-4-hydroxycinnamic acid and 4,4'-dibenzamidostilbene-2,2'-disulphonic acid. In contrast, amiloride and 4,4'-di-isothiocyanostilbene-2,2'-disulphonate were without effect on hypoxia-induced acid shifts. The results indicate the presence of a lactate-proton co-transporter in rat peripheral nerves. This transport system and not Na+/H+ or Cl-/HCO3- exchange seems to be the dominant mechanism responsible for interstitial acidification during nerve hypoxia.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Transport of lactate and other monocarboxylates across mammalian plasma membranes.

Transport of L-lactate across the plasma membrane is of considerable importance to almost all mammalian cells. In most cells a specific H(+)-monocarboxylate cotransporter is largely responsible for this process; the capacity of this carrier is usually very high, to support the high rates of production or utilization of L-lactate. The best characterized H(+)-monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates, including pyruvate and the ketone bodies acetoacetate and beta-hydroxybutyrate. This carrier is inhibited by alpha-cyanocinnamate derivatives and some stilbene disulfonates and has been identified as a protein of 35-50 kDa on the basis of purification and specific labeling experiments. Other cells possess similar alpha-cyanocinnamate-sensitive H(+)-linked monocarboxylate transporters, but in some cases there are significant differences in the properties of these systems, sufficient to suggest the existence of a family of such carriers. In particular, cardiac muscle and tumor cells have transporters that differ in their Km values for certain substrates (including stereoselectivity for L- over D-lactate) and in their sensitivity to inhibitors. Mitochondria, bacteria, and yeast also possess H(+)-monocarboxylate transporters that share some properties in common with those in the mammalian plasma membrane but are adapted to their specific roles. However, there are distinct Na(+)-monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which enable active uptake of lactate, pyruvate, and ketone bodies in these tissues. This article reviews the properties of these transport systems and their role in mammalian metabolism.

Animals↗
PubMed1992

Purification and N-terminal sequencing of peptidyl-prolyl cis-trans-isomerase from rat liver mitochondrial matrix reveals the existence of a distinct mitochondrial cyclophilin.

1. Rat liver mitochondrial matrix peptidyl-prolyl cis-trans-isomerase (PPIase) has been purified. The major form of the enzyme has a molecular mass of 18.6 kDa, with a minor active component of 17.6 kDa. 2. The second-order rate constant for cyclosporin A binding to the enzyme was determined from the time-dependence of the inhibition of PPIase by low concentrations of cyclosporin A and found to be 0.9 microM-1.s-1 at 10 degrees C. 3. The Ki for cyclosporin A inhibition of the enzyme was 3.6 nM, and the half-life for dissociation of the enzyme-inhibitor complex was 3.6 min. 4. From the specific activity of the pure enzyme it can be calculated that isolated liver mitochondria contain approx. 45 pmol of enzyme per mg of total mitochondrial protein. Higher values estimated previously [Halestrap & Davidson (1990) Biochem. J. 268, 153-160] are explained by the use of a short (30 s) preincubation period of the enzyme with cyclosporin, which is insufficient to allow full equilibration of the binding of the inhibitor to the PPIase. 5. N-Terminal sequencing of the 18.6 and 17.5 kDa forms of PPIase show the presence of mitochondrial presequences of 13 and three amino acids respectively, with the remaining sequence having a strong sequence similarity to other cyclophilins. 6. Parallel purification and N-terminal sequencing of rat cytosolic PPIase showed the two proteins to have significant differences, implying that they are probably products of separate genes.

Amino Acid Isomerases↗
PubMed1992

Identification and partial purification of the erythrocyte L-lactate transporter.

1. Intact erythrocytes were incubated with 100 microM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS), a concentration sufficient to inhibit lactate transport irreversibly by 65%. DIDS-labelled proteins were detected by immunoblotting of erythrocyte membrane proteins with an anti-DIDS antibody. Labelled polypeptides of 35-45 kDa in rat erythrocytes, and of 40-50 kDa in rabbit and guinea pig erythrocytes, were detected by this technique. In human erythrocytes, which have 10-fold less transport activity, no labelled polypeptide in this molecular mass range was detected. 2. Labelling of these 35-50 kDa polypeptides was decreased markedly in the presence of the specific inhibitors of lactate transport alpha-cyano-4-hydroxycinnamate and 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), which compete with DIDS for binding to the transporter. However, the weakly bound inhibitor 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS) afforded little protection against labelling by DIDS. 3. The lactate transporter from rat erythrocytes was solubilized with decanoyl-N-methyl glucamide (MEGA-10) and partially purified by Mono-Q anion-exchange chromatography, with transport activity eluting at 0.1-0.15 M-NaCl. The 35-45 kDa DIDS-labelled polypeptide from rat erythrocytes was eluted in the same peak of protein as lactate transporter activity during Mono-Q chromatography. 4. These observations provide strong evidence that the lactate transporter is a polypeptide of 35-45 kDa in rat erythrocytes and of 40-50 kDa in rabbit and guinea pig erythrocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Inhibition of L-lactate transport and band 3-mediated anion transport in erythrocytes by the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl-4,4'-diaminostilbene-2,2'-disulpho nat e (TBenzDS).

(1) The synthesis of the novel stilbenedisulphonate N,N,N',N'-tetrabenzyl- 4,4'-diaminostilbene-2,2'-disulphonate (TBenzDS) is described, and its interaction with the lactate transporter and band 3 protein of erythrocytes investigated. At 10% haematocrit the IC50 (concn. required for 50% inhibition) for inhibition of transport of 0.5 mM L-lactate into rat erythrocytes at 7 degrees C was approx. 1.6 microM, as low as any other inhibitor of the transporter. In human erythrocytes at 10% haematocrit the IC50 value was increased from approx. 3 microM to 9 microM upon raising the temperature from 7 degrees C to 25 degrees C. (2) TBenzDS inhibited transport of L-lactate into rat erythrocytes in a manner that was competitive with the substrate, as is the case for some other stilbene disulphonate derivatives (Poole, R.C. and Halestrap, A.P. (1991) Biochem. J. 275, 307-312). (3) Increasing the haematocrit from 5 to 20% caused a 3-fold increase in the IC50 value for inhibition of L-lactate transport in rat erythrocytes. (4) TBenzDS was found to bind to erythrocyte membranes, with a partition coefficient (Pm) of 6000-7000 under all conditions tested. (5) TBenzDS also inhibited band 3-mediated sulphate transport in rat erythrocytes; 50% inhibition required approx. 2.5 microM TBenzDS for cells at 10% haematocrit. (6) TBenzDS is fluorescent, and an enhancement of this fluorescence occurs upon addition of BSA or erythrocyte membranes. The fluorescence enhancement caused by erythrocyte membranes is due to binding of the inhibitor to the band 3 protein at the same site as the stilbenedisulphonate 4,4'-diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS).

Animals↗
PubMed1991

Calcium-dependent opening of a non-specific pore in the mitochondrial inner membrane is inhibited at pH values below 7. Implications for the protective effect of low pH against chemical and hypoxic cell damage.

1. The rate of opening of the Ca(2+)-induced non-specific, cyclosporin A-inhibited, pore of the mitochondrial inner membrane of rat heart and liver mitochondria at pH 6.0 was less than 10% of that at pH 7.4. 2. The effect could not be explained by inhibition of Ca2+ uptake into the mitochondria, or of the matrix peptidyl-prolyl cis-trans isomerase (PPIase), or of the Ca(2+)-induced conformational change of the adenine nucleotide translocase. 3. It is suggested that the proposed interaction of matrix PPIase with the 'c' conformation of the adenine nucleotide carrier in the presence of Ca2+ [Griffiths & Halestrap (1991) Biochem. J. 274, 611-614] is inhibited by low pH. 4. The relevance of this to the protective effect of low pH on hypoxic and chemical-induced cell damage is discussed.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes. Identification of some new high-affinity inhibitors.

1. Inhibition of L-lactate transport into rat erythrocytes by stilbenedisulphonates was studied under conditions which allowed the contribution of reversible and irreversible inhibition to be assessed. 2. At low temperatures (7 degrees C), 4,4'-di-isothiocyanostilbene-2,2'-disulphonate (DIDS) and other stilbenedisulphonates were found to inhibit lactate transport instantaneously, in a manner which was fully reversible. The most potent reversible inhibitors were 4,4'-dibenzamidostilbene-2,2'-disulphonate (DBDS), DIDS and 4-acetamido-4'isothiocyanostilbene-2,2'-disulphonate (SITS), for which apparent Ki values at 0.5 mM-L-lactate were approx. 36, 53 and 130 microM respectively. 3. DIDS and DBDS were competitive inhibitors with respect to L-lactate, with Ki values of approx 40 microM and 22 microM respectively. 4. After incubation for 1 h at 37 degrees C with DIDS or its dihydro derivative (H2DIDS), which contain the amino-reactive isothiocyanate group, most of the inhibition observed was irreversible. Under these conditions the IC50 value (concn. causing 50% inhibition) for irreversible inhibition by both compounds was approx 100 microM. SITS was much less potent as an irreversible inhibitor of L-lactate transport, approx. 20% inhibition being obtained at 100 microM. 5. The reversible inhibitor DBDS (1 mM) afforded protection against irreversible inhibition by DIDS and H2DIDS (100 microM); protection was 60 and 65% respectively after a 60 min incubation. This indicates that specific binding of the irreversible inhibitors is required before covalent modification can take place. 6. These compounds may be useful high-affinity probes for lactate transport in other tissues and might act as affinity labels for the transport protein(s).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Further evidence that cyclosporin A protects mitochondria from calcium overload by inhibiting a matrix peptidyl-prolyl cis-trans isomerase. Implications for the immunosuppressive and toxic effects of cyclosporin.

The Ki values of cyclosporins A, G and H for the peptidyl-prolyl cis-trans isomerase (PPIase) of liver and heart mitochondria are about 2, 20 and 500 nM respectively. This parallels their profile as inhibitors of non-specific pore opening of mitochondria induced by supraphysiological Ca2+ concentrations. The novel immunosuppressant FK-506 gave little inhibition of either process at 5 microM. These data support our previous hypothesis [Halestrap & Davidson (1990) Biochem. J. 268, 153-160] that pore opening involves an interaction between matrix PPIase and the adenine nucleotide translocase. It is suggested that this model may help to clarify the mechanism of action of cyclosporin as an immunosuppressant and its toxic effects on the liver and kidney following prolonged therapy.

Adjuvants, Immunologic↗
PubMed1990

Substrate and inhibitor specificity of monocarboxylate transport into heart cells and erythrocytes. Further evidence for the existence of two distinct carriers.

A range of short-chain aliphatic monocarboxylates, both unsubstituted and substituted with hydroxy, chloro and keto groups, were shown to inhibit transport of L-lactate and pyruvate into both guinea-pig cardiac myocytes and rat erythrocytes. The carrier of heart cells exhibited a higher affinity (approx. 10-fold) for most of the monocarboxylates than did the erythrocyte carrier. A notable exception was L-lactate, whose Km for both carriers was similar. The K1 values of the two carriers for inhibitors such as phenylpyruvate and alpha-cyanocinnamate derivatives were also different. The high affinity of the heart cell carrier for ketone bodies and acetate may be physiologically important, since these substrates are used as fuels by the heart.

Animals↗
PubMed1990

Partial inhibition by cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+], but not by butyrate. Evidence for two distinct swelling mechanisms.

1. The effects of cyclosporin A on the increase in matrix PPi and consequent swelling of energized liver mitochondria incubated with 1 mM-butyrate, 30 microM-bongkrekic acid or 0.1-35 microM-Ca2+ [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382] were studied. 2. Cyclosporin (1 microM) had no significant effect on the swelling induced by butyrate, bongkrekic acid or Ca2+ at concentrations of less than 0.3 microM. 3. At higher [Ca2+] (greater than 0.3 microM), swelling became progressively inhibited by cyclosporin, although the increase in matrix PPi was slightly greater in the presence than in the absence of cyclosporin. 4. Titration with cyclosporin indicated that there are 128 pmol of relevant cyclosporin-binding sites per mg of mitochondrial protein, with a Ki of about 5 nM. 5. The decrease in light-scattering by hepatocytes induced by butyrate [Davidson & Halestrap (1988) Biochem. J. 254, 379-384] was unaffected by cyclosporin, whereas that induced by vasopressin was inhibited by 20-30% without a significant change in cellular PPi content. 6. It is suggested that there are two mechanisms for the increase in mitochondrial volume induced by Ca2+: a PPi-mediated mechanism that is insensitive to cyclosporin and an additional Ca2(+)-mediated effect that is inhibited by cyclosporin. The nature of these pathways and their inter-relationship is discussed in the following paper [Halestrap & Davidson (1990) Biochem. J. 268, 153-160].

Animals↗
PubMed1990

Inhibition of Ca2(+)-induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase.

1. Isolated rat liver and heart mitochondria incubated in 150 mM-KSCN or sucrose medium in the presence of respiratory-chain inhibitors showed a large increase in swelling when exposed to 250 microM-Ca2+. Swelling was inhibited by bongkrekic acid and cyclosporin A in both media and by ADP in KSCN medium; the effect of ADP was reversed by carboxyatractyloside. These results demonstrate that this is a suitable technique with which to study the opening of the Ca2(+)-induced non-specific pore of the mitochondrial inner membrane and implicate the adenine nucleotide carrier in this process. 2. Titration of the rate of swelling with increasing concentrations of cyclosporin showed the number of cyclosporin-binding sites (+/- S.E.M.) in liver and heart mitochondria to be respectively 113.7 +/- 5.0 (n = 9) and 124.3 +/- 11.2 (n = 10) pmol/mg of protein, with a Ki of about 5 nM. 3. Liver and heart mitochondrial-matrix fractions were prepared free of membrane and cytosolic contamination and shown to contain cyclosporin-sensitive peptidyl-prolyl cis-trans isomerase (cyclophilin) activity. Titration of isomerase activity with cyclosporin gave values (+/- S.E.M.) of 110.6 +/- 10.1 (n = 5) and 165.4 +/- 15.0 (n = 3) pmol of enzyme/mg of liver and heart mitochondrial protein respectively, with a Ki of 2.5 nM. The similarity of these results to those from the swelling experiments suggest that the isomerase may be involved in the Ca2(+)-induced swelling. 4. The rapid light-scattering change induced in energized heart mitochondria exposed to submicromolar Ca2+ [Halestrap (1987) Biochem. J. 244, 159-164] was inhibited by ADP and bongkrekic acid, the former effect being reversed by carboxyatractyloside. These results suggest an interaction of Ca2+ with the adenine nucleotide carrier when the 'c' conformation. 5. A model is proposed in which mitochondrial peptidyl-prolyl cis-trans isomerase interacts with the adenine nucleotide carrier in the presence of Ca2+ to cause non-specific pore opening. The model also explains the involvement of the adenine nucleotide translocase in the PPi-mediated cyclosporin-insensitive increase in K+ permeability described in the preceding paper [Davidson & Halestrap (1990) Biochem. J. 268, 147-152]. 6. The physiological and pathological implications of the model are discussed in relation to reperfusion injury and cyclosporin toxicity.

Adenosine Diphosphate↗