Biomedical subjects

A P Halestrap

Publications and source records attributed to A P Halestrap.

At least 37 records · Page 2Linked to original sources

PubMed1999

The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.

Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location.

Amino Acid Sequence↗

PubMed1999

Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes.

Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevis oocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H(+) gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a K(m) value of 0.74+/-0.07 mM was determined at pH 7.0. For the other substrates, K(i) values between 100 microM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by alpha-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.

Animals↗

PubMed1999

Lactate transport in skeletal muscle - role and regulation of the monocarboxylate transporter.

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibres also use lactic acid as a respiratory fuel. The stereoselective transport of L-lactic acid across the plasma membrane of muscle fibres has been shown to involve a proton-linked monocarboxylate transporter (MCT) similar to that described in erythrocytes and other cells. This transporter plays an important role in the pH regulation of skeletal muscle. A family of eight MCTs has now been cloned and sequenced, and the tissue distribution of each isoform varies. Skeletal muscle contains both MCT1 (the only isoform found in erythrocytes but also present in most other cells) and MCT4. The latter is found in all fibre types, although least in more oxidative red muscles such as soleus, whereas expression of MCT1 is highest in the more oxidative muscles and very low in white muscles that are almost entirely glycolytic. The properties of MCT1 and MCT2 have been described in some detail and the latter shown to have a higher affinity for substrates. MCT4 has been less well characterized but has a lower affinity for L-lactate (i.e. a higher Km of 20 mM) than does MCT1 (Km of 5 mM). MCT1 expression is increased in response to chronic stimulation and either endurance or explosive exercise training in rats and humans, whereas denervation decreases expression of both MCT1 and MCT4. The mechanism of regulation is not established, but does not appear to be accompanied by changes in mRNA concentrations. However, in other cells MCT1 and MCT4 are intimately associated with an ancillary protein OX-47 (also known as CD147). This protein is a member of the immunoglobulin superfamily with a single transmembrane helix, whose expression is known to be increased in a range of cells when their metabolic activity is increased.

Amino Acid Sequence↗

PubMed1999

The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury.

The mitochondrial permeability transition (mPT) involves the opening of a non-specific pore in the inner membrane of mitochondria, converting them from organelles whose production of ATP sustains the cell, to instruments of death. Here, I first summarize the evidence in favour of our model for the molecular mechanism of the mPT. It is proposed that the adenine nucleotide translocase (ANT) is converted into a non-specific pore through a calcium-mediated conformational change. This requires the binding of a unique cyclophilin (cyclophilin-D, CyP-D) to the ANT, except when matrix [Ca2+] is very high. Binding of CyP-D is increased in response to oxidative stress and some thiol reagents which sensitize the mPT to [Ca2+]. Matrix adenine nucleotides decrease the sensitivity of the mPT to [Ca2+] by binding to the ANT. This is antagonized by carboxyatractyloside (an inhibitor of the ANT) and by modification of specific thiol groups on the ANT by oxidative stress or thiol reagents; such treatments thus enhance the mPT. In contrast, decreasing intracellular pH below 7.0 greatly desensitizes the mPT to [Ca2+]. Conditions which sensitize the mPT towards [Ca2+] are found in hearts reperfused after a period of ischaemia, a process that may irreversibly damage the heart (reperfusion injury). We have demonstrated directly that mPT pores open during reperfusion (but not ischaemia) using a technique that involves entrapment of [3H]deoxyglucose in mitochondria that have undergone the mPT. The mPT may subsequently reverse in hearts that recover from ischaemia/reperfusion, the extent of resealing correlating with recovery of heart function. A variety of agents that antagonize the mPT protect the heart from reperfusion injury, including cyclosporin A, pyruvate and propofol. Mitochondria that undergo the mPT and then reseal may cause cytochrome c release and thus initiate apoptosis in cells subjected to stresses less severe than those causing necrosis. An example is the apoptotic cell death in the hippocampus that occurs several days after insulin-induced hypoglycaemia, and can be prevented by prior treatment with cyclosporin A.

Animals↗

PubMed1999

Differences in the activation of the mitochondrial permeability transition among brain regions in the rat correlate with selective vulnerability.

Mitochondria from different regions of the brain were prepared, and the activation of the mitochondrial permeability transition (MPT) by calcium was investigated by monitoring the associated mitochondrial swelling. In general, the properties of the MPT in brain mitochondria were found to be qualitatively similar to those observed in liver and heart mitochondria. Thus, swelling was inhibited by adenine nucleotides (AdNs) and low pH (<7.0), whereas thiol reagents and alkalosis facilitated swelling. Cyclosporin A and its nonimmunosuppressive analogue N-methyl-Val-4-cyclosporin A (PKF 220-384) both inhibited swelling and prevented the translocation of cyclophilin D from the matrix to the membranes of cortical mitochondria. However, the calcium sensitivity of the MPT differed in mitochondria from three brain regions (hippocampus > cortex > cerebellum) and is correlated with the susceptibility of these regions to ischemic damage. Depleting mitochondria of AdNs by treatment with pyrophosphate ions sensitized the MPT to [Ca2+] and abolished regional differences, implying regional differences in mitochondrial AdN content. This was confirmed by measurements showing significant differences in AdN content among regions (cerebellum > cortex > hippocampus). Our data add to recent evidence that the MPT may be involved in neuronal death.

Adenine Nucleotides↗

PubMed1999

Effect of high-intensity exercise training on lactate/H+ transport capacity in human skeletal muscle.

The present study examined the effect of high-intensity exercise training on muscle sarcolemmal lactate/H+ transport and the monocarboxylate transporters (MCT1 and MCT4) as well as lactate and H+ release during intense exercise in humans. One-legged knee-extensor exercise training was performed for 8 wk, and biopsies were obtained from untrained and trained vastus lateralis muscle. The rate of lactate/H+ transport determined in sarcolemmal giant vesicles was 12% higher (P < 0.05) in the trained than in untrained muscle (n = 7). The content of MCT1 and MCT4 protein was also higher (76 and 32%, respectively; n = 4) in trained muscle. Release of lactate and H+ from the quadriceps muscle at the end of intense exhaustive knee-extensor exercise was similar in the trained and untrained leg, although the estimated muscle intracellular-to-interstitial gradients of lactate and H+ were lower (P < 0.05) in the trained than in the untrained muscle. The present data show that intense exercise training can increase lactate/H+ transport capacity in human skeletal muscle as well as improve the ability of the muscle to release lactate and H+ during contractions.

Acid-Base Equilibrium↗

PubMed1999

Reversal of permeability transition during recovery of hearts from ischemia and its enhancement by pyruvate.

We have used mitochondrial entrapment of 2-deoxy-D-[3H]glucose (2-DG) to demonstrate that recovery of Langendorff-perfused rat hearts from ischemia is accompanied by reversal of the mitochondrial permeability transition (MPT). In hearts loaded with 2-DG before 40 min of ischemia and 25 min of reperfusion, 2-DG entrapment [expressed as 10(5) x (mitochondrial 2-[3H]DG dpm per unit citrate synthase)/(total heart 2-[3H]DG dpm/g wet wt)] increased from 11.1 +/- 1.3 (no ischemia, n = 4) to 32.5 +/- 1.9 (n = 6; P < 0.001). In other experiments, 2-DG was loaded after 25 min of reperfusion to determine whether some mitochondria that had undergone the MPT during the initial phase of reperfusion subsequently "resealed" and thus no longer took up 2-DG. The reduction of 2-DG entrapment to 20. 6 +/- 2.4 units (n = 5) confirmed that this was the case. Pyruvate (10 mM) in the perfusion medium increased recovery of left ventricular developed pressure from 57.2 +/- 10.3 to 98.9 +/- 10.8% (n = 6; P < 0.05) and reduced entrapment of 2-DG loaded preischemically and postischemically to 23.5 +/- 1.5 (n = 4; P < 0. 001) and 10.5 +/- 0.5 (n = 4; P < 0.01) units, respectively. The presence of pyruvate increased tissue lactate content at the end of ischemia and decreased the effluent pH during the initial phase of reperfusion concomitant with an increase in lactate output. We suggest that pyruvate may inhibit the MPT by decreasing pHi and scavenging free radicals, thus protecting hearts from reperfusion injury.

Adenine Nucleotides↗

PubMed1998

Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition.

A fusion protein between cyclophilin-D (CyP-D) and glutathione S-transferase (GST) was shown to bind to purified liver inner mitochondrial membranes (IMMs) in a cyclosporin A (CsA)-sensitive manner. Binding was enhanced by diamide treatment of the IMMs. Immobilized GST-CyP-D avidly bound a single 30 kDa protein present in Triton X-100-solubilized IMMs; immunoblotting showed this to be the adenine nucleotide translocase (ANT). Binding was prevented by pretreatment of the CyP-D with CsA, but not with cyclosporin H. Purified ANT also bound specifically to GST-CyP-D, but porin did not, even in the presence of ANT.

Animals↗

PubMed1998

Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart.

First, we present a summary of the evidence for our model of the molecular mechanism of the permeability transition (MPT). Our proposal is that the MPT occurs as a result of the binding of mitochondrial cyclophilin (CyP-D) to the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane. This binding is enhanced by thiol modification of the ANT caused by oxidative stress or other thiol reagents. CyP-D binding enhances the ability of the ANT to undergo a conformational change triggered by Ca2+. Binding of ADP or ATP to a matrix site of the ANT antagonises this effect of Ca2+; modification of other ANT thiol groups inhibits ADP binding and sensitises the MPT to [Ca2+]. Increased membrane potential changes the ANT conformation to enhance ATP binding and hence inhibit the MPT. Our most recent data shows that a fusion protein of CyP-D and glutathione-S-transferase immobilised to Sepharose specifically binds the ANT from Triton-solubilised inner mitochondrial membranes in a cyclosporin A (CsA) sensitive manner. Second we summarise the evidence for the MPT being a major factor in the transition from reversible to irreversible injury during reperfusion of a heart following a period of ischaemia. We describe how in the perfused heart [3H]deoxyglucose entrapment within mitochondria can be used to measure the opening of MPT pore in situ. During ischaemia pore opening does not occur, but significant opening does occur during reperfusion, and recovery of the heart is dependent on subsequent pore closure. Pore opening is inhibited by the presence in the perfusion medium of pyruvate and the anaesthetic propofol which both protect the heart from reperfusion injury. Third we discuss how the MPT may be involved in determining whether cell death occurs by necrosis (extensive pore opening and ATP depletion) or apoptosis (transient pore opening with maintenance of ATP).

Adenosine Triphosphate↗

PubMed1998

Cyclosporin A, but not FK 506, protects mitochondria and neurons against hypoglycemic damage and implicates the mitochondrial permeability transition in cell death.

Induction of the mitochondrial permeability transition (MPT) has been implicated in cellular apoptosis and in ischemia-reperfusion injury. During MPT, a channel in the inner mitochondrial membrane, the mitochondrial megachannel, opens and causes isolated mitochondria to swell. MPT and mitochondrial swelling is inhibited by cyclosporin A (CsA), which may also inhibit apoptosis in some cells. Treatment with CsA (50 mg/kg, i.v.) showed a robust reduction of brain damage when administered 30 min before insulin-induced hypoglycemic isoelectricity of 30 min duration. Ultrastructural examination of the dentate gyrus revealed a marked swelling of dendrites and mitochondria during the hypoglycemic insult. In CsA-treated animals, mitochondria resumed a normal and contracted appearance during and after the hypoglycemic insult. Treatment with FK 506 (2 mg/kg, i.v.), a compound with immunosuppressive action similar to that of CsA, was not protective. Studies on the swelling kinetics of isolated mitochondria from the hippocampus showed that CsA, but not FK 506, inhibits calcium ion-induced MPT. We conclude that CsA treatment during hypoglycemic coma inhibits the MPT and reduces damage and that mitochondria and the MPT are likely to be involved in the development of hypoglycemic brain damage in the rat.

Animals↗

PubMed1998

Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3.

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40-60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Animals↗

PubMed1998

Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past.

Measurement of monocarboxylate transport kinetics in a range of cell types has provided strong circumstantial evidence for a family of monocarboxylate transporters (MCTs). Two mammalian MCT isoforms (MCT1 and MCT2) and a chicken isoform (REMP or MCT3) have already been cloned, sequenced and expressed, and another MCT-like sequence (XPCT) has been identified. Here we report the identification of new human MCT homologues in the database of expression sequence tags and the cloning and sequencing of four new full-length MCT-like sequences from human cDNA libraries, which we have denoted MCT3, MCT4, MCT5 and MCT6. Northern blotting revealed a unique tissue distribution for the expression of mRNA for each of the seven putative MCT isoforms (MCT1-MCT6 and XPCT). All sequences were predicted to have 12 transmembrane (TM) helical domains with a large intracellular loop between TM6 and TM7. Multiple sequence alignments showed identities ranging from 20% to 55%, with the greatest conservation in the predicted TM regions and more variation in the C-terminal than the N-terminal region. Searching of additional sequence databases identified candidate MCT homologues from the yeast Saccharomyces cerevisiae, the nematode worm Caenorhabditis elegans and the archaebacterium Sulfolobus solfataricus. Together these sequences constitute a new family of transporters with some strongly conserved sequence motifs, the possible functions of which are discussed.

Amino Acid Sequence↗

PubMed1997

Lactate transport in heart in relation to myocardial ischemia.

In this article, the importance of lactic acid transport into and out of heart cells is described and the properties of the monocarboxylate transporters (MCTs) responsible are presented. These are monocarboxylate/proton symporters with a broad substrate specificity that includes L-lactate, pyruvate, and the ketone bodies acetate, acetoacetate, and beta-hydroxybutyrate. Although it is unlikely that lactic acid transport constrains heart metabolism under most conditions, it may do so during severe hypoxia or ischemia. The transporter plays a critical role in maintaining intracellular pH because it removes the protons that are produced stoichiometrically with lactate during glycolysis. The kinetics and substrate and inhibitor specificities of the transport process have been determined in cell suspensions using a radiotracer technique and in single cells using a fluorescent measurement of the decrease in intracellular pH that accompanies transport. The results of these experiments suggest the presence of 2 different transporter isoforms in heart cells, at least one of which is different from the cloned MCT1 and MCT2. Immunofluorescence microscopy shows that MCT1 expression is restricted to the intercalated disk region, yet the rate of lactate transport in this region is slower than in the center of the cell, where there is no MCT1. New cDNA sequences with strong homology to MCT1 have been found in human cDNA libraries and Northern blots show that the corresponding mRNA is expressed in rat heart. Expressions of these new MCT isoforms have yet to be demonstrated and their properties and cellular distribution defined.

Animals↗

PubMed1997

Interaction of the erythrocyte lactate transporter (monocarboxylate transporter 1) with an integral 70-kDa membrane glycoprotein of the immunoglobulin superfamily.

Treatment of intact erythrocytes with 4,4'-diisothiocyanostilbene-2, 2'-disulfonate (DIDS) causes irreversible inhibition and chemical labeling of the lactate transporter, monocarboxylate transporter 1 (MCT1) (Poole, R. C., and Halestrap, A. P. (1992) Biochem. J. 283, 855-862). In rat erythrocytes DIDS also causes cross-linking of MCT1 to another protein in the membrane to give a product of 130 kDa on SDS-polyacrylamide gel electrophoresis. Cross-linking is markedly reduced by those compounds that protect against irreversible inhibition of lactate transport by DIDS and enhanced by imposition of a pH gradient across the plasma membrane to recruit the substrate binding site of MCT1 to an exofacial conformation. These data indicate that DIDS cross-linking is via the same site on MCT1 as is responsible for inhibition of transport. Antibodies raised against the cross-linked conjugate react with proteins of approximately 40 kDa (MCT1) and 70 kDa on Western blots of erythrocyte membranes and an additional band of 130 kDa after treatment of erythrocytes with 100 microM DIDS. The 70-kDa protein that is cross-linked to MCT1 was purified and shown to contain N-linked carbohydrate; the apparent core molecular mass is 40 kDa. Amino acid sequencing showed that the protein is the rat equivalent of the membrane-spanning mouse teratocarcinoma glycoprotein GP-70, a member of the immunoglobulin superfamily related to basigin (Ozawa, M., Huang, R. P., Furukawa, T. , and Muramatsu, T. (1988) J. Biol. Chem. 263, 3059-3062). Possible implications of the specific interaction between MCT1 and this protein are discussed.

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

PubMed1997

Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation.

The cDNA for the monocarboxylate transporter MCT2 from rat testis has been cloned and sequenced. The derived protein sequence shows 82% identity with that from hamster. Rat MCT2 has a relative insertion of five amino acids in the N-terminal sequence preceding the first predicted transmembrane segment. MCT2 appears to be less highly conserved between species than MCT1. Using Northern blotting of RNA from rat and mouse tissues, MCT2 message was demonstrated to be abundant in the testis where a smaller, less abundant MCT2 transcript was also present. Low levels of a slightly different-sized transcript were found in rat and mouse liver, and mouse kidney. In hamster, only one-size transcript was detected at relatively high abundance in all the tissues examined. Antibodies were raised against a peptide derived from the extreme C-terminus of rat MCT2, and Western blotting with these detected MCT2 in membrane fractions prepared from rat testis, liver and brain but not those from heart or skeletal muscle. In hamster, MCT2 was detected in liver, heart and testis but not in brain [Garcia, Brown, Pathak, and Goldstein (1995) J. Biol. Chem. 270, 1843-1849]. For both rat MCT1 and MCT2 there were marked differences between the relative abundance of their respective messages and the amount of protein in membrane fractions from different tissues. This suggests that expression of both of these transporters in different tissues may be species-specific and regulated post-transcriptionally. The different-sized MCT2 transcripts may arise from alternative splicing. Starvation of rats for up to 48 h did not lead to any change in MCT1 or MCT2 expression in the liver, as determined by either Northern or Western blotting.

Amino Acid Sequence↗

PubMed1997

cDNA cloning of rat mitochondrial cyclophilin.

Human cyclophilin-3 (hCyp-3) cDNA was used to screen a rat skeletal muscle cDNA library which led to the isolation of a full-length cDNA encoding the rat homologue. The derived amino acid sequence showed 89.5% identity with the human sequence, with major differences being restricted to the mitochondrial targeting sequence. The N-terminal sequence of the purified rat liver mitochondrial cyclophilin (CyP-D) corresponded to that derived from the cDNA following 30 amino acids of targeting sequence. This confirms that hCyp-3 encodes mitochondrial matrix CyP (CyP-D), which plays a crucial role in the mitochondrial permeability transition. CyP-D mRNA of a single sized (1.5 kb) was shown by Northern blotting to be present in liver, heart, skeletal muscle and brain.

Amino Acid Isomerases↗

PubMed1997

Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase.

Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 microM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 microM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 microM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.

Adenosine Diphosphate↗

PubMed1997

Butyrate can act as a stimulator of growth or inducer of apoptosis in human colonic epithelial cell lines depending on the presence of alternative energy sources.

In vivo, butyrate is a major energy source for the colonic epithelium and is thought to stimulate proliferation. In contrast, butyrate in vitro has been shown to inhibit proliferation and induce differentiation and apoptosis in colonic epithelial cells. Most colon cell cultures are grown in medium containing high concentrations of glucose, whereas in vivo, the main energy source used by the colon cells is butyrate. The aim of this study was to determine whether the apparent contrasting roles of butyrate in vivo and in vitro could be as a consequence of differences in glucose availability. The sensitivity of two human colorectal tumour cell lines, one adenoma (S/RG/C2) and one carcinoma (HT29) to butyrate-induced growth inhibition and apoptosis was investigated to determine whether these cellular effects were altered under glucose depleted culture conditions. Glucose depletion resulted in increased apoptosis in both cell lines in the absence of butyrate. Butyrate in standard culture conditions (containing 25 mM glucose and 1 mM pyruvate) inhibited growth and induced apoptosis in both cell lines. However, low concentrations of butyrate in glucose depleted culture conditions (i.e. standard growth medium without glucose and pyruvate supplements) were found to reduce apoptosis induced by glucose deprivation and increase cell yield in both cell lines. The results show that in glucose depleted culture conditions, butyrate at low concentrations (0.5 mM for S/RG/C2, and 0.5 and 2 mM for HT29 cells) was found to be growth stimulatory whereas in the presence of glucose, these same concentrations of butyrate induced apoptosis. Thus, whether butyrate is growth stimulatory or growth inhibitory may depend on the availability of other energy sources. These observations may, in part, provide an explanation for the apparent opposite effects of butyrate on proliferation reported in vivo and in vitro.

Adenoma↗