Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis.

AIMS/HYPOTHESIS: ATP-sensitive K(+) (K(ATP)) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca(2+) concentration or pH. METHODS: To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca(2+) were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin. RESULTS: SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca(2+) uptake into mitochondria, and K(ATP) channel regulators had no significant effect on intravesicular free Ca(2+) concentrations or vesicular pH. CONCLUSIONS/INTERPRETATION: A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.

Calcium, mitochondria and reperfusion injury: a pore way to die.

When mitochondria are exposed to high Ca2+ concentrations, especially when accompanied by oxidative stress and adenine nucleotide depletion, they undergo massive swelling and become uncoupled. This occurs as a result of the opening of a non-specific pore in the inner mitochondrial membrane, known as the MPTP (mitochondrial permeability transition pore). If the pore remains open, cells cannot maintain their ATP levels and this will lead to cell death by necrosis. This article briefly reviews what is known of the molecular mechanism of the MPTP and its role in causing the necrotic cell death of the heart and brain that occurs during reperfusion after a long period of ischaemia. Such reperfusion injury is a major problem during cardiac surgery and in the treatment of coronary thrombosis and stroke. Prevention of MPTP opening either directly, using agents such as cyclosporin A, or indirectly by reducing oxidative stress or Ca2+ overload, provides a protective strategy against reperfusion injury. Furthermore, mice in which a component of the MPTP, CyP-D (cyclophilin D), has been knocked out are protected against heart and brain ischaemia/reperfusion. When cells experience a less severe insult, the MPTP may open transiently. The resulting mitochondrial swelling may be sufficient to cause release of cytochrome c and activation of the apoptotic pathway rather than necrosis. However, the CyP-D-knockout mice develop normally and show no protection against a range of apoptotic stimuli, suggesting that the MPTP does not play a role in most forms of apoptosis.

Cross-reinnervation changes the expression patterns of the monocarboxylate transporters 1 and 4: An experimental study in slow and fast rat skeletal muscle.

The monocarboxylate transporters 1 and 4 are expressed in brain as well as in skeletal muscle and play important roles in the energy metabolism of both tissues. In brain, monocarboxylate transporter 1 occurs in astrocytes, ependymocytes, and endothelial cells while monocarboxylate transporter 4 appears to be restricted to astrocytes. In muscle, monocarboxylate transporter 1 is enriched in oxidative muscle fibers whereas monocarboxylate transporter 4 is expressed in all fibers, with the lowest levels in oxidative fiber types. The mechanisms regulating monocarboxylate transporter 1 and monocarboxylate transporter 4 expression are not known. We hypothesized that the expression of these transporters would be sensitive to long term changes in metabolic activity level. This hypothesis can be tested in rat skeletal muscle, where permanent changes in activity level can be induced by cross-reinnervation. We transplanted motor axons originally innervating the fast-twitch extensor digitorum longus muscle to the slow-twitch soleus muscle and vice versa. Four months later, microscopic analysis revealed transformation of muscle fiber types in the cross-reinnervated muscles. Western blot analysis showed that monocarboxylate transporter 1 was increased by 140% in extensor digitorum longus muscle and decreased by 30% in soleus muscle after cross-reinnervation. In contrast, cross-reinnervation induced a 62% decrease of monocarboxylate transporter 4 in extensor digitorum longus muscle and a 1300% increase in soleus muscle. Our findings show that cross-reinnervation causes pronounced changes in the expression levels of monocarboxylate transporter 1 and monocarboxylate transporter 4, probably as a direct consequence of the new pattern of nerve impulses. The data indicate that the mode of innervation dictates the expression of monocarboxylate transporter proteins in the target cells and that the change in monocarboxylate transporter isoform profile is an integral part of the muscle fiber transformation that occurs after cross-reinnervation. Our findings support the hypothesis that the expression of monocarboxylate transporter 1 and monocarboxylate transporter 4 in excitable tissues is regulated by activity.

Highly differential expression of the monocarboxylate transporters MCT2 and MCT4 in the developing rat brain.

Monocarboxylate transporters (MCTs) play an important role in the metabolism of all cells. They mediate the transport of lactate and pyruvate but also some other substrates such as ketone bodies. It has been proposed that glial cells release monocarboxylates to fuel neighbouring neurons. A key element in this hypothesis is the existence of neuronal MCTs. Amongst the three MCTs known to be expressed in the brain (MCT1, 2 and 4) only MCT2 has been found in neurons. Here we have studied the expression pattern of MCT2 during postnatal development. By use of immunoperoxidase and double immunofluorescence microscopy we report that neuronal MCT2 occurs in most brain areas, including the hippocampus and cerebellum, from birth to adult. MCT2 is also expressed in specific subpopulations of astrocytes. Neuronal MCT2 is most abundant in the first 3 postnatal weeks and thereafter decreases toward adulthood. In contrast to MCT2, MCT4 is exclusively present in astroglia during all stages of development. Furthermore, MCT4 expression is very low at birth and reaches adult level by P14. Our results are consistent with previous data suggesting that in the immature brain much of the energy demand is met by monocarboxylates and ketone bodies.

Increases in muscle MCT are associated with reductions in muscle lactate after a single exercise session in humans.

To investigate the effects of a single session of prolonged cycle exercise [60% peak O2 uptake (VO2 peak) for 5-6 h] on metabolic adaptations in working vastus lateralis muscle, nine untrained males (peak O2 uptake = 47.2 +/- 1.1 ml x kg(-1) x min(-1), means +/- SE) were examined before (Pre) and at 2 (Post-2), 4 (Post-4), and 6 (Post-6) days after the training session. On the basis of 15 min of cycle exercise at 59% VO2 peak, it was found that training reduced (P < 0.05) exercise muscle lactate (mM) at Post-2 (6.65 +/- 0.69), Post-4 (7.74 +/- 0.63), and Post-6 (7.78 +/- 1.2) compared with Pre (10.9 +/- 1.3). No effect of training was observed on exercise ATP, phosphocreatine, and glycogen levels. After the single session of training, plasma volumes were elevated (P < 0.05) at Post-2 (6.7 +/- 1.7%), Post-4 (5.86 +/- 1.9), and Post-6 (5.13 +/- 2.5). The single exercise session also resulted in elevations (P < 0.05) in the monocarboxylate transporters MCT1 and MCT4 throughout the 6 days after exercise. Although epinephrine and norepinephrine both increased with exercise, only norepinephrine was reduced (P < 0.05) with training and only at Post-4. These results indicate that regulation of cellular lactate levels occurs rapidly and independently of other metabolic adaptations. It is proposed that increases in MCT and plasma volume are at least partly involved in the lower muscle lactate content observed after the training session by increasing lactate membrane transport and removal, respectively.

Tumour necrosis factor-alpha activation of protein kinase B in WEHI-164 cells is accompanied by increased phosphorylation of Ser473, but not Thr308.

Tumour necrosis factor-alpha (TNF-alpha) may activate both cell survival and cell death pathways. In the murine fibrosarcoma cell line WEHI-164, physiological concentrations (1 ng/ml) of TNF-alpha induced wortmannin-sensitive cell ruffling characteristic of the phosphoinositide 3-kinase (PI3-kinase) activation associated with cell survival. Wortmannin also enhanced cell death induced by TNF-alpha in the presence of actinomycin D, confirming that TNF-alpha activates a transcription-independent survival pathway requiring PI3-kinase activity. Both TNF-alpha and insulin-like growth factor 1 (IGF-1) caused a 6-10-fold wortmannin-sensitive increase in protein kinase B (PKB) activity within 5 min. For IGF-1, this was associated with an increase in phosphorylation of both Thr(308) and Ser(473), whereas for TNF-alpha only phosphorylation of Ser(473) was increased, even in the presence of okadaic acid to inhibit protein phosphatases 1 and 2A. TNF-alpha did not decrease the phosphorylation of Thr(308) induced by IGF-1, implying that TNF-alpha neither inhibits phosphoinositide-dependent kinase 1 (PDK1) nor activates an opposing phosphatase. In WEHI cells overexpressing a form of PKB, IGF-1 increased phosphorylation of Ser(473) on PKB, but not its kinase activity, whereas TNF-alpha failed to induce Ser(473) phosphorylation or kinase activation of either overexpressed T308A or wild-type PKB (where T308A is the mutant bearing the substitution Thr(308)-->A). IGF-1 caused translocation of green-fluorescent-protein-tagged ADP-ribosylation factor nucleotide-binding site opener (ARNO) to the plasma membrane of WEHI cells, but this was not detected with TNF-alpha. We conclude that, at physiological concentrations, TNF-alpha activates endogenous PKB by stimulating PDK2 (increase in Ser(473) phosphorylation) in a PI3-kinase-dependent (wortmannin-sensitive) manner, without causing detectable stimulation of PDK1 (no increase in Thr(308) phosphorylation) or ARNO translocation. Possible explanations of these observations are discussed.

Upregulation of the cardiac monocarboxylate transporter MCT1 in a rat model of congestive heart failure.

BACKGROUND: Cardiac metabolism becomes more dependent on carbohydrates in congestive heart failure (CHF), and lactate may be used as an important respiratory substrate. Monocarboxylate transporter 1 (MCT1) promotes cotransport of lactate and protons into and out of heart cells and conceivably flux of lactate between cells, because it is abundantly present in the intercalated disk. METHODS AND RESULTS: Six weeks after induction of myocardial infarction (MI) in Wistar rats, left ventricular end-diastolic pressures were >15 mm Hg, signifying CHF. MCT1 and connexin43 protein levels in CHF were 260% and 20%, respectively, of those in sham-operated animals (Sham), and the corresponding mRNA signals were 181% and not significantly changed, respectively. Confocal laserscan immunohistochemistry and quantitative immunogold cytochemistry showed that MCT1 density was much higher in CHF than in Sham both at the surface membrane and in the intercalated disk. In CHF, a novel intracellular pool of MCT1 appeared to be associated with cisternae, some close to the T tubules. In contrast, connexin43 particles, seen exclusively at gap junctions, were substantially fewer. Maximum lactate uptake was 107+/-15 mmol. L(-1). min(-1) in CHF and 42+/-6 mmol. L(-1). min(-1) in Sham cells (P<0.05). The K(m) values were between 7 and 9 mmol/L (P=NS). CONCLUSIONS: In cardiomyocytes from CHF rats, (1) the amount of functional MCT1 in the sarcolemma, including in the intercalated disk, is increased several-fold; (2) a new intracellular pool of MCT1 appears; (3) another disk protein, connexin43, is much reduced; and (4) increased reliance on lactate and other monocarboxylates (eg, pyruvate) could provide tight metabolic control of high-energy phosphates.

Mitochondria: a target for myocardial protection.

The ischemic heart requires reperfusion using clinical interventions, such as coronary artery bypass graft surgery, in order to recover. Despite recent developments in myocardial protection techniques, reperfusion damage still occurs, and significant morbidity remains a problem. Therefore, the search continues for techniques that will limit myocardial damage and that will enhance recovery upon reperfusion. Mitochondria are known to be intimately involved in the processes that lead to cell death following reperfusion, in both necrotic and apoptotic forms of cell death, and so are potential targets for protective intervention. In this review, we consider several aspects of mitochondrial function that we believe to be possible targets for myocardial protection; namely, mitochondrial Ca(2+) transport, the permeability transition pore, and improved mitochondrial substrate supply. We discuss work by ourselves and others in these areas, and also consider the recently proposed role of mitochondrial ATP-dependent K(+) channels in mediating myocardial protection by ischemic preconditioning. Finally, we describe use of cardioplegic solutions in the clinical setting, and discuss how improved understanding of the aspects of mitochondrial function summarised above may lead to better protective strategies in the future.

A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with delta-glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses.

Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber-Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the delta2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.

Expression and distribution of lactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.

Transport of lactate across the plasma membrane of pancreatic islet beta-cells is slow, as described by Sekine et al. (J Biol Chem 269:4895-4902, 1994), which is a feature that may be important for normal nutrient-induced insulin secretion. Although eight members of the monocarboxylate transporter (MCT) family have now been identified, the expression of these isoforms within the exocrine and endocrine pancreas has not been explored in detail. Using immunocytochemical analysis of pancreatic sections fixed in situ, we demonstrated three phenomena. First, immunoreactivity of the commonly expressed lactate transporter isoform MCT1 is near zero in both alpha- and beta-cells but is abundant in the pancreatic acinar cell plasma membrane. No MCT2 or MCT4 was detected in any pancreatic cell type. Second, Western analysis of purified beta- and non-beta-cell membranes revealed undetectable levels of MCT1 and MCT4. In derived beta-cell lines, MCT1 was absent from MIN6 cells and present in low amounts in INS-1 cell membranes and at high levels in RINm5F cells. MCT4 was weakly expressed in MIN6 beta-cells. Third, CD147, an MCT-associated chaperone protein, which is closely colocalized with MCT1 on acinar cell membranes, was absent from islet cell membranes. CD147 was also largely absent from MIN6 and INS-1 cells but abundant in RINm5F cells. Low expression of MCT1, MCT2, and MCT4 contributes to the enzymatic configuration of beta-cells, which is poised to ensure glucose oxidation and the generation of metabolic signals and may also be important for glucose sensing in islet non-beta-cells. MCT overexpression throughout the islet could contribute to deranged hormone secretion in some forms of type 2 diabetes.

Characterisation of human monocarboxylate transporter 4 substantiates its role in lactic acid efflux from skeletal muscle.

Monocarboxylate transporter (MCT) 4 is the major monocarboxylate transporter isoform present in white skeletal muscle and is responsible for the efflux of lactic acid produced by glycolysis. Here we report the characterisation of MCT4 expressed in Xenopus oocytes. The protein was correctly targeted to the plasma membrane and rates of substrate transport were determined from the rate of intracellular acidification monitored with the pH-sensitive dye 2', 7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In order to validate the technique, the kinetics of monocarboxylate transport were measured in oocytes expressing MCT1. Km values determined for L-lactate, D-lactate and pyruvate of 4.4, > 60 and 2.1 mM, respectively, were similar to those determined previously in tumour cells. Comparison of the time course of [14C]lactate accumulation with the rate of intracellular acidification monitored with BCECF suggests that the latter reflects pH changes close to the plasma membrane associated with transport, whilst the former may include diffusion-limited movement of lactate into the bulk cytosol. Km values of MCT4 for these substrates were found to be 28, 519 and 153 mM, respectively, and for a range of other monocarboxylates values were at least an order of magnitude higher than for MCT1. Vmax values appeared to be similar for all substrates. K0.5 values of MCT4 (determined at 30 mM L-lactate) for inhibition by alpha-cyano-4-hydroxycinnamate (991 microM), phloretin (41 microM), 5-nitro-2-(3-phenylpropylamino)benzoate (240 microM), p-chloromercuribenzene sulphonate (21 microM) and 3-isobutyl-1-methylxanthine (970 microM, partial inhibition) were also substantially higher than for MCT1. No inhibition of MCT4 by 2 mM 4,4'-diisothiocyanostilbene-2,2'-disulphonate was observed. The properties of MCT4 are consistent with published data on giant sarcolemmal vesicles in which MCT4 is the dominant MCT isoform, and are appropriate for the proposed role of MCT4 in mediating the efflux from the cell of glycolytically derived lactic acid but not pyruvate.

Isoform-specific regulation of the lactate transporters MCT1 and MCT4 by contractile activity.

We examined the isoform-specific regulation of monocarboxylate transporter (MCT)1 and MCT4 expression by contractile activity in red and white tibialis anterior muscles. After 1 and 3 wk of chronic muscle stimulation (24 h/day), MCT1 protein expression was increased in the red muscles (+78%, P < 0.05). In the white muscles, MCT1 was increased after 1 wk (+191%) and then was decreased after 3 wk. In the red muscle, MCT1 mRNA accumulation was increased only after 3 wk (+21%; P < 0.05). In the white muscle, MCT1 mRNA was increased after 1 wk (+30%; P < 0.05) and 3 wk (+15%; P < 0.05). MCT4 protein was not altered in either the red or white muscles after 1 or 3 wk. MCT4 mRNA was transiently lowered (approximately 15%) in both muscles in the 1st wk, but MCT4 mRNA levels were back to control levels after 3 wk. In conclusion, chronic contractile activity induces the expression of MCT1 but not MCT4. This increase in MCT1 alone was sufficient to increase lactate uptake from the circulation.

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression.

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.

Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain.

Although metformin is widely used for the treatment of non-insulin-dependent diabetes, its mode of action remains unclear. Here we provide evidence that its primary site of action is through a direct inhibition of complex 1 of the respiratory chain. Metformin(50 microM) inhibited mitochondrial oxidation of glutamate+malate in hepatoma cells by 13 and 30% after 24 and 60 h exposure respectively, but succinate oxidation was unaffected. Metformin also caused time-dependent inhibition of complex 1 in isolated mitochondria, whereas in sub-mitochondrial particles inhibition was immediate but required very high metformin concentrations (K(0.5),79 mM). These data are compatible with the slow membrane-potential-driven accumulation of the positively charged drug within the mitochondrial matrix leading to inhibition of complex 1. Metformin inhibition of gluconeogenesis from L-lactate in isolated rat hepatocytes was also time- and concentration-dependent, and accompanied by changes in metabolite levels similar to those induced by other inhibitors of gluconeogenesis acting on complex 1. Freeze-clamped livers from metformin-treated rats exhibited similar changes in metabolite concentrations. We conclude that the drug's pharmacological effects are mediated, at least in part, through a time-dependent, self-limiting inhibition of the respiratory chain that restrains hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues. Lactic acidosis, an occasional side effect, canal so be explained in this way.

Abundance and subcellular distribution of MCT1 and MCT4 in heart and fast-twitch skeletal muscles.

The expression of two monocarboxylate transporters (MCTs) was examined in muscle and heart. MCT1 and MCT4 proteins are coexpressed in rat skeletal muscles, but only MCT1 is expressed in rat hearts. Among six rat fast-twitch muscles (red and white gastrocnemius, plantaris, extensor digitorum longus, red and white tibialis anterior) there was an inverse relationship between MCT1 and MCT4 (r = -0.94). MCT1 protein was correlated with MCT1 mRNA (r = 0.94). There was no relationship between MCT4 mRNA and MCT4 protein. MCT1 (r = -0.97) and MCT4 (r = 0.88) protein contents were correlated with percent fast-twitch glycolytic fiber. When normalized for their mRNAs, MCT1 but not MCT4 was still correlated with the percent fast-twitch glycolytic fiber composition of rat muscles (r = -0.98). MCT1 and MCT4 were also measured in plasma membranes (PM), triads (TR), T tubules (TT), sarcoplasmic reticulum (SR), and intracellular membranes (IM). There was an intracellular pool of MCT4 but not of MCT1. The MCT1 subcellular distribution was as follows: PM (100%) > TR (31.6%) > SR (15%) = TT (14%) > IM (1.7%). The MCT4 subcellular distribution was considerably different [PM (100%) > TR (66.5%) > TT (36%) = SR (43%) > IM (24%)]. These studies have shown that 1) the mechanisms regulating the expression of MCT1 (transcriptional and posttranscriptional) and MCT4 (posttranscriptional) are different and 2) differences in MCT1 and MCT4 expression among muscles, as well as in their subcellular locations, suggest that they may have different roles in muscle.

Mitochondria and cell death.

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.

Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites.

Percoll-purified rat liver mitochondria were shown to contain BAX dimer and rapidly (<2 min) release 5-10% of their cytochrome c when incubated in a standard KCl incubation medium under energized conditions. This release was not accompanied by release of adenylate kinase (AK), another intermembrane protein, and was not inhibited by Mg(2+), dATP, inhibitors of the permeability transition or ligands of the peripheral benzodiazepine receptor. However, release was greatly reduced by the presence of 5% (w/v) dextran (40 kDa), which caused a decrease in the light scattering (A(520)) of mitochondrial suspensions. Dextran also inhibited both mitochondrial oxidation of exogenous ferrocytochrome c in the presence of rotenone and antimycin, and respiratory-chain-driven reduction of exogenous ferricytochrome c. Hypo-osmotic medium or digitonin treatment of mitochondria caused a large additional release of both cytochrome c and AK that was not blocked by dextran. Polyaspartate, which stabilizes the low conductance state of the voltage-dependent anion channel (VDAC), increased cytochrome c release. VDAC and BAX are both found at the contact sites between the inner and outer membranes and dextran is known to stabilize these contact sites in isolated mitochondria. Thus our data suggest that regulation of a specific permeability pathway for cytochrome c may be mediated by changes in protein-protein interactions within contact sites. The adenine nucleotide translocase is known to bind to VDAC and thus provides an additional link between the specific cytochrome c release pathway and the permeability transition.

Expression of the lactate transporter MCT1 in macrophages.

This study evaluated macrophage expression of the stereospecific lactate transporter, MCT1, and the effects of lipopolysaccharide (LPS), tumor necrosis factor (TNFa), or nitric oxide (NO) on MCT1 mRNA and protein levels and lactate transporter activity. Peritoneal and J774.1 macrophages were treated with either saline, LPS (10 or 100 ng/mL), dexamethasone (100 nM), and dexamethasone + LPS. Cells were harvested at 4, 8, and 16 h after treatment and processed for total RNA and protein isolation. LPS treatment significantly increased macrophage MCT1 mRNA expression at 4 and 8 h compared with the saline-treated cells. Dexamethasone did not alter MCT1 mRNA levels. Treatment of J774.1 macrophages with TNFalpha (1 ng/mL) or nitric oxide (DETA-NO, 100 microM) also significantly increased MCT1 mRNA levels at 4 and 8 h after treatment. LPS and TNFalpha treatment significantly increased MCT1 protein levels at 8 and 16 h after treatment. Lactate transporter activity in J774.1 macrophages was measured by uptake of 14C-labeled lactate. LPS and TNFalpha treatment significantly augmented lactate uptake, 12 h after administration; however, nitric oxide treatment did not affect lactate uptake. Thus, our data demonstrated that stimulated peritoneal and J774.1 macrophages express the lactate transporter, MCT, and that LPS and TNFalpha regulate MCT1 mRNA and protein levels.