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Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial. There have been a number of studies suggesting that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen et al. provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of 3-day old Caenorhabditis elegans correlated inversely with lifespan. The interpretation of ‘mitoflashes’ as ‘bursts of superoxide’ radicals assumes that circularly permuted yellow fluorescent protein (cpYFP) is a reliable indicator of mitochondrial superoxide. This interpretation has been criticised because experiments and theoretical considerations both show that changes in cpYFP fluorescence are due to alterations in pH, not superoxide-. We now provide direct evidence that purified cpYFP is completely unresponsive to superoxide. Therefore ‘mitoflashes’ do not reflect superoxide generation and are not evidence for a link between mitochondrial free radical dynamics and lifespan.

A novel disease mechanism based on the failure of cell-specific transcriptional silencing of a gene that is highly expressed in other tissues is described.

Urocortin (UCN) protects hearts against ischemia and reperfusion injury whether given before ischemia or at reperfusion. Here we investigate the roles of PKC, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute UCN treatment improved hemodynamic recovery during reperfusion after 30 min of global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of 2-[[sup.3]H]deoxyglucose). UCN pretreatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that UCN decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 h) treatment with UCN reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells that had been pretreated with UCN, suggesting that this is the mechanism by which UCN desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKC-[epsilon] or PKC-[alpha] translocate to the mitochondria following acute UCN treatment, inhibition of PKC with chelerythrine eliminated the effect of UCN on oxidative stress. Our data suggest that acute UCN treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress. mitochondria; oxygen radicals; peptide hormones

Intense exercise training can increase lactate/H+ transport capacity in human skeletal muscle and improve the muscle's ability to release lactate and H+ during contractions. This was found in a study aimed at examining whether high-intensity exercise training can induce changes in muscle lactate/H+ transport capacity and lactate/H+ transporter proteins in humans. The increase in the sarcolemmal lactate/H+ transport capacity and an enhanced content of the monocarboxylate transporters MCT1 and MCT4 in human skeletal muscle were associated with similar release rates of lactate and H+ during intense exercise before and after training.

The recovery of Langendorff-perfused rat hearts from ischemia was demonstrated to be accompanied by reversal of the mitochondrial permeability transition (MPT) through the use of mitochondrial entrapment of 2-deoxy-D[3H]glucose. The presence of pyruvate increased tissue lactate content at the end of ischemia and decreased effluent pH during the initial phase of reperfusion concomitant with an increase in lactate output. This suggests that pyruvate may inhibit the MPT by decreasing internal acidity and scavenging free radicals to effectively protect the heart.

Research was conducted to examine whether chronic stimulation of red and white rat muscles increased the concentrations of the monocarboxylate transporter MCT1. Red and white tibialis anterior (RTA and WTA, respectively) and extensor digitorum longus (EDL) muscles were chronically stimulated via the peroneal nerve for 7 days. Stimulated and contralateral muscles were analyzed for MCTI content, L-lactate uptake, lactate dehydrogenase (LDH) isoforms and muscle fiber composition. Results show that chronic muscle contraction increases MCT1 in white and red skeletal muscles.

The skeletal muscles are the major site of lactate formation. Transport of lactate in and out of these muscles are carried out by lactate transporters such as the monocarboxylate transporter (MCT1). To characterize MCT1, an antibody was constructed against the MCT1 protein and was analyzed for its expression. Results showed higher MCT1 expression in red than in white muscles. Expression was correlated with oxidative fiber content and lactate uptake by the muscles. Lactate dehydrogenase activity in the heart was also closely correlated with MCT1, suggesting that MCT1 may be expressed with heart forms of LDH.

The detection of the bound mitochondrial cyclophilin (CyP) by Western blotting is very much improved by fixing the CyP to the blotting membrane with glutaraldehyde. The sensitivity of Ca2+ of the mitochondrial permeability transition pore (MTP) in deenergized mitochondria was vastly improved in KSCN medium in contrast with the KCl medium. Binding of CyP to the mitochondrial membrane was enhanced by treatment with tert-butylhydroperoxide, phenylarsine oxide, diamide and by hypoosmotic KCl medium.

Kinetic studies using the intracellular pH (pHi)-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescin help determine the rate of sarcolemmal lactate transport in single cardiac myocytes. Measurement of the rate of pHi recovery upon removing external lactate helps determine the kinetics of lactate efflux that exhibits a higher Michaelis constant and maximal rate than influx. Sarcolemmal lactate carrier limits the rate of extrusion of lactate under hypoxic conditions and two different lactate carriers coexist in the same cell.

The physiologic transport of lactate and other monocarboxylates were reviewed by studying human erythrocytes. This biologic transport process is mainly performed by an H+-monocarboxylate cotransporter which is highly specific for lactate. The carrier mechanism is shown to be inhibited by stilbene disulfonates and alpha-cyanocinnamate derivatives. It is also responsible for the transport of other aliphatic monocarboxylates and is likewise present in other mammalian tissues and organisms. This transport system is vital for cellular metabolism.