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Pyruvate oxidation was measured in cardiac mitochondria from euthyroid and hypothyroid rats fed diets enriched with either omega-6 or omega-3 fatty acids. Both State 4 and State 3 rates of pyruvate-dependent respiration were markedly reduced in hypothyroid mitochondria, regardless of diet consumed, compared to euthyroid controls. Respiratory control ratios and ADP/O ratios were the same under all treatments. While there was no significant effect of diet on respiration in euthyroid mitochondria, pyruvate oxidation was 28% higher in hypothyroid mitochondria from animals fed the omega-3 diet compared to those fed the omega-6 diet. Depressed respiration in the hypothyroid state was correlated with 18% more phosphatidylcholine in the inner mitochondrial membrane whereas phosphatidylethanolamine was 17% lower and cardiolipin 32% lower compared to controls. The total phospholipid fatty acid composition was not affected by the hypothyroid state. However, enhanced respiration in hypothyroid animals fed the omega-3 diet was associated with a 3-fold increase in monounsaturated fatty acids in the cardiolipin fraction, and a 12-fold increase in omega-3 fatty acids, primarily 22:5(omega-3) and 22:6(omega-3). The data suggest that membrane levels of cardiolipin and its omega-3 fatty acid content modulate pyruvate transport in hypothyroid mitochondria.

Myocardial pyruvate oxidation is work- or calcium-load-related, but control of pyruvate dehydrogenase (PDH) by the specific mitochondrial pyruvate transporter has also been proposed. To test the transport hypothesis distribution of pyruvate across the cell membrane as well as rates of mitochondrial pyruvate net transport plus oxidation were examined in isolated perfused but stable and physiologically working guinea-pig hearts. 150 microM-1.2 mM alpha-cyanohydroxycinnamate proved to specifically block mitochondrial pyruvate uptake in these hearts. When perfusate glucose as cytosolic pyruvate precursor was supplied in combination with octanoate (0.2 or 0.5 mM) as diffusible alternative fatty acid substrate, alpha-cyanohydroxycinnamate produced up to 20- and 3-fold increases in pyruvate and lactate efflux, respectively. Cinnamates did not alter myocardial hemodynamics nor sarcolemmal pyruvate and lactate export. In contrast the tested concentrations of cinnamate produced reversible, dose-dependent decreases in 14CO2 production from [1-14C]pyruvate or [U-14C]glucose by inhibiting mitochondrial pyruvate uptake. Linear least-squares estimates of available cinnamate-sensitive total pyruvate transport potential yielded rates close to 110 mumol/min per g dry mass at S0.5 approximately 120 microM, which compared reasonably well with literature values from isolated cardiac mitochondria. This transport potential was severalfold larger than total extractable myocardial PDH activity of approximately 32 mumol/min per g dry mass at 37 degrees C. Even when cytosolic pyruvate levels were in the lower physiologic range of about 90 microM, pyruvate oxidation readily kept pace with mitochondrial respiration over a wide range of workload and inotropism. Furthermore, dichloroacetate, a selective activator of PDH, stimulated pyruvate oxidation without affecting myocardial O2 consumption, regardless of the metabolic or inotropic state of the hearts. Consequently, little or no regulatory function with regard to pyruvate oxidation could be assigned to the native mitochondrial pyruvate carrier of the working heart. Therefore, mitochondrial pyruvate-H+ symport was the normal, highly efficient (rather than controlling) mechanism for pyruvate entry into the mitochondria where PDH regulation controlled pyruvate oxidation.

1. 1. A method has been developed for measuring the facilitated diffusion of pyruvate anions across the isolated human erythrocyte membrane. Ghosts were resealed in the presence of an excess of lactate dehydrogenase and NADH so that the influx of pyruvate could be followed by a continuous spectrophotometric recording of the oxidation of NADH (Rice, W.R. and Steck, T.L. (1976) Biochim. Biophys. Acta 433, 39–53). This approach has now been extended to sealed inside-out vesicles derived from the same membrane. 2. 2. Transport across inside-out and right-side-out membranes was similar in certain respects. In both cases, the influx of pyruvate was greatly stimulated by increasing the ionic strength in the external medium. Salts of transported anions (such as chloride) but not non-transported anions (such as malate or acetate) were inhibitory at high concentrations, presumably a competitive effect. The non-competitive inhibitors of anion flux, salicylate, 4-acetamido-4′-isothiocyanato-2,2′-stilbene disulfonate (SITS) and 1-fluoro-2,4-dinitrobenzene, reduced pyruvate transport from both sides of the membrane. 3. 3. Characteristic asymmetries in the transport of pyruvate were noted. Probenecid was found to act only at the cytoplasmic surface, although it inhibited flux in both directions. Mixtures of o- phenanthroline and CuSO 4 catalyzed the disulfide cross-linking of band 3, the purported anion transport protein, only at the membrane's cytoplasmic surface. This treatment stimulated pyruvate flux into resealed ghosts, but inhibited flux into inside-out vesicles. 4. 4. The binding of glyceraldehyde-3-phosphate dehydrogenase and aldolase to the cytoplasmic surface of the membrane did not significantly alter pyruvate flux in either direction, even though these enzymes are known to associate specifically with band 3. Proteolytic excision of the cytoplasmic domain of band 3, amounting to approximately 40% of its mass, also had no effect on pyruvate flux.

The effect of pH and substrates on the binding of radiolabelled alpha-cyanocinnamate to mitochondria and submitochondrial particles has been investigated. It has been found that the binding is strongly influenced by the pH of the medium (it decreases on increasing the pH of the medium). The inhibition of pyruvate oxidation by this inhibitor follows the same pH dependence. The pH affects only the affinity of the alpha-cyanocinnamate binding site without changing their total number. A similar pH dependence has been found in inside-out submitochondrial particles where the binding sites are directly accessible. The quantitative parameters of the binding of alpha-cyanocinnamate in submitochondrial particles have been determined. The binding can be prevented or displaced by pyruvate and other substrates of the carrier. The turnover number for pyruvate transport in rat-heart mitochondria has been determined.

1. 1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 °C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. 3. Differences between pyruvate, propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.

1. 1. Various methods to measure the rate of accumulation of [3- 14 C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane.

The characteristics of renal transport of pyruvate by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule were studied. It was found that the uptake of pyruvate in these vesicle preparations occurred by means of multiple transport systems. An electrogenic and Na+-requiring system confined to pars convoluta, exists for transport of pyruvate with an intermediate affinity, KA = 0.71 +/- 0.08 mM. In vesicles from pars recta, the uptake of pyruvate was mediated by a dual transport system with a high (KA1 = 0.30 +/- 0.05 mM) and low affinity (KA2 = 5.75 +/- 0.82 mM). The relation of these three pyruvate transport systems to the transport of other monocarboxylates and dicarboxylates was determined by examination of the inhibitory effect of L-lactate, beta-hydroxybutyrate and L-malate on the renal uptake of pyruvate. It was found that L-malate efficiently reduced the uptake of pyruvate by vesicles from pars convoluta, while addition of monocarboxylates (L-lactate, beta-hydroxybutyrate) has no effect on the transport of pyruvate in this region of proximal tubule. Furthermore, it was observed that Na+-dependent uptake of L-malate was specifically inhibited to about the same extent by 1 mM pyruvate. These findings strongly suggest that pyruvate and L-malate are taken up by the same transport system in vesicles from pars convoluta, which is different from that of L-lactate and beta-hydroxybutyrate. Experiments designed to investigate the substrate specificity for the high and low affinity system for pyruvate in pars recta, revealed that monocarboxylates (L-lactate, beta-hydroxybutyrate) substantially inhibit pyruvate transport at the low, but not the high substrate concentration, indicating that all monocarboxylates compete for the same high-affinity transport system. By contrast, L-malate at low concentrations preferentially inhibited the low-affinity system for pyruvate. This observation led us to suggest that L-malate and pyruvate share a common transport system in pars recta with low affinity for pyruvate and high affinity for L-malate.

The binding of alpha-cyanocinnamate to rat-heart mitochondrial membrane was investigated using alpha-cyano[14C]cinnamate. The binding was correlated to the inhibition of pyruvate transport. The results obtained demonstrate that both these functions reach saturation at the same titre of the inhibitor. Quantitative parameters of alpha-cyano[14C]cinnamate binding have been determined. The binding can be prevented by pyruvate and other substrates of the carrier but not by acetate. Pyruvate decreases the affinity of alpha-cyanocinnamate binding, leaving the maximum number of binding unchanged. It is concluded that rat-heart mitochondria contain a specific site at which alpha-cyanocinnamate binds which is directly involved in the inhibition of pyruvate transport.

Tumour-cell mitochondria contain a pyruvate-transporting system exhibiting the same general properties as those described in rat liver mitochondria. The Km for net pyruvate uptake in tumour-cell mitochondria is practically similar to that measured in rat liver mitochondria but the V is lower. This difference is also shown by swelling experiments. The possible implication of these observations in the context of lactate accumulation in tumour-cell is discussed.

1. (1) The kinetics and substrate specificity of the pyruvate translocator in rat liver mitochondria have been studied by both influx and efflux experiments. 2. (2) Pre-exposure of mitochondria to unlabelled pyruvate or 2-oxobutyrate caused marked decrease of the K m of pyruvate uptake but no change in the V . 3. (3) Mitochondrial [ 14 C]pyruvate exchanged with saturation kinetic with specific oxomonocarboxylates. The kinetic parameters of the various [ 14 C]pyruvate-monocarboxylate exchanges were determined. All the reactions exhibited the same V value but different K m values. 4. (4) The initial rate of [ 14 C]pyruvate uptake by mitochondria was inhibited by the presence in the external medium of the oxomonocarboxylates which in the efflux experiments were found to exchange with mitochondrial [ 14 C]pyruvate. Dicarboxylates had, on the other hand, no effect on pyruvate transport. The inhibition of pyruvate uptake by oxomonocarboxylates was of competitive type. The K i values for the various monocarboxylic oxoacids competing for pyruvate uptake were determined and found to be practically equal to the K m values measured in the exchange experiments. 5. (5) Besides oxomonocarboxylates, the initial rate of pyruvate uptake was inhibited by various halogenated monocarboxylic acids. Also these acids behaved as purely competitive inhibitors of pyruvate uptake. 6. (6) The nature of the inhibition of pyruvate transport by α-cyanocinnamate has been studied. α-Cyanocinnamate acted as a purely competitive inhibitor of pyruvate uptake by both unloaded and 2-oxobutyrate preloaded mitochondria. 7. (7) The results presented provide evidence for the existence in the pyruvate translocator of a single substrate-binding site. On the basis of the structural requirements of the binding site a possible chemical mechanism for the interaction between the substrate and the functional groups of the translocator is proposed. The mechanism of the inhibition of pyruvate transport by α-cyanocinnamate and other SH-reagents is discussed in terms of two types of functional thiol groups in the translocator.