Robert C. Poole

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.

Cloning and sequencing of the monocarboxylate transporter from mouse Ehrlich Lettré tumour cell confirms its identity as MCT1 and demonstrates that glycosylation is not required for MCT1 function

Lactate transport is mediated in most tissues by H+-monocarboxylate-- cotransporters (MCTs). We have cloned and sequenced the lactate transporter from Ehrlich Lettré tumour cells by using the polymerase chain reaction (PCR) to amplify MCT1-related sequence from cDNA. The sequence is 93% and 87% identical to MCT1 from Chinese hamster and human respectively and so represents mouse MCT1. Most differences between MCT1 from Chinese hamster and mouse are conservative substitutions, located in hydrophilic parts of the molecule. Specific antipeptide antibodies confirm the presence of MCT1 protein in membranes from Ehrlich Lettré tumour cells. One difference between the mouse and Chinese hamster MCT1 is the absence of a predicted external consensus sequence for N-linked glycosylation in the mouse sequence. Using N-glycanase-F treatment and an in vitro translation system, we provide evidence that this glycosylation site is not actually utilised in Chinese hamster MCT1. These results are discussed in relation to current understanding of the roles of glycosylation of membrane proteins.

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 α‐cyanoeinnamamides (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.

Derivatives of cinnamic acid interact with the nucleotide binding site of mitochondrial aldehyde dehydrogenase: Effects on the dehydrogenase reaction and stimulation of esterase activity by nucleotides

A wide variety of cinnamic acid derivatives are inhibitors of the low Km mitochondrial aldehyde dehydrogenase. Two of the most potent inhibitors are alpha-cyano-3,4-dihydroxythiocinnamamide (Ki0.6 microM) and alpha-cyano-3,4,5-trihydroxycinnamonitrile (Ki2.6 microM). With propionaldehyde as substrate the inhibition by these compounds was competitive with respect to NAD+. alpha-Fluorocinnamate was a much less effective inhibitor of the enzyme, with mixed behaviour towards NAD+, but with a major competitive component. These cinnamic acid derivatives were ineffective as inhibitors of the aldehyde dehydrogenase-catalysed hydrolysis of p-nitrophenyl acetate, but inhibited the ability of NAD+ and NADH to activate this activity. Inhibition of the stimulation of esterase activity was competitive with respect to NAD+ and NADH, and the derived Ki values were the same as for inhibition of dehydrogenase activity. NAD+, but not acetaldehyde, could elute the low Km aldehyde dehydrogenase from alpha-cyanocinnamate-Sepharose, to which the enzyme binds specifically (Poole RC and Halestrap AP, Biochem J 259: 105-110, 1989). The cinnamic acid derivatives have little effect on lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase or a high Km aldehyde dehydrogenase present in rat liver mitochondria. It is concluded that some cinnamic acid derivatives are potent inhibitors of the low Km aldehyde dehydrogenase, by competing with NAD+/NADH for binding to the enzyme. They are much less effective as inhibitors of other NAD(+)-dependent dehydrogenases.