Abstract. Both normal (PTN1A) and cancer (PC3) prostate cells produce high levels of L-lactate because of a low energy supply via the citric cycle and oxidative phosphorylation. Since some mammalian mitochondria possess a mitochondrial L-lactate dehydrogenase (mLDH), we investigated whether prostate cells can take up L-lactate and metabolize it in the mitochondria. We report here that externally added L-lactate can enter both normal and cancer cells and mitochondria, as shown by both the oxygen consumption and by the increase in fluorescence of NAD(P)H which occur as a result of L-lactate addition. In both cell types L-lactate enters mitochondria in a carrier-mediated manner, as shown by the inhibition of swelling measurements due to the non-penetrant thiol reagent mersalyl. An L-lactate dehydrogenase exists in mitochondria of both cell types located in the inner compartment, as shown by kinetic investigation and by immunological analysis. The mLDHs proved to differ from the cytosolic enzymes (which themselves differ from one another) as functionally investigated with respect to kinetic features and pH profile. Normal and cancer cells were found to differ from one another with respect to mLDH protein level and activity, being the enzyme more highly expressed and of higher activity in PC3 cells. Moreover, the kinetic features and pH profiles of the PC3 mLDH also differ from those of the PNT1A enzyme, this suggesting the occurrence of separate isoenzymes.

According to the Davies-Roberts hypothesis, plants primarily respond to oxygen limitation by a burst of l-lactate production ([1] and refs there in). The acidification of the cytoplasm during the first phase of anaerobiosis arising from lactic fermentation results in inhibition of lactate dehydrogenase (LDH) and activation of pyruvate decarboxylase [2]. As a result, a switch from lactic to ethanolic fermentation occurs. In those organisms that cannot switch to ethanolic fermentation, when oxygen falls below 1%, glycolysis is stimulated and l-lactate accumulates [3], leading to decreased cytoplasmic pH and cell death [4,5]. Thus, according to the DaviesRoberts concept, cytoplasmic acidification potentially induces damage and death of intolerant plants.Because of the damage that can arise from l-lactate accumulation, a cellular safety valve to minimize that damage is to be expected. It has been consistently reported that metabolism of l-lactate in potato after a period of anoxia is accompanied by a two-fold increase in LDH activity and by the induction of two LDH isozymes [6]. These observations related to l-lactate metabolism occurring in the cytoplasm involved pyruvate formation via LDH, and further pyruvate metabolism, both in mitochondria and in the cytoplasm. There is reason to suspect, however, that mitochondria themselves may be involved in l-lactate metabolism. This is based on our previous work, which has shown that l-lactate is transported into the organelles isolated from both rat We investigated the metabolism of l-lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal antilactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X-100. The addition of l-lactate to potato tuber mitochondria caused: (a) a minor reduction of intramitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an l-lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium l-lactate in a stereospecific manner, thus showing that l-lactate enters mitochondria by a proton-compensated process. Externally added l-lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on l-lactate concentration and was inhibited by phenylsuccinate. Hence, potato tuber mitochondria possess a non-energy-competent l-lactate ⁄ pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of l-lactate plays a previousl...