Anatolii A. Nikiforov

Stimulation of weak organic acid uptake in rat renal tubules by cadmium and nystatin

The uphill uptake of a weak organic acid, fluorescein, in superficial proximal tubules of the rat kidney was stimulated by CdCl2 (0.1 mM) or nystatin (20 microM) in the absence of metabolic substrates in the incubation medium. The stimulation could be observed during the initial period of incubation (up to 30 min) only and was prevented completely by ouabain (0.1 mM), fluoroacetate (1 mM), malonate (10 mM), alpha-cyano-4-hydroxycinnamate (0.1 mM), phenylpyruvate (1 mM), D-malate (2 mM) or phenazine methosulfate (20 microM). In the renal cortex fragment suspension, both Cd2+ and nystatin increased the ouabain-sensitive, basal oxygen consumption and inhibited the rate of glucose production from pyruvate, but not from lactate. In the presence of lactate (0.5-5 mM) in the incubation medium, Cd2+ and nystatin rather inhibited fluorescein uptake, while externally added pyruvate did not influence their stimulatory effects. Taken together, these data suggest that both activation of the tricarboxylic acid cycle and export of reducing equivalents from the mitochondria to the cytosol are necessary for the stimulatory effects of Cd2+ and nystatin on the weak organic acid uptake to develop.

Effects of inhibitors of gluconeogenesis on weak organic acid uptake in rat renal tubules.

Using inhibitors of gluconeogenesis (phenylpyruvate, alpha-cyano-4-hydroxycinnamate, quinolinate, D-malate, aminooxyacetate), we analysed mechanisms by which the gluconeogenic substrates, lactate and pyruvate, as well as a short-chain fatty acid, acetate, stimulate the uptake of a weak organic acid, fluorescein, in the rat kidney. We have shown that these inhibitors modified both the rate of glucose production from lactate and pyruvate in the renal cortex fragment suspension and the stimulatory effects of the metabolic substrates on fluorescein uptake in superficial proximal tubules in the renal cortex slices. The peculiarities of the effects of lactate and pyruvate on the uptake were correlated with the partial divergence of the pathways of gluconeogenesis from these precursors. The linkage of the weak organic acid uptake with gluconeogenesis is interpreted in terms of the hypothesis that the uptake is controlled by the cytoplasmic pyridine nucleotide redox potential, which is maintained with the participation of certain processes involved in glucose synthesis.

Inhibitory effect of valproate on weak organic acid uptake in rat renal proximal tubules

The effects of an antiepileptic drug, valproic acid (VPA), on transport mechanisms involved in renal excretion of anionic xenobiotics were investigated on rat renal proximal tubules in vitro. It was found that VPA (0.1-1 mM) dose dependently inhibited the baseline uptake of a marker organic anion, fluorescein, in the tubules. The inhibition could not be exclusively accounted for by competition between VPA and fluorescein. Taking into account a proposed relationship between the weak organic anion uptake and ammoniagenesis, the influence of VPA (0.5 mM) on the effects of glutamine and glutamate (both at 5 mM) on fluorescein uptake and ammonia production were examined. Glutamine stimulated ammonia production by the tubules, with the glutamine-induced ammoniagenesis being further augmented by VPA, while glutamate failed to affect the basal ammoniagenesis. Both glutamine (5 mM) and glutamate (5 mM) slightly inhibited fluorescein uptake, with the inhibitory effects not modified by VPA. Thus, there was no coincidence in the effects of VPA on organic anion uptake and renal ammoniagenesis. At the same time, the inhibitory effect of VPA (0.5 mM) on fluorescein uptake was largely overcome by addition of pyruvate (5 mM) to the incubation medium. In addition, VPA strongly inhibited glucose production from pyruvate. A known modulator of pyruvate metabolism, dichloroacetic acid (DCA, 1 mM), also inhibited fluorescein uptake, although its inhibitory effect was less pronounced than that of VPA. Both inhibitors failed to alter the tissue content of alpha-ketoglutarate or lactate but did slightly augment the pyruvate level. The inhibitory effects of VPA and DCA on the baseline fluorescein uptake were not additive, suggesting their similar intracellular targeting. It is assumed that the inhibitory effect of VPA on baseline fluorescein uptake in rat renal proximal tubules in vitro may be associated with its action on pyruvate metabolism.

Reversible metabolic depression in lamprey hepatocytes during prespawning migration: dynamics of mitochondrial membrane potential.

The lamprey (Lampetra fluviatilis L.) is an extant representative of the ancient vertebrate group of Agnathans. During the prespawning migration (the river period of life from autumn until spring) lamprey hepatocytes exhibit widely different energy states: a high-energy state in autumn and spring, corresponding to a normal physiological standard, and a low-energy state in winter, which is provoked by prolonged starvation and profound metabolic arrest. In spring the restoration of energy status (return to an active state) is associated with hormonally induced lipolysis of the lipid droplets stored in the cells. Lamprey hepatocytes demonstrate an aerobic metabolism based on oxidation of free fatty acids. The dynamics of mitochondrial membrane potential (MMP) were measured throughout the prespawning migration. Pharmacological inhibition of the electron transport chain decreased the MMP and caused extensive depletion of cellular ATP without loss of cell viability. The potential molecular mechanisms responsible for winter metabolic depression in lamprey hepatocytes are discussed.

Stimulatory effect of ethanol on weak organic acid uptake in rat renal tubules

Ethanol at relatively low concentrations (10-40 mM) significantly stimulated the uphill uptake of a weak organic acid, fluorescein, in the superficial proximal tubules of rat renal cortex slices, but it did not affect the rate of glucose production from lactate or pyruvate in rat renal cortex fragment suspension. In a low Na+ medium, ethanol failed to stimulate fluorescein uptake, although under the conditions employed in the present study, the baseline weak organic acid uptake was not dependent on external Na+. The stimulation of fluorescein uptake by ethanol (20 mM) was abolished by an inhibitor of alcohol dehydrogenase (EC 1.1.1.1), pyrazole (1 mM), or an inhibitor of aldehyde dehydrogenase (EC 1.2.1.3), cyanamide (0.3 mM), suggesting that oxidation of ethanol mediated its effect on the uptake. Among gluconeogenesis inhibitors tested, only D-malate (2 mM) abolished the stimulatory effect of ethanol, while the rest either did not affect (quinolinate) or even slightly augmented (alpha-cyano-4-hydroxycinnamate and phenylpyruvate) it. The effect of ethanol was markedly increased by an inhibitor of the tricarboxylic acid cycle, fluoroacetate. It was concluded that the stimulation by ethanol of weak organic acid uptake in rat renal tubules was mediated by the production of acetate.

Mitochondrial and lysosomal pathways of lamprey ( Lampetra fluviatilis L.) hepatocyte death

Mechanisms of mitochondrial and lysosomal pathways of natural death of lamprey hepatocytes are described at the spring period of the prespawn migration. The mitochondrial pathway (release of cytochrome c from mitochondria into cytosol and activation of caspases) is realized by the classic scheme of apoptosis. Comparatively recently, the lysosomal pathway of cell death associated with cathepsin B activation has been revealed in cells in pathologies, specifically in obstruction of gallbladder and bile ducts. A peculiarity of lamprey hepatocytes consists in that in the adult animal liver there takes place biliary atresia (the absence of gallbladder and bile ducts. Thereby, the lamprey hepatocytes are an excellent object for study of this new pathway of cell death. We have revealed development of the mitochondrial and the lysosomal pathways of cell death of lamprey hepatocytes.

ATP-consuming processes in hepatocytes of river lamprey Lampetra fluviatilis on the course of prespawning starvation.

The work was performed to establish which of the major ATP-consuming processes is the most important for surviving of hepatocytes of female lampreys on the course of prespawning starvation. The requirements of protein synthesis and Na(+)-K(+)-ATPase for ATP in the cells were monitored by the changes in mitochondrial membrane potential (MMP) in the presence of corresponding inhibitors from the peak of metabolic depression (January-February) to the time of recovery from it (March-April) and spawning (May). Integrity of lamprey liver cells was estimated by catalytic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood plasma. In January-February, the share of ATP necessary for protein synthesis was 20-22%, whereas before spawning it decreased to 8-11%. Functioning of Na(+)-K(+)-pump required 22% of cellular ATP at the peak of metabolic depression, but 38% and 62% of ATP in March-April and May, respectively. Progression of prespawning period was accompanied by 3.75- and 1.6-fold rise of ALT and AST activities in blood plasma, respectively, whereas de Ritis coefficient decreased from 2.51±0.34 to 0.81±0.08, what indicates severe damage of hepatocyte membranes. Thus, the adaptive strategy of lamprey hepatocytes to develop metabolic depression under conditions of energy limitation is the selective production of proteins necessary for spawning, most probably vitellogenins. As spawning approaches, the maintenance of transmembrane ion gradients, membrane potential and cell volume to prevent premature cell death becomes the priority cell function.

Accumulation of sodium and potassium ions in oocytes of the river lamprey Lampetra fluviatilis at the prespawning period

Accumulation of Na+ and K+ ions in oocytes of the river lamprey Lampetra fluviatilis and their transport across the plasma membrane is realized by two main mechanisms-Na,K-pump and Na,K,Cl-cotransport. At the prespawning period from December to May, the intracellular Na+ concentration was observed to increase from 10 to 25 mM and the K+ concentration-from 28 to 45 mM. Results obtained on isolated oocytes with aid of radioactive labels 22Na and 204Tl have shown that contributions of the Na,K-pump and Na,K,Cl-cotransport to potassium accumulations were close until March. In spring, the total K+ inflow almost doubled owing to activation of the Na,K-pump, whereas contribution of Na,K,Cl-cotransport did not change. It seems that an increase of the intracellular content of the main inorganic cations in oocytes resulted in parallel activation of the Na,K-pump and probably of Na/H-exchange. The biological significance of activation of these mechanisms of ion transport at the prespawning period might be due to a necessity of accumulation of Na+ and K+ ions in concentrations optimal for subsequent embryonic development.