Anthony G. Dawson

Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules.

The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4 In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.

Inhibition of respiration and gluconeogenesis by paracetamol in rat kidney preparations

Abstract Paracetamol, at concentrations up to 10 mM, caused a reversible, concentration-dependent inhibition of respiration in isolated rat-kidney tubules metabolizing glucose, glutamine, lactate or glutamate. It also strongly inhibited the synthesis of glucose from glutamine or lactate and brought about a significant fall in the cell ATP level. Paracetamol lowered both coupled and uncoupled respiration in isolated kidney mitochondria oxidizing glutamate, but had no effect on respiration supported by succinate. Experiments with submitochondrial particles revealed that the drug did not influence the activity of NADH dehydrogenase but slowed the rate at which electrons were transferred from reduced NADH dehydrogenase to cytochrome b. The implications of these findings for paracetamol cytoxicity are discussed.

Ethanol oxidation in systems containing soluble and mitochondrial fractions of rat liver: Regulation by acetaldehyde

Systems containing soluble fraction of rat liver, with or without mitochondrial fraction, oxidised [1-14C] ethanol to acetaldehyde, 14CO2 and non-volatile 14C-products of which acetate was the principal, and possibly the only, component. Ethanol oxidation was stimulated by pyruvate which served as an electron sink thereby allowing rapid regeneration of NAD. When no mitochondria were present acetaldehyde accumulated, rapidly at first but eventually reaching a plateau. The rate of ethanol oxidation in these systems was much lower than the measured maximum activity of alcohol dehydrogenase (ADH) and it was concluded that ADH was inhibited by the accumulated acetaldehyde. Mitochondria, because of their relatively high aldehyde dehydrogenase (ALDH) activity, prevented the accumulation of acetaldehyde, or quickly removed acetaldehyde already accumulated. This action was accompanied by a sharp increase in the rate of ethanol oxidation, presumably due to the deinhibition of ADH. Cyanamide, an inhibitor of mitochondrial ALDH, blocked the stimulatory effect of mitochondria on ethanol oxidation. It was concluded that, in the reconstituted systems, acetaldehyde played a dominant role in controlling the rate of ethanol oxidation. The possible importance of acetaldehyde in governing ethanol oxidation in vivo is discussed.

Effects of acetylsalicylate on gluconeogenesis in isolated rat kidney tubules

Abstract The effect of acetylsalicylate on gluconeogenesis in isolated rat kidney tubules was investigated. It was found that acetylsalicylate, at a concentration of 2 mM, inhibited tthe formation of glucose from several potentially glucogenic substrates including 2-oxoglutarate, succinate and d -fructose. Addition of butyrate to the incubation medium partially reversed or, in the case of oxoglutarate, eliminated the inhibition. Acetylsalicylata also caused an increase in the respiratory rate of isolated tubules and brought about a decrease in the intracellular ATP level. The effects of acetylsalicylate on gluconeogenesis and respiration were almost identical to the effects of 2,4-dinitrophenol on these processes. It is proposed that, in isolated rat kidney tubules, the fall in the intracellular level of ATP, caused by acetylsalicylate, is a consequence of the uncoupling of respiratory chain phosphorylation. The decreased rate of gluconeogenesis is considered to be a response to the lowered ATP level.

Effects of indomethacin on respiration and the α-glycerolphosphate shuttle in rat kidney mitochondria

Abstract The anti-inflammatory drug indomethacin was found to stimulate State 4 respiration in rat kidney mitochondria, indicating an uncoupler activity which was maximal at a concentration of 0.1–0.2mM. Indomethacin also inhibited State 3 respiration in mitochondria oxidizing glutamate or succinate, but not in mitochondria oxidizing ascorbate together with tetramethylphenylene diamine. This inhibition was not relieved by 2,4-dinitrophenol and suggested that indomethacin directly inhibited electron transport along the respiratory chain at a point prior to cytochrome c . At concentrations one order of magnitude lower than that required for substantial uncoupling or respiratory inhibition, indomethacin severely restricted the transfer of reducing equivalents from extramitochondrial NADH to the respiratory chain via a reconstructed α-glycerolphosphate shuttle. It was found that the drug exerted a strong inhibitory effect on mitochondrial α-glycerolphosphate dehydrogenase activity, and that this inhibition was relatively specific since indomethacin had little effect on the activity of succinate dehydrogenase, another FAD-linked enzyme. The inhibition of the α-glycerolphosphate shuttle is discussed in relation to the previously observed effects of indomethacin on glucose metabolism in isolated rat kidney tubules.

Inhibition of respiration by phenacetin in isolated tubules and mitochondria of rat kidney

Abstract Phenacetin. an analgesic drug thought to exert nephrotoxic effects in vivo , was found to inhibit respiration in isolated rat kidney tubules metabolizing endogenous substrate or exogenous glutamine, glucose or lactate. With isolated rat kidney mitochondria the oxidation of glutamate or succinate was strongly inhibited by phenacetin; in each case State 3 respiration and State 3u (uncoupled) respiration were affected to the same extent, indicating that phenacetin exerted its influence directly on the respiratory chain. The effects of phenacetin on the oxidation of NADH and succinate by submitochondrial particles in the presence of various electron acceptors suggested that at least two oxidoreduction reactions of the respiratory chain were susceptible to inhibition by phenacetin. One of these reactions was that catalysed by succinate dehydrogenase, while the other probably lay between reduced NADH dehydrogenase and coenzyme Q. The possibility that impairment to the oxygen-metabolising capacity of the kidney cell might contribute to the perceived cytotoxicity of phenacetin is discussed.

Increased ketogenesis in hyperthyroid rats metabolizing ethanol

The levels of various metabolites were measured in freeze-clamped samples of liver from triiodothyronine-treated and control rats to which either saline or ethanol (2.5 g/kg body weight) had been administered 2 hours earlier. It was found that ethanol led to a sharp increase in the hepatic acetate concentration in both hyperthyroid and euthyroid rats whereas lactate and pyruvate concentrations were lowered in both groups. The lactate/pyruvate ratio rose significantly in euthyroid animals that had received ethanol but the ratio remained relatively low in hyperthyroid rats. The adenine nucleotide phosphorylation potential, already low in hyperthyroid rats, was further lowered by ethanol. However, the most remarkable difference between the responses of euthyroid and hyperthyroid rats to ethanol was in the hepatic concentrations of ketone bodies, particularly 3-hydroxybutyrate. In control animals, administration of ethanol did not affect either the acetoacetate or 3-hydroxybutyrate concentration but, although the level of ketone bodies in the livers of hyperthyroid rats that had not received ethanol was the same as that of controls, there was a greater than fivefold increase in the 3-hydroxybutyrate level when ethanol was given. While this increase in ethanol-dependent ketogenesis is not explicable at this stage, hyperthyroidism did not increase the activity of cytoplasmic acetyl-CoA synthetase, an enzyme that is probably involved in the formation of ketone bodies from ethanol-derived acetate.