Edwin S. Higgins

Succinylacetone effects on renal tubular phosphate metabolism: a model for experimental renal Fanconi syndrome.

Abstract Phosphaturia is a prominent component of the renal Fanconi syndrome associated with the autosomal recessive disease, hereditary tyrosinemia. Succinylacetone (SA), the metabolic by-product of the enzyme deficiency, can be shown to produce multiple adverse effects on rat renal epithelial cell function in vitro. With the use of this compound, we have examined its interaction with Pi handling by the renal tubule cell in order to form a basis for understanding the effects of endogenously generated SA in causing phosphaturia in the genetically affected kidney. In this report we have shown complete inhibition of sodium-dependent phosphate uptake by renal brush border membrane vesicles, decreased ATP production by the SA-exposed renal tubule, and reversible inhibition of State 3 oxidation of glutamate by isolated renal mitochondria. We conclude that the phosphaturia observed in hereditary tyrosinemia results from multiple metabolic effects of SA on the renal tubule which are additive and lead to intracellular Pi depletion and diminished ATP production.

Effects of succinylacetone on methyl α-d-glucoside uptake by the rat renal tubule

Succinylacetone, a catabolic end-product of tyrosine, is excreted in large quantities in urine from individuals with hereditary tyrosinemia and the Fanconi syndrome. Succinylacetone inhibits rat renal tubular concentrative uptake of the glucose transport analogue, methyl alpha-D-glucoside, in a noncompetitive and reversible fashion. This compound also depresses oxygen consumption by the rat renal tubule without fine structural damage to mitochondria. It is concluded that succinylacetone may be a useful probe in elucidation of the biochemical mechanism underlying the human Fanconi syndrome.

Hydrazine stress in the diabetic: Ornithine decarboxylase activity

Streptozotocin-induced diabetes of 7 weeks duration increased male Sprague-Dawley rat kidney ornithine decarboxylase activity by 4.8-fold but did not affect the liver enzyme. Hydrazine treatment of 4 hr duration stimulated equally kidney ornithine decarboxylase activities of nondiabetic and diabetic rats. Hydrazine treatment increased liver ornithine decarboxylase activity in the nondiabetic rat but did not increase it in the diabetic rat. Since hydrazine stimulates ornithine decarboxylase activity prior to polyamine and protein syntheses, we speculate that the lack of hydrazine stimulation of ornithine decarboxylase in the diabetic liver may be related in part to the unrestrained gluconeogenesis and depressed Krebs cycle activity: the latter being required for protein synthesis.

Effects of Cetyltrimethylammonium and Dodecylnicotinamide Halides on Functional States of Mitochondria

Summary Cetyltrimethylammonium bromide and N-1-dodecylnicotinamide chloride were members of a group of 12 organic cations which were previously shown to depress mitochondrial respiratory control (2). In the present study, the influence of these two substances on high amplitude volume changes, latent ATPase and resting and active respiratory velocities of mitochondria were examined and compared with similar information obtained with a related lipophilic cation, tetrabutylammonium bromide. These data verified that cetyltrimethylammonium bromide was an uncoupling agent and N-1-dodecylnicotinamide chloride was an inhibitor of phosphorylating oxidation. At relatively high concentrations (above 30 μM) the pyridinium derivative also possessed uncoupling activity. The expert technical assistance of Wilson H. Friend, Mrs. Regina Foster and Brenda Dunlavey is gratefully acknowledged.

Cognate effects of ethanol, hydrazine and tissue regeneration on hepatic mitochondrial activities

Some of the early hepatic responses to a single dose of ethanol or a subconvulsant dose of hydrazine are similar to those extant in regenerating liver. For example, in all three cases gluconeogenesis is inhibited, lipid accumulates, and varying degrees of structural change occur in mitochondria. The present communication establishes that, in addition, the concurrent mitochondrial generation of ATP is stimulated and that in each case such functions as activated respiration (state 3), phosphorylation rate, respiratory control and ADP:0 ratios with both a flavin-linked and a pyridine nucleotide-linked mitochondrial substrate markedly exceed the corresponding activities in both fed and fasted control animals. Thus was observed the unusual consequence of a group of experimental treatments which increased rather than decreased the efficiency of intracellular energy retention as ATP. Such auxiliary mitochondrial activities as spontaneous and activated ATPases and high amplitude volume changes were minimally or not at all affected. It is proposed that elevated ATP generation was related to permeability changes of the organelles and that augmented ATP synthesis may be a concomitant of increased mitochondrial turnover. Such an explanation could account for the quite different mitochondrial response to chronic ethanol consumption and to typical hepatotoxic substances.

Stimulation of Phosphogluconate Pathway in Rat Brain Mince by Ethanol

Summary Administration of a moderately intoxicating dose of ethanol to rats resulted in an increase in that portion of glucose metabolized via the phosphogluconate pathway in minced preparations of cerebral cortex.

Ethidium Bromide Inhibits Mitochondrial Phosphorylating Oxidation

Summary Ethidium bromide, in addition to combination with mitochondrial nucleic acids, is a phosphorylation inhibitor during glutamate and succinate respiration by mitochondria. Exhaustive washing of ethidium bromide-treated mitochondria did not relieve the inhibition nor significantly decrease the amount of bound dye. Dialysis against a cation exchange resin at 3° for 17 hr removed about 97% of bound dye. This restored phosphorylating capacity to that of untreated mitochondria which had also been dialyzed against the resin. Since state 3 respiration was diminished and state 4 was unaffected by the presence of the acridine dye, and since neither swelling of mitochondria nor release of latent ATPase was observed, then ethidium bromide was not an electron transport inhibitor nor an uncoupler of oxidative phosphorylation. Inhibition of metabolic processes by ethidium bromide may be due in part to depressed generation of mitochondrial ATP. This work was supported in part by an American Cancer Society Institutional Grant No. IN-105.

In Vitro and in Vivo Effects of Dimethylnitrosamine on Mouse Liver Mitochondrial Function

Summary The in vivo and in vitro effects of the carcinogen dimethylnitrosamine (DMN) on the metabolic activity of mitochondria were studied. Oxygen consumption by mitochondria in state 3 and state 4 was determined polarographically following the addition of succinate or glutamate as substrates. The control of respiration by ADP is indicated by the ratio of state 3 to state 4 oxygen utilization (RCR), and is indicative of the tightness of coupling of the oxidative phosphorylation and electron transport systems. DMN in concentrations of 37.8 μM and higher significantly decreased state 3 oxidation of glutamate. State 4 oxidation of glutamate was inhibited significantly only at DMN concentrations of 75, 151, and 226 μM (5, 10, and 15 μg! ml). The RCR was decreased corresponding to the decrease in state 3 oxidation. Similar results, but to a lesser magnitude, were obtained using succinate as substrate. In mitochondria from mice treated with DMN (25 mg/kg ip) state 3 oxygen consumption was decreased and the RCR was significantly different from control with either substrate. The results indicate that DMN inhibits oxidative phosphorylation both in vivo and in vitro.

Respiratory control depression by tetraalkylammonium bromides in rat liver mitochondria

Six different lipophilic (hydrophobic) organic cations, tetraethyl-, tetrapropyl, tetrabutyl-, tetrapentyl-, tetrahexyl-, and tetraheptylammonium bromide, depressed respiratory control in rat liver mitochondria. Evaluation of mitochondrial responses in terms of a quadratic equation in log P (an index of lipophilicity) indicated that the NADH dehydrogenase receptor site for inhibitor (diminution of control of glutamate, alpha-ketoglutarate, and beta-hydroxybutyrate respiration) was more lipophilic than receptor sites for flavin-linked substrates (reduction of control of succinate, choline and alpha-glycerophosphate respiration). The succinate dehydrogenase receptor site for inhibition by the tetraalkylammonium bromides was more hydrophillic (less lipophilic) than the choline or alpha-glycerophosphate dehydrogenase receptor sites. Depression of respiratory control may be a function of charge density and of lipophilicity at specific inner membranal sites and the susceptible site may differ for different respiratory substrates.

Uncoupling by sodium alkyl sulfates of energy-linked functions of rat liver mitochondria.

Summary Sodium octyl, decyl, dodecyl, and tetradecyl sulfates were tested for uncoupling activity against energy-linked functions of rat liver mitochondria. All four detergents diminished respiratory control with either succinate or L-glutamate as substrate. The alkyl sulfates augmented mitochondrial swelling and, at high concentrations, the detergent depressed energized (ATP) contraction. The latter incompetency may be partially due to lysis of the organelle by the detergent. The potency of the detergents for both depression of respiratory control and inhibition of energized recovery from swelling by 40% [log(l/I 40) and log (l/R 40), respectively] was a function of the number of methylene groups in the alkyl sulfate. This potency was equated with the lipophilicity of the molecules as measured by their partitioning across the interfacial barrier of n-octanol and water. Uncoupling and inhibition of recovery from swelling required lesser amounts of a molecule that was lipophilic than one that was hydrophilic.