George C. Tremblay

Cloning and characterization of salmon hsp90 cDNA: Upregulation by thermal and hyperosmotic stress

Accumulating evidence suggests that glucocorticoids are essential for development of hypoosmoregulatory capacity in salmon during adaptation to seawater. Heat shock protein (hsp)90 has been reported to function in signal transduction and the maturation and affinity of glucocorticoid receptors. We sought to determine whether this hsp might be upregulated by thermal and hyperosmotic stress in salmon, a species that migrates between the freshwater and marine environments. A 2625-bp cDNA cloned from a salmon cDNA library was found to code for a protein of 722 amino acids exhibiting a high degree of identity with zebra fish (92%) and human (89%) hsp90beta. Accumulation of hsp90 mRNA was observed in isolated branchial lamellae incubated under hyperosmotic conditions and in branchial lamellae of salmon exposed to hyperosmotic stress in vivo. In contrast, exposure of kidney to hyperosmotic stress in vitro and in vivo failed to elicit an increase in the quantity of hsp90 mRNA. By way of comparison, accumulation of hsp90 mRNA was observed in both branchial lamellae and kidney tissue subjected to thermal stress in vitro and in vivo. Western blot analyses of proteins isolated from tissues under identical conditions in vitro revealed that the pool of hsp90 increased with thermal stress but not with osmotic stress. The results suggest that accumulation of hsp90 mRNA in response to osmotic stress is unrelated to cellular protein denaturation and that synthesis of hsp90 may be regulated at both the level of transcription and translation.

Hsp70 and a 54 kDa protein (Osp54) are induced in salmon (Salmo salar) in response to hyperosmotic stress.

Hsp70 and a 54 kDa osmotic stress protein (osp54) were induced in isolated tissues of anadromous Atlantic salmon (Salmo salar) upon exposure to hyperosmotic conditions. Incubation of branchial lamellae, hepatic tissue, and erythrocytes in medium supplemented with 200-600 mM NaCl dramatically reduced protein synthesis. Although general protein synthesis remained depressed following transfer of tissues from 450 mM supplemental NaCl to iso-osmotic medium, hsp70 was prominently induced in branchial lamellae and hepatic tissue. Accumulation of hsp70 mRNA and a decrease in actin mRNA suggest preferential upregulation of the hsp70 gene. Induction of osp54 was observed in branchial lamellae and erythrocytes, but not in hepatic tissue, during exposure to 75-125 mM supplemental NaCl. Use of glycerol in place of NaCl to create hyperosmotic conditions stimulated induction of hsp70 in branchial lamellae. Substitution with mannitol resulted in induction of osp54 in both branchial lamellae and erythrocytes. The solute-specific and temporal patterns of response suggest that hsp70 and osp54 might function in concert to restore osmotic homeostasis and renature proteins destabilized or denatured during the early stages of osmotic shock.

Thermal shock of salmon in vivo induces the heat shock protein hsp 70 and confers protection against osmotic shock

Abstract Salmon transferred from freshwater hatcheries to seawater netpens are subjected to osmotic shock which can disrupt biochemical processes and cause stunted growth or death. The present study aimed to determine if heat shock proteins (hsps) induced by exposure of living animals to thermal shock might confer protection against this osmotic challenge. A 66 kDa protein which cross reacted with a monoclonal antibody to mammalian hsp 70, was induced in hepatic and branchial tissue of juvenile salmon subjected to a 15 min heat shock at 26°. De novo synthesis of this protein did not continue beyond the first 3 h of recovery from heat shock, but the newly synthesized protein was stable for at least 12 h. Heat shocked salmon were better able to survive a subsequent severe osmotic challenge (45 ppt). Cross protection against osmotic shock was observed only during a two month period coincident with parr–smolt transformation, when branchial Na + /K + ATPase activity was elevated. This report is the first to demonstrate protection against osmotic challenge by heat shock in a living animal.

Orotic acid biosynthesis in rat liver: studies on the source of carbamoylphosphate.

Abstract Measurements of the incorporation of radiolabeled precursors into orotic acid in tissue slices and minces provided evidence of the participation of the intramitochondrial carbamoylphosphate synthetase (CPSase-I) in the de novo biosynthesis of pyrimidines in rat liver. Ammonia, the only nitrogen source utilized by CPSase-I, markedly stimulated the incorporation of NaH14CO3 into orotic acid in liver slices, and ornithine, which enhances the intramitochondrial consumption of carbamoylphosphate (CP) in citrulline synthesis, antagonized the stimulation by ammonia. Sensitivity of the incorporation of NaH14CO3 into orotic acid to stimulation by ammonia was found to increase with age in concert with the emergence of CPSase-I in the liver during late fetal and neonatal development. Tissues lacking in CPSase-I activity did not exhibit the responses to ammonia and ornithine observed with the adult rat liver. While the occurrence of CPSase-I in the liver contributes extensively toward the exceptionally high capacity of that tissue for the de novo biosynthesis of orotic acid, our results also indicate that the physiological rate of orotic acid biosynthesis in rat liver is approximately one-third of capacity; the incorporation of NaH14CO3 into orotic acid averaged 488 nmol/g of tissue in 3 h in the presence of toxic levels of ammonia, but declined to 160 nmol/g of tissue in 3 h when physiological levels of both ammonia and ornithine were provided. However, the rate of orotic acid biosynthesis observed with physiological concentrations of ammonia and ornithine could be reduced further, to about one-quarter of the physiological rate, by providing additional ornithine; thus, physiological levels of ornithine do not prevent the escape of intramitochondrial CP into the cytoplasm. Finally, over 80% of the incorporation of NaH14CO3 into orotic acid at physiological levels of ammonia and ornithine was found to be ammonia dependent, and all but a small fraction of the ammonia-dependent incorporation could be blocked by providing ornithine in amounts in excess of physiological. These results indicate that CPSase-I is the major source of CP in the biosynthesis of hepatic pyrimidines under normal (physiological) conditions as well as in ammonia toxicity.

The biochemistry and toxicology of benzoic acid metabolism and its relationship to the elimination of waste nitrogen

Detoxification of sodium benzoate by elimination as a conjugate with glycine, a nonessential amino acid, provides a pathway for the disposal of waste nitrogen. Since 1979, sodium benzoate has been widely used in the therapeutic regimen to combat ammonia toxicity in patients born with genetic defects in the urea cycle. Although the clinical use of benzoate is associated with improved outcome, the search for biochemical evidence in support of the rationale for benzoate therapy has produced conflicting results. This review begins with an historical account leading to elucidation of the biochemistry of benzoate detoxification and early work indicating the potential utility of the pathway for elimination of waste nitrogen. An introduction to contemporary efforts at employing benzoate to treat hyperammonemia is followed by a detailed review of studies on benzoate metabolism and resultant toxic interactions with other major metabolic pathways. With this background, the several metabolic routes by which benzoate is thought to promote the disposal of waste nitrogen are then examined, followed by a consideration of alternative mechanisms by which benzoate might combat ammonia toxicity.

On the availability of intramitochondrial carbamoylphosphate for the extramitochondrial biosynthesis of pyrimidines

Abstract Carbamoylphosphate synthesized inside the mitochondria is not compartmentally isolated but, rather, is readily available for extramitochondrial reactions and may constitute a major source of carbamoylphosphate for the biosynthesis of pyrimidines.

The effect of sodium benzoate on ammonia toxicity in rats

At 9.5 mmoles/kg body weight, sodium benzoate sharply increased mortality in rats subsequently challenged with ammonia. Fasted animals were less sensitive to potentiation of ammonia toxicity by benzoate than were fed animals. At 2.5 mmoles/kg body weight, benzoate was observed to protect fasted animals against ammonia toxicity. Measurements of ammonia disappearance, urea formation, and hippurate synthesis in suspensions of isolated hepatocytes indicate that benzoate potentiates ammonia toxicity by inhibiting the urea cycle.

On the mechanism of inhibition of gluconeogenesis and ureagenesis by sodium benzoate

Synthesis of glucose from lactate and generation of urea from ammonia were inhibited when sodium benzoate was added to suspensions of rat hepatocytes. Assays with isolated mitochondria suggested pyruvate carboxylase and the N-acetyl-L-glutamate (NAG)-dependent carbamoylphosphate synthetase (CPS-I) as potential sites of inhibition for both pathways, owing to a shared dependency on aspartate efflux from the mitochondria and its subsequent conversion to oxaloacetate in the cytosol. Assays with isolated hepatocytes indicated inhibition to be initiated by accumulation of benzoyl CoA with a resultant depletion of free CoA and acetyl CoA. Measurements of adenine nucleotides showed that benzoate metabolism did not sufficiently alter energy status to account for the observed inhibition. Consistent with these interpretations, acceleration of the conversion of benzoyl CoA to hippurate by the addition of glycine restored the levels of free CoA and acetyl CoA and the rates of gluconeogenesis and ureagenesis. Reduction of the levels of aspartate and glutamate, presumably by interference with the anapleurotic function of pyruvate carboxylase, most likely accounted for inhibition of gluconeogenesis by benzoate. Whether reduced flux through the urea cycle also contributed to inhibition of gluconeogenesis (by diminishing cytosolic conversion of aspartate to oxaloacetate) requires further study. Depression of glutamate and acetyl CoA to levels at or below the Km for NAG synthetase probably accounted for the observed inhibition of ureagenesis. Rates of urea production were observed to vary with changes in the levels of NAG, suggesting NAG-dependent CPS-I to be the primary site of inhibition of ureagenesis by benzoate.

Inhibition of pyruvate carboxylase by sequestration of coenzyme A with sodium benzoate

Pyruvate-dependent CO2 fixation by isolated mitochondria was strongly inhibited by sodium benzoate. Pyruvate carboxylase was identified as a site of inhibition by limiting flux measurements to assays of pyruvate carboxylase coupled with malate dehydrogenase. Benzoate reduced pyruvate-dependent incorporation of [14C]KHCO3 into malate and pyruvate-dependent malate accumulation by 74 and 72%, respectively. Aspartate-dependent malate accumulation was insensitive to benzoate, ruling out malate dehydrogenase as a site of action. Inhibition by benzoate was antagonized by glycine, which sharply accelerated conversion of benzoate to hippurate. Assays of coenzyme A and its acyl derivatives revealed inhibition to correlate with depletion of acetyl CoA and accumulation of benzoyl CoA. Depletion of acetyl CoA was sufficient to account for greater than 50% reduction in pyruvate carboxylase activity. Competition between acetyl CoA and benzoyl CoA for the activator site on pyruvate carboxylase was insignificant. Results support the interpretation that the observed inhibition of pyruvate carboxylase occurred primarily by depletion of the activator, acetyl CoA, through sequestration of coenzyme A during benzoate metabolism.

Insulin-like effects of a physiologic concentration of carnitine on cardiac metabolism

Pharmacologic (millimolar) levels of carnitine have been reported to increase myocardial glucose oxidation, but whether physiologically relevant concentrations of carnitine affect cardiac metabolism is not known. We employed the isolated, perfused rat heart to compare the effects of physiologic levels of carnitine (50 μM) and insulin (75 mU/l [0.5 nM]) on the following metabolic processes: (1) glycolysis (release of 3H2O from 5-3H-glucose); (2) oxidation of glucose and pyruvate (production of 14CO2 from U-14C-glucose, 1-14C-glucose, 3,4-14C-glucose, 1-14C-pyruvate, and 2-14C-pyruvate); and (3) oxidation of palmitate (release of 3H2O from 9,10-3H-palmitate). We found that addition of carnitine (50 μM) to a perfusate containing both glucose (10 mM) and palmitate (0.5 mM) stimulated glycolytic flux by 20%, nearly doubled the rate of glucose oxidation, and inhibited palmitate oxidation by 20%. These actions of carnitine were uniformly similar to those of insulin. When carnitine and insulin were administered together, their effects on the oxidation of glucose and palmitate, but not on glycolysis, were additive. When pyruvate (1 mM) was substituted for glucose, neither carnitine nor insulin influenced the rate of oxidation of pyruvate or palmitate. In combination, however, carnitine and insulin sharply suppressed pyruvate oxidation (75%) and doubled the rate of palmitate oxidation. None of the responses to carnitine or insulin was affected by varying the isotopic labeling of glucose or pyruvate. The results show that carnitine, at normal blood levels, exerts insulin-like effects on myocardial fuel utilization. They also suggest that plasma carnitine in vivo may interact with insulin both additively and permissively on the metabolism of carbohydrates and fatty acids