Effect of phenylpyruvate on enzymes involved in fatty acid synthesis in rat brain

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of ¹⁴CO₂ from [1-¹⁴C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K_i of 100μm. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.     

Effect of GABA ascorbinate on epileptiform reactions of cortical neurons and some metabolic consequences of hypoxia in the rat brain

In the first series of experiments on immobilized anesthetized rats we studied the effects of GABA ascorbinate (A-GABA) in combination with sodium thiopental on the epileptiform reactions of cortical neurons. It is demonstrated that when combined with a classic anticonvulsant, barbiturate, A-GABA effectively suppressed neuronal epileptiform reactions. This makes it possible to considerably decrease the dose of inected barbiturate. In the second experimental series, we studied the effects of acute cerebral hypoxia on the content of overall macroergic phosphates (MP) and Na, K-ATPase activity in the cortex. We also tested the possibility of corecting the hypoxia-related shifts of the above indices by treatment with GABA, A-GABA, or their combination with a vitamin complex (VC). A successive increase in the efficacy of the above agents from GABA to A-GABA+ VC was observed with respect to the above two indices. The mechanisms of the A-GABA effects on GABA receptor-channel complexes and the prospects for using A-GABA in combination with barbiturates and VC for treatment of various brain pathologies are discussed.     

Effect of hypoxia on tyrosine and tryptophan hydroxylation in unanaesthetized rat brain

T he T yrosine and tryptophan hydroxylase enzymes have been proposed as rate-limiting steps in the biosynthesis of catecholamines and 5-hydroxytryptamine (5-HT), respectively; thus under normal physiological conditions the rate of amine synthesis appears to be controlled by the activity of these hydroxylase enzymes (see U denfriend , 1966; L ovenberg , J equier and S joerdsma , 1968). Subtle changes in neuronal activity may result not in changes in the levels of the amine neurotransmitters, but rather in alteration in their production and metabolism without measurable change in their levels. Previous studies of the effect of hypoxia on monoamines have dealt with amine levels, but there have been no studies of the effect of lowered oxygen on the synthesis of these substances.     

Effect of undernutrition on some enzymes involved in the salvage pathway of purine nucleotides in different regions of developing rat brain

The effect of undernutrition on the activity of two key enzymes of purine salvage pathway, namely hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) and adenine phosphoribosyltransferase (APRTase), in cerebral hemispheres, cerebellum and brain stem of rats at different days of postnatal development was studied. The activity of HGPRTase and of APRTase is significantly lower in all brain regions of undernourished animals at 5 days after birth; between 10 and 15 days of age there is a recovery of the enzymatic activity which is particularly evident in the cerebellum. Successively both enzymatic activities decrease reaching at 30 days of age values quite similar to those of controls. These results indicate that undernutrition during fetal and postnatal development, impairs and delays the activity of the enzymes of purine salvage pathway.     

Effect of acute hypoxia on respiration and brain stem blood flow in the piglet

Changes in local brain stem perfusion that alter extracellular fluid Pco2 and/or [H+] near central chemoreceptors may contribute to the decrease in respiration observed during hypoxia after peripheral chemoreceptor denervation and to the delayed decrease observed during hypoxia in the newborn. In this study, we measured the changes in respiration and brain stem blood flow (BBF) during 2-4 min of hypoxic hypoxia in both intact and denervated piglets and calculated the changes in brain stem Pco2 and [H+] that would be expected to occur as a result of the changes in BBF. All animals were anesthetized, spontaneously breathing, and between 2 and 7 days of age. Respiratory and other variables were measured before and during hypoxia in all animals, and BBF (microspheres) was measured in a subgroup of intact and denervated animals at 0, 30, and 260 s and at 0 and 80 s, respectively. During hypoxia, minute ventilation increased and then decreased (biphasic response) in the intact animals but decreased only in the denervated animals. BBF increased in a near linear fashion, and calculated brain stem extracellular fluid Pco2 and [H+] decreased over the first 80 s both before and after denervation. We speculate that a rapid increase in BBF during acute hypoxia decreases brain stem extracellular fluid Pco2 and [H+], which, in turn, negatively modulate the increase in respiratory drive produced by peripheral chemoreceptor input to the central respiratory generator.     

Effect of acute hypoxia on microcirculatory and tissue oxygen levels in rat cremaster muscle.

Repeated exposure to brief periods of hypoxia leads to pathophysiological changes in experimental animals similar to those seen in sleep apnea. To determine the effects of such exposure on oxygen levels in vivo, we used an optical method to measure PO2 in microcirculatory vessels and tissue of the rat cremaster muscle during a 1-min step reduction of inspired oxygen fraction from 0.21 to 0.07. Under control conditions, PO2 was 98.1 +/- 1.9 Torr in arterial blood, 52.2 +/- 2.8 Torr in 29.0 +/- 2.7-microm arterioles, 26.8 +/- 1.7 Torr in the tissue interstitium near venous capillaries, and 35.1 +/- 2.6 Torr in 29.7 +/- 1.9-microm venules. The initial fall in PO2 during hypoxia was significantly greater in arterial blood, being 93% complete in the first 10 s, whereas it was 68% complete in arterioles, 47% at the tissue sites, and 38% in venules. In the 10- to 30-s period, the fall in normalized tissue and venular PO2 was significantly greater than in arterial PO2. At the end of hypoxic exposure, PO2 at all measurement sites had fallen very nearly in proportion to that in the inspired gas, but tissue oxygen levels did not reach critical PO2. Significant differences in oxyhemoglobin desaturation rate were also observed between arterial and microcirculatory vessels during hypoxia. In conclusion, the fall in microcirculatory and tissue oxygen levels in resting skeletal muscle is significantly slower than in arterial blood during a step reduction to an inspired oxygen fraction of 0.07, and tissue PO2 does not reach anaerobic levels.     

Differential metabolic adaptation to acute and long-term hypoxia in rat primary cortical astrocytes

Brain astrocytes provide structural and metabolic support to surrounding cells during ischemia. Glucose and oxygen are critical to brain function, and glucose uptake and metabolism by astrocytes are essential to their metabolic coupling to neurons. To examine astrocyte metabolic response to hypoxia, cell survival and metabolic parameters were assessed in rat primary cortical astrocytes cultured for 3 weeks in either normoxia or in either 1 day or 3 weeks sustained hypoxia (5% O2). Although cell survival and proliferation were not affected by the mildly hypoxic environment, substantial differences in glucose consumption and lactate release after either acute or prolonged hypoxia suggest that astrocyte metabolism may contribute to their adaptation. Hypoxia over a period of 1 day increased glucose uptake, lactate release, and glucose transporter 1 (GLUT1) and monocarboxylate transporter 1 (MCT1) expression, whereas hypoxia over a period of 3 weeks resulted in a decrease of all parameters. Furthermore, increased glucose uptake at 1 day of hypoxia was not inhibited by cytochalasin B suggesting the involvement of additional glucose transporters. We uncovered hypoxia-regulated expression of sodium-dependent glucose transporters (SGLT1) in astrocytes indicating a novel adaptive strategy involving both SGLT1 and GLUT1 to regulate glucose intake in response to hypoxia. Overall, these findings suggest that although increased metabolic response is required for the onset of astrocyte adaptation to hypoxia, prolonged hypoxia requires a shift to an energy conservation mode. These findings may contribute to the understanding of the relative tolerance of astrocytes to hypoxia compared with neurons and provide novel therapeutic strategies aimed at maintaining brain function in cerebral pathologies involving hypoxia.     

Effect of leucine on enzymes of the tryptophan-niacin metabolic pathway in rat liver and kidney

Dietary excess of leucine affects tryptophan–niacin metabolism adversely and has thus been implicated in the etiology of pellagra. To understand the biochemical basis of leucine-induced changes in tryptophan–niacin metabolism the effect of leucine on enzymes of tryptophan–niacin metabolism was investigated. Excess of leucine in the diet had no effect on rat liver 3-hydroxyanthranilate oxygenase and nicotinate phosphoribosyltransferase but significantly decreased the activity of quinolinate phosphoribosyltransferase of rat liver and kidney. The activities of tryptophan oxygenase in liver and picolinate carboxylase in kidney were significantly higher in leucine-fed animals than in the controls. Also, oxidation of [U-¹⁴C]tryptophan in vivo was higher in leucine-fed animals. Increased picolinate carboxylase and decreased quinolinate phosphoribosyltransferase activities would result in a decrease in NAD formation from dietary tryptophan. Lowered NAD formation from tryptophan particularly when the niacin concentrations in the diet are marginal would result in a state of conditioned niacin deficiency.     

Activity of gluconeogenetic enzymes of rat kidney cortex during acute hypoxia]

The activities of gluconeogenic enzymes of the rat kidney cortex was studied after exposure to lowered atmospheric pressure (200 mm Hg) for 3 hours. The hypoxic stress was found to cause an increase in the activities of phosphoenolpyruvate carboxykinase and alanine aminotransferase, but failed to affect significantly the activities of fructose-1,6-diphosphatase, glucose-6-phosphatase, and aspartate aminotranspherase. The ratio of glucose-6-phosphatase/hexokinase activities was increased under these conditions.     

Effect of thyroid hormone on metabolic compartmentation in the developing rat brain

1. The effects of treatment with thyroid hormone (tri-iodothyronine) and of neonatal thyroidectomy on the cerebral metabolism of [U-¹⁴C]leucine were investigated during the period of functional maturation of the rat brain extending from 9 to 25 days after birth. 2. Age-dependent changes in the labelling of brain constituents under normal conditions appear to depend on changes in the availability of blood-borne [¹⁴C]leucine resulting from differential rates of growth of body and brain; but developmental changes in the pool size of free leucine and in the rates of protein synthesis and oxidation of leucine are also involved. 3. Treatment with thyroid hormone had no significant effect on the conversion of leucine carbon into proteins and lipids; and the age-dependent changes in the concentration and specific radioactivity of leucine were similar to controls. On the other hand there was an acceleration in the conversion of leucine carbon into amino acids associated with the tricarboxylic acid cycle. These observations indicate that leucine oxidation was the process mainly affected. 4. The specific radioactivity of glutamine relative to that of glutamate was used as an index of metabolic compartmentation in brain tissue. Treatment with thyroid hormone advanced the development of metabolic compartmentation. 5. Neonatal thyroidectomy led to a marked decrease in the conversion of leucine carbon into proteins and lipids and to a significant increase in the amount of ¹⁴C combined in the amino acids associated with the tricarboxylic acid cycle. The age-dependent increase in the glutamate/glutamine specific-radioactivity ratio was strongly retarded. 6. The increased conversion of leucine carbon into cerebral amino acids applied to glutamate and aspartate, but not to glutamine and γ-aminobutyrate. This observation facilitated the understanding of the effects of thyroid deprivation on brain metabolism and provided new evidence for the allocation of morphological structures to the metabolic compartments in brain tissue. 7. In contrast with the marked effects of the thyroid state on metabolic compartmentation, it had relatively little effect on the developmental changes in the concentration of amino acids in the brain. 8. The rate of conversion of leucine carbon into the `cycle amino acids' both under normal conditions and in thyroid deficiency indicated a special metabolic relationship between glutamate and aspartate on the one hand, and glutamine and γ-aminobutyrate on the other.     

Effect of hypoxia on percent of arteriolar and capillary beds perfused in the rat brain

The effects of moderate and severe hypoxia on quantitative regional morphometric indexes of the total and perfused arteriolar and capillary network were studied in the rat brain to determine whether diffusion distances were reduced in hypoxia. Fluorescein isothiocyanate (FITC)-labeled dextran was injected into the femoral vein of conscious control and hypoxic rats. After 20 s, the animal was decapitated and the head was frozen in liquid N2. Sections from eight brain regions were photographed to detect the perfused microvessels and then stained for alkaline phosphatase to visualize the total vascular network. There were significant increases in percent perfused arteriolar and capillary morphology between the two groups of hypoxic animals and control animals. In control rats, the percent of capillaries perfused averaged 45.6 +/- 0.6% (mean +/- SE). In moderate hypoxia 63.4 +/- 1.8% of the vessels were perfused and in severe hypoxia 89.4 +/- 0.1% were perfused. The percentage of arterioles perfused changed similarly. There were no significant differences in any index of total or percent perfused arteriolar or capillary morphometry among the regions within any group. During severe hypoxia, a greater percentage of the capillary reserves was utilized. These results demonstrate a uniform response to hypoxia in the capillary and arteriolar network of the conscious rat brain.     

Effect of arginine on the activity of proteolytic enzymes in the rat brain and liver

The influence of arginine on autolysis and proteolysis was studied. Arginine at the concentration of 0.5 and 1.0 microM/ml was added to the incubation mixture. Proteolytic processes were studied in the acid, neutral and alkaline media (pH 4.5; 7.4; 8.5). Autolysis was determined by incubation of the brain and liver homogenates and proteolysis by the use of bovine serum albumin as a substrate. Autolytic and proteolytic activities were calculated as an increase of Folin positive compounds or amino nitrogen in the samples. It was established that the influence in vitro of arginine on the proteolytic processes depended on pH, type of the peptide-hydrolases, to a lesser extent, on the arginine concentration and did not depend on the tissue type. Arginine displayed its regulative action in the brain and liver by the same way. The addition of arginine had an effect on autolysis and proteolysis in the neutral and alkaline media. Determination of autolytic and proteolytic activities by Folin positive compounds has shown that arginine addition into the samples decreased autolysis and proteolysis. At the same time determination of autolysis and proteolysis by amino nitrogen in the presence of arginine has shown that autolytic and proteolytic activities increased.     

Stability of enzymes in post-mortem rat brain.

Enzyme activities were measured in rat brains kept at room temperature for various intervals after death by decapitation. Tytosine hydroxylase, monoamine oxidase, choline acetyltransferase, and acetylcholinesterase show a substantial decline in activity over 14h reaching 64, 78, 60 and 58%, respectively. of the zero-time activity. DOPA decarboxylase, glutamic acid decarboxylase, lactate dehydrogenase, Na-K-ATPase and Mg-ATPase show less than a 15% decline in activity. The activities of most of the enzymes studied show little change time period when human brain specimens are likely between the 6th and 14th hour after death, the to be obtained for biochemical studies     

Effect of glycerol-induced acute renal failure and di-2-ethylhexyl phthalate on the enzymes involved in biotransformation of xenobiotixs.

The effects of di-(2-ethylhexyl)-phthalate (DEPH) on the levels of cytochrome P-450 and b5 monooxygenases were studied in the rat kidney and liver in acute renal failure induced by glycerol. Intramuscular injection of glycerol (50%,10 ml x kg(-1)) to rats produced proximal tubular damage and acute renal failure. The indicators of renal function, serum urea and creatinine significantly increased (480 and 350 percent, respectively). In control and glycerol-treated animals DEPH had no significant effect on the concentrations of serum urea and creatinine. Twenty-four hours after glycerol injection the total amount of cytochrome P-450 and b5 significantly decreased in renal but increased in liver microsomal fractions. Moreover, 48 and 72 hours after glycerol injection the level of cytochrome P-450 and b5 significantly increased in both organs. A single dose of DEPH (2 ml x kg(-1), i.p.) also elevated the total cytochrome P-450 and b5 in control animals. This enhancing effect of DEPH was additive to that of glycerol in glycerol-induced acute renal failure. These results indicate that DEPH and glycerol evoked pathological changes may affect the metabolism of xenobiotics plus endogenous hormones in the liver and in kidney.