Placental permeability and energy metabolism enzymes in fetuses of lipemic rats

A model of maternal lipemia without hyperglycemia, in the rat, produced by high-fat feedings, was developed to study the effects of an abnormal maternal lipid homeostasis on placental transport of nutrients and possible alterations of key enzymes of energy metabolism in the liver and brain of the fetuses. Pregnant rats fed lower concentrations of fat served as controls. All studies were carried out in dams and fetuses one day prior to delivery. The dietary treatment of the dams and fetuses produced in the fetuses ketonemia as well as lipemia. Following a bolus of ¹⁴C-3-0-methyl-D-glucose to the dams, the levels of the tracer remained higher in the blood and brain of lipemic than in control fetuses. By contrast, there was a decrease in the fluxes of ¹⁴C--amino-isobutryic acid in the fetuses of lipemic dams as compared to controls. Among enzymes of energy metabolism, fetal liver glucose-6-phosphatase and succinic dehydrogenase were enhanced by lipemia. Fetal brain glucose-6-phosphatase was depressed. Thus, lipemia, as occuring in poorly controlled maternal diabetes, may be a factor in determining the access to the fetus of essential, neutral amino acids and alter the normal activity of energy metabolism enzymes in the fetus.     

Direct effects of CO on cerebral energy metabolism in bloodless rats

Cerebrocortical b-cytochromes have been found to be sensitive to reduction in the presence of CO and O2 in vivo. CO-mediated cytochrome b reduction responses in "bloodless" rats were correlated in this study with changes in concentrations of high energy and glycolytic intermediates measured in cortex after rapid brain freezing. Cytochrome redox state and metabolite concentrations also were compared with cerebral blood flow (CBF) and cerebral metabolic rate for O2 (CMRo2) measured before and after CO administration. No definite biochemical evidence of energy limitation was found in parietal cortex after the fluorocarbon-for-blood exchange; however, CO had direct effects on brain metabolite concentrations. Fifteen-minute CO exposures at inspired CO/O2 of 0.003-0.06 increased cerebrocortical phosphocreatine and ADP and decreased creatine concentration. CO exposure produced no significant changes in either ATP concentration or CMRo2, although CBF increased slightly. These findings may be interpreted to indicate that CO binding to cytochrome aa3 at low CO/O2 in vivo increases extramitochondrial pH relative to that within the mitochondrial matrix. In the process, cytochrome b reduction levels increase, possibly signaling an increased efficiency of oxidative phosphorylation relative to O2 uptake by unblocked respiratory chains.     

Tyrosine protein kinases in membrane fractions from rat cerebral cortex

Specific activities of tyrosine tubulin kinase in the particulate fractions from rat cerebellum, medulla oblongata, hypothalamus, striatum, midbrain, and cerebral cortex ranged within 30% of each other and more than 3 times higher than those in the soluble fractions. In the cerebral cortex, tyrosine protein kinase activity toward tubulin and tyrosine-glutamate (1:4) copolymers was mainly distributed in the plasma membrane and the microsome fractions. The kinase activity in cerebral cortex particulate fractions was quantitatively solubilized and separated into two peaks, kinase I and kinase II, by Sephacryl S-300 gel filtration in the presence of 0.2% Nonidet P-40 and 0.2 M NaCl. Kinases I and II were each resolved into 5 active peaks (I-1----5 and II-1----5) by casein-Sepharose column chromatography. The molecular weights of these kinases were estimated from the s20,w values to be 59,000-65,000. The Km values of II-1----5 for tubulin were nearly 10 times higher than those of I-1----5. However, the Km values of the two groups of kinases for tyrosine-glutamate copolymers were not so significantly different. About 60% of the copolymers kinase activity in I-3, I-4, II-3, and II-4 was immunoprecipitable with a saturating amount of monoclonal antibody against pp60c-src. Incubation of the immunoprecipitates with ATP resulted in the autophosphorylation of a 60 kDa protein in I-3 and I-4, and a 52 kDa protein in II-3 and II-4. Immunoblotting also indicated I-3 and I-4 as 60 kDa bands and II-3 and II-4 as 52 kDa bands on SDS-polyacrylamide gels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypanosoma evansi: Activities of adenine nucleotide degradation enzymes in cerebral cortex of infected rats

This study aimed to evaluate the activities of the ectoenzymes NTPDase and 5′-nucleotidase in synaptosomes from cerebral cortex of rats experimentally infected with Trypanosoma evansi. The animals were divided in four groups (n = 10) according to the time and degree of parasitemia (groups A, B, C and D). The animals from group A were euthanized on day 3 (low parasitemia), group B on day 5 (high parasitemia) and group C on day 15 (low parasitemia). Group D consisted of healthy rats (not-infected, n = 15) and were divided in three periods (n = 5) in order to compare with the infected groups. After euthanasia, cerebral cortex was removed for the preparation of synaptosomes and enzymatic assays. Group A showed no changes in enzymatic activities compared with control. The hydrolysis of ATP, ADP and AMP by the enzymes NTPDase and 5′-nucleotidase were increased (P < 0.05) in group B (38%, 140% and 61%, respectively) when compared with control. In the group C it was observed a decreased (22%) hydrolysis of ATP when compared with control group. The activities of NTPDase and 5′-nucleotidase in synaptosomes alters the acute phase of the disease when the number of circulating parasites is high, thus the change observed is probably due to the parasitemia.     

Guanine nucleotide- and muscarinic agonist-dependent phosphoinositide metabolism in synaptoneurosomes from cerebral cortex of immature rats

Guanine nucleotide-, neurotransmitter-, and fluoride-stimulated accumulation of [³H]inositol phosphates ([³H]InsPs) was measured in [³H]inositol-labeled synaptoneurosomes from cerebral cortex of immature (7-day-old) and adult rats, in order to clarify the role of GTP-binding proteins (G-proteins) in modulating phosphoinositide (PtdIns) metabolism during brain development. GTP(S) [Guanosine 5-O-(3-thio)triphosphate] time- and concentration-dependently stimulated PtdIns hydrolysis. Its effect was potentiated by full (carbachol, metacholine) and partial (oxotremorine) cholinergic agonists through activation of muscarinic receptors. The presence of deoxycholate was required to demonstrate agonist protentiation of the guanine nucleotide effect. The response to GTP(S) was higher in adult than in immature rats, while the effect of cholinergic agonists was similar at the two ages examined. At both ages, histamine potentiated the effect of GTP(S), while norepinephrine was ineffective. At both ages, guanosine 5-O-(2-thio)diphosphate [GDP(S)] and pertussis toxin significantly decreased GTP(S)-induced [³H]InsPs formation. The phorbol ester phorbol 12-myristate 13-acetate (PMA), on the other hand, did not inhibit the guanine nucleotide response in synaptoneurosomes from immature rats. NaF mimicked the action of GTP(S) in stimulating PtdIns hydrolysis. Its effect was not affected by carbachol and was highly synergistic with that of AlCl₃, according to the concept that fluoroaluminate (AlF₄ ^–) is the active stimulatory species. No quantitative differences were found in the response to these salts between immature and adult animals. These results provide evidence that, in both the immature and adult rat brain, neuroreceptor activation is coupled to PtdIns hydrolysis through modulatory G-proteins.     

Oxidative metabolism of the cerebral cortex in young animals

Intensity of glucose, leucine and palmitate oxidation in the cortex of pigs increases during the first days after their birth. Fasting of pigs during 24 hours after birth intensifies glucose catabolism in the cortex through the pentose phosphate way. Intensity of palmitate and leucine oxidation in the cortex of fasting pigs is at the low level. Ketonic bodies participate in the power supply of cortex functions of satisfied one-day pigs but their contribution to the nerve tissue power of fasting one-and five-day pigs is insignificant.     

Evidence that quinolinic acid severely impairs energy metabolism through activation of NMDA receptors in striatum from developing rats

In the present study we investigated the effect of intrastriatal administration of 150 nmol quinolinic acid to young rats on critical enzyme activities of energy production and transfer, as well as on 14CO2 production from [1-14C]acetate at distinct periods after quinolinic acid injection. We observed that quinolinic acid injection significantly inhibited complexes II (50%), III (46%) and II-III (35%), as well as creatine kinase (27%), but not the activities of complexes I and IV and citrate synthase in striatum prepared 12 h after treatment. In contrast, no alterations of these enzyme activities were observed 3 or 6 h after quinolinic acid administration. 14CO2 production from [1-14C]acetate was also significantly inhibited (27%) by quinolinic acid in rat striatum prepared 12 h after injection. However, no alterations of these activities were observed in striatum homogenates incubated in the presence of 100 microm quinolinic acid . Pretreatment with the NMDA receptor antagonist MK-801 and with creatine totally prevented all inhibitory effects elicited by quinolinic acid administration. In addition, alpha-tocopherol plus ascorbate and the nitric oxide synthase inhibitor l-NAME completely abolished the inhibitions provoked by quinolinic acid on creatine kinase and complex III. Furthermore, pyruvate pretreatment totally blocked the inhibitory effects of quinolinic acid injection on complex II activity and partially prevented quinolinic acid-induced creatine kinase inhibition. These observations strongly indicate that oxidative phosphorylation, the citric acid cycle and cellular energy transfer are compromised by high concentrations of quinolinic acid in the striatum of young rats and that these inhibitory effects were probably mediated by NMDA stimulation.     

Inhibition of astrocytic energy metabolism by D-lactate exposure impairs memory

Bead discrimination learning in day-old chicken was inhibited by bilateral injection into the intermediate medial mesopallium (IMM), a homolog of the mammalian brain cortex, of the poorly metabolized enantiomer of L-lactate, D-lactate. The window of vulnerability extended from 10 min before training to 20 min after training. Unilateral injection 10 min before training inhibited only in the left IMM, whereas 10 min after training injection was only inhibitory if made into the right hemisphere. The pre-training administration caused memory loss from the earliest time tested whereas memory was maintained for another 20 min when D-lactate was injected 10 min post-training. The ability of acetate, an astrocyte-specific substrate, injected into the IMM to counteract the inhibitory effect was tested. Following D-lactate injection 10 min before training, rescue of memory immediately after training was achieved by acetate as long as aspartate, an oxaloacetate precursor, was also present. This suggests that pyruvate carboxylation is necessary for net synthesis of glutamate, which is known to occur at this time [Gibbs, M.E., Lloyd, H.G.E., Santa, T., Hertz, L., 2007. Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: biochemical and behavioral evidence. J. Neurosci. Res., 85, 3326-3333]. However, acetate alone rescued memory 20 min post-training (following d-lactate injection 10 min after training), indicating that pyruvate at this time is used for energy production, consistent with memory inhibition by dinitrophenol. These findings suggest that D-lactate acts by inhibiting uptake of L-lactate into astrocytes (an extracellular effect) or metabolism of pyruvate in astrocytic mitochondria (an intracellular effect). An apparent lag phase between the administration of d-lactate and its inhibition of learning favors the latter possibility. Thus, under the present experimental conditions D-lactate acts as an astrocytic metabolic inhibitor rather than as an inhibitor of neuronal L-lactate uptake, as has occasionally been suggested. Analogously, a rare reversible neurological syndrome with memory deficits, D-lactate encephalopathy, may mainly or exclusively be due to astrocytic malfunction.     

Differences in prolyl-leucyl-glycinamide metabolism by hypothalamus,pituitary and cerebral cortex of male and female rats

l-Prolyl-l-leucyl-glycinamide is rapidly hydrolyzed by hypothalamic, hypophyseal and cortical homogenates from male or female rats. The peptidase activity is higher in the pituitary followed in decreasing order by the hypothalamus and the cerebral cortex. It is mostly localized in the supernatant fraction of a 100,000 g centrifugation and is inhibited by bacitracin.Tissues from female rats are half as active as those from male rats and show variations during the estrous cycle, with very low PLG metabolism at diestrus 1 in pituitary and hypothalamus. In contrast, the cerebral cortex at proestrus and estrus has significant lower hydrolyzing activity than at diestrus. No change of the peptidase activity is observed in tissues from ovariectomized animals after treatment with estrogen or progesterone.The results obtained suggest the existence of a correlation between peptidasic activity and melanotropin secretion.     

Proteomics analysis of cerebral cortex in Wistar rats

To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality. Eighty-four protein spots were identified, includ-ing metabolic enzymes, skeleton proteins, heat shock pro-teins, antioxidant proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins. The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models.     

Proteomics analysis of cerebral cortex in Wistar rats

To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality. Eighty-four protein spots were identified, including metabolic enzymes, skeleton proteins, heat shock proteins, antioxidant proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins. The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models. __________ Translated from Acta Biophysica Sinica, 2007, 23 (1): 151–156 [译自: 生物物理学报]     

Age-related dynamics of energy metabolism enzymes of neurons and microvessels of the brain in spontaneously hypertensive rats

The age changes in the activity of some enzymes in neurons and in microvessels, revealed histochemically, as well as the volume of microvessels in spontaneously hypertensive (SH) rats differ from these changes in the controls. At the age of 3 months the activity of these enzymes and the number of active microvessels in SH rats increased. At the age of 6 months the activity of studied enzymes in SH rats decreased, while the number of active microvessels remained constant. The correlation between the morpho-functional characteristics of brain tissue in SH rats and its greater ischemic vulnerability is assumed.     

Regional cerebral energy metabolism in bicuculline induced seizures

In order to assess the early regional changes in energy metabolism in bicuculline induced seizures, mice were injected and sacrificed before the onset of overt seizure activity, and shortly after clonic-tonic seizures began. The energy metabolites glucose, ATP, and phosphocreatine were measured in layers of the motor cortex and the cerebellar vermis. Results showed minimal metabolite changes in the cerebellum, whereas changes in energy metabolism in the motor cortex were largely localized to the layers containing pyramidal cells. These results are in agreement with previous studies showing a relative sparing of the cerebellum, and suggest early cortical changes occur in pyramidal cells.