In situ measurement of glutamate concentrations in the periportal, intermediate, and pericentral zones of rat liver

We developed a quantitative histochemical assay for measurement of local glutamate concentrations in cryostat sections of rat liver. Deamination of glutamate by glutamate dehydrogenase (GDH) was coupled to the production of formazan and formazan precipitation was used for colorimetric visualization. The method was tested and validated with gelatin model sections with known glutamate concentrations. Calibration graphs showed linear relationships with high correlation coefficients (> 96%) between glutamate concentrations or section thickness and absorbance values. The method was reproducible, with a constant percentage of 60 +/- 5% of glutamate being converted in gelatin model sections containing glutamate concentrations of 2 mM and higher. Glutamate concentrations were estimated in periportal, intermediate, and pericentral zones of liver lobules that contain low, intermediate, and high GDH activity, respectively. In fed adult male rat livers, periportal zones contained the highest concentrations of glutamate (approximately 14 mM) and intermediate and pericentral zones approximately 13 and 9 mM, respectively. On starvation, glutamate concentrations increased only in the small rim of pericentral cells that express glutamine synthetase, to approximately 15 mM. In livers of fetal and newborn rats, glutamate was homogeneously distributed, with a concentration of approximately 5 mM. In suckling rat liver, distribution of glutamate was still homogeneous but the concentration was increased to approximately 8 mM. These glutamate distribution patterns were in agreement with those detected immunohistochemically.     

Discrimination between periportal and pericentral necrosis of rat liver by determination of glutamine synthetase and other enzyme activities in serum

Periportal or pericentral necrosis of rat liver was produced by injection of allyl-alcohol or bromobenzene, respectively. Activities of predominantly periportal and perivenous enzymes were determined in serum during maximal necrosis. Aspartate aminotransferase, which is more or less homogeneously distributed in the liver acinus, exhibited similar activities in serum after periportal and pericentral injury. Serum activities of the mainly periportal enzymes alanine aminotransferase and fructose 1,6-bisphosphatase were 1.5- to 2-fold higher after periportal as compared to pericentral necrosis. Serum activity of the mainly pericentral glutamate dehydrogenase was 3-fold higher after pericentral than after periportal damage. However, due to individual variations necrosis could not be definitively localized in any case by measurement of these enzyme activities. Better discrimination between periportal and pericentral necrosis was achieved by the serum activity of the exclusively pericentral enzyme glutamine synthetase, which was 8-fold higher after pericentral as compared to periportal necrosis. Conclusive discrimination was obtained by the activity ratio fructose 1,6-bisphosphatase/glutamine synthetase in serum.     

Split hand/split foot malformation, deafness, and mental retardation with a complex cytogenetic rearrangement involving 7q21.3

Using 98 plasma samples from cancer patients undergoing antineoplastic chemotherapy, we compared the activities of aspartate aminotransferase and alanine aminotransferase measured by two different methods, with and without the addition of pyridoxal-5'-phosphate to the assay medium. Pyridoxal-5'-phosphate caused an increase of 1 to 20 U/l in aspartate aminotransferase and alanine aminotransferase activity in 90 and 78 patient plasma samples, respectively. Increases of aspartate aminotransferase and alanine aminotransferase activity of more than 20 U/l were observed in 8 and 20 samples, respectively. In 8 cases, the increase in alanine aminotransferase activity was greater than 50 U/l, whereas a similar increase in aspartate aminotransferase activity was decreased in only 2 cases. The considerable pyridoxal-5'-phosphate activation in aminotransferase activity observed in the plasma of a significant number of patients suggests that the use of the method with pyridoxal-5'-phosphate is advisable for a correct measurement of the catalytic concentration of aminotransferases in the plasma of patients undergoing chemotherapy.     

Significance of terminal rinse for rat liver preservation

A terminal rinse (TR) is standard practice in liver preservation with University of Wisconsin solution (UW) to avoid a potassium load. The fact that sodium lactobionate sucrose solution (SLS) is an effective organ preservation solution with a low potassium provided an opportunity to evaluate rat liver preservation without the TR step. Its importance was investigated in 122 rat liver preservation experiments. In study 1, UW and a hydroxyethyl starch-free, modified UW (UWm) were used for 20-hr liver preservation followed by either no TR or Ringer's lactate TR. The 1-week survival was: UW-TR, 2/14; UW-no TR, 1/6; UWm-TR, 0/6; UWm-no TR, 5/5 (P < 0.01). In study 2, livers were stored for 30 hr in SLS, UW, UWm, and UWm + chlorpromazine 5 mg/L, all without a TR. Nine of 11 rats survived 7 days after SLS, but there were no survivors in the other groups (P < 0.05). Study 3 compared no TR with TR with SLS, Ringer's lactate (RL), or a modified Carolina rinse (CRm) after 30-hr SLS preservation. Survival, serum aspartate aminotransferase and alanine aminotransferase, and histology were assessed. One-week survival of 9/11 rats in no TR was significantly better than in the other groups (3/14 in TR-SLS, 0/8 in TR-RL, and 0/14 in TR-CRm, P < 0.01). The values of aspartate aminotransferase (mean +/- SE) 3 hr after transplantation were 1862 +/- 439 U/L, 3334 +/- 817 U/L, 6591 +/- 1944 U/L, and 7028 +/- 1704 U/L, respectively, in no TR, TR-SLS, TR-RL, and TR-CRm. There were significant differences both in aspartate aminotransferase and alanine aminotransferase between no-TR and each of TR-RL and TR-CRm (P < 0.05). Liver specimens from rats killed 3 hr after OLT showed only mild injury in the no TR group and severe injury in the remaining groups. We conclude that a terminal rinse is harmful in rat liver preservation.     

Ontogeny of FAD-linked glycerophosphate dehydrogenase in rat pancreatic islets

The activities of the mitochondrial FAD-linked glycerophosphate dehydrogenase (m-GDH), glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, glutamate-pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase were measured in islet and liver homogenates from fetal, neonatal, adult male, adult female, pregnant and lactating rats. Either parallel or dissociated ontogenic changes were observed in islet and liver homogenates. The activity of islet m-GDH was slightly, albeit not significantly, lower in neonates than in adult rats, comparable in male and female adult animals, unaffected by pregnancy, and increased during lactation. It was much higher in fetal or adult islets cultured for 7 days than in freshly isolated islets from adult rats. In cultured islets from adult rats, the increase in m-GDH activity coincided with a dramatic decrease of GPT activity, a situation the mirror image of that found in several animal models of non-insulin-dependent diabetes mellitus. The intrinsic properties of m-GDH, as judged by comparison of measurements made by either a radioisotopic or a colorimetric procedure, were not identical in islet and liver homogenates and differed between fetal and adult islets, suggesting the existence of distinct iso-enzymes. These findings illustrate adaptive changes of islet enzymes, with exclusive or partial mitochondrial location, in ontogenic situations characterized by a remodelling of fuel homeostasis.     

The development of creatine kinase in rat skeletal muscle. Changes in isoenzyme ratio, protein, RNA and DNA during development

The activity of cytosolic creatine kinase in rat skeletal muscle rises stepwise during development. The increases occur simultnaeously with transient increases in DNA content. The second increase is accompanied by a rise in total protein, soluble sarcoplasmic protein and RNA/DNA ratio. Such changes are not observed at 20 days after birth, when creatine kinase finally accumulates to the adult level. Transient higher amounts of the MB and BB isoenzymes are observed after the first and second stepwise increase. The increase in creatine kinase activity observed after birth is predominantly due to an activation of the M gene. The BB isoenzyme is still present in adult skeletal muscle, but contributes little to the total activity.     

Serum enzyme activities in the African elephant (loxodonta africana)

The serum activities of aspartate aminotransferase, alanine aminotransferase, alpha-hydroxybutyrate dehydrogenase and creatine phosphokinase have been measured in the African elephant. In general, the values were broadly comparable with those of man except that alanine aminotransferase was much lower and creatine phosphokinase higher. No variation due to age, sex, season or location was observed.     

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.     

Instrumental and metabolic evaluation of patients affected by peripheral arterial occlusive disease (PAOD) following surgical revascularization surgery

Experiments were performed on eight subjects affected by peripheral arterial occlusive disease (PAOD) of the lower limbs. Each patient was submitted to Ecodoppler, angiography and the "Treadmill test". Two bioptic muscle of these patients. A sample was used for the spectrophotometric and spectrophotofluorimetric determinations of: glycogen, pyruvate, lactate, citrate, alpha-ketoglutarate, malate, aspartate, glutamate, AMP, ADP, ATP and creatine phosphate (CP). The other bioptic sample was used to determine the following enzyme activities: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, malate dehydrogenase, total NADH cytochrome c reductase, cytochrome oxidase, aspartate aminotransferase and alanine aminotransferase. Patients showed an increase in lactate dehydrogenase, total NADH cytochrome c reductase and succinate dehydrogenase activities, a decrease in glycogen, ATP and CP concentrations. Telethermographic data showed patient muscle thermic emission quantitatively different from control group. The telethermographic test can be used as an additional diagnostic tool to determine and monitor the efficiency of a muscle undergoing metabolic failure.     

Muscle damage induced by experimental hypoglycemia

To determine organ damage due to hypoglycemia, we studied the effects of insulin dose and hypoglycemia duration on serum enzyme activity in rabbits. Thirty rabbits were randomly divided into five groups according to hypoglycemia duration and insulin dose: A2, hypoglycemia for 30 minutes with 2 U/kg insulin; A10, hypoglycemia for 30 minutes with 10 U/kg insulin; B2, hypoglycemia for 60 minutes with 2 U/kg insulin; B10, hypoglycemia for 60 minutes with 10 U/kg insulin; and C, no hypoglycemia with 10 U/kg insulin and 50% glucose. Insulin-induced hypoglycemia was reversed by intravenous injection of glucose. Alterations in serum enzyme activity and creatine kinase (CK) isoenzyme distribution were determined before and after insulin injection. Serum CK activity increased significantly in all hypoglycemic groups compared with preinjection values, and tended to remain high for 24 hours in both groups A10 and B10. Serum activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) increased only in group B10. In addition, the level of band 4 of serum CK isoenzymes, which exists predominantly in skeletal muscle and myocardium, increased significantly in group B10. These results suggest that the increase in both serum enzyme and CK band 4 isoenzyme activities during hypoglycemia is primarily due to damage in muscle rather than liver, and that the hypoglycemia duration and insulin dosage may influence the extent of organ damage.     

Evidence for negative charge in the conduction pathway of the cardiac ryanodine receptor channel provided by the interaction of K+ channel N-type inactivation peptides

1. Precision-cut liver slices represent a suitable and convenient in vitro preparation for studying metabolism and toxicity mechanisms of drugs and toxic chemicals. Particularly in the case of human liver slices, cryopreservation would enable more efficient utilization of this scarce and irregularly available tissue. 2. Liver slices from consecutive human livers were cryopreserved using a method previously developed for rat and monkey liver slices. This procedure involves incubation in 12% dimethyl sulphoxide for 30 min on ice and direct immersion into liquid nitrogen. 3. Functional integrity of cryopreserved human liver slices, as compared with that of fresh liver slices, was maintained at 66 +/- 8% (alanine aminotransferase activity retained in the slices), 78 +/- 7% (urea synthesis), 88 +/- 14% (testosterone hydroxylation), 84 +/- 7% (N-deethylation of lidocaine) and 88 +/- 10% (total O-deethylation of 7-ethoxycoumarin). The ratios of testosterone metabolites did not change on cryopreservation. 4. These results show that the cryopreserved human liver slices retained the measured drug metabolism activities. Therefore, this cryopreservation method is suitable for storing liver slices to be used for comparing drug metabolism patterns, at least qualitatively, between species.     

A new treatment for penile conservation in penile carcinoma: a preliminary study of combined laser hyperthermia, radiation and chemotherapy

We have previously shown that the four promoters of the IGF2 gene are under a tight but dynamic control during human liver development, whereby P3 and P1 are reciprocally active before and after birth respectively while the P2 and P4 promoters are constitutively active at a relatively lower level. In this study, we investigated the methylation status of the promoters P1 and P3 of IGF2 and the promoter region of the H19 gene in developing human livers ranging from fetal to late adult. A region of about 300 bp immediately upstream of the IGF2 exon 5 was found to be subjected to a developmental-specific methylation and this may correlate to the P3 promoter activity. The P1 domain of IGF2 was also found to be methylated in a developmentally-specific pattern. The promoter region of the H19 gene displayed different methylation patterns in different development stages showing decreased general methylation with increase of age. Therefore, regional- and developmental-specific DNA methylation is displayed in the promoter regions of the IGF2 and H19 genes. This may be an important factor involved in gene regulation in the developing human liver.     

A study of the capacity of soils to induce nodules in Alnus glutinosa (L.) Gaertn. and Myrica gale L., with special reference to the specificity of the endophytes

1. Several ring-substituted derivatives of diphenyleneiodonium catalyse the exchange of Cl- and OH- ions across the inner membrane of rat liver mitochondria. They also inhibit state 3 and state 3u oxidations of glutamate plus malate in the presence of Cl- more than in its absence. Most have activities similar to diphenyleneiodonium, although 2,4-dichlorodiphenyleneiodonium is up to 50 times more active. 2. Diphenyleneiodonium inhibits soluble rat liver NADH dehydrogenase and NADH oxidation by rat liver sub-mitochondrial particles directly; 2,4-dichlorodiphenyleneiodonium is only about twice as inhibitory. 3. Liver mitochondria contain two classes of binding sites for diphenylene[125I]iodonium, namely high-affinity sites with an affinity constant of 3 X 10(5) M-1 (1--2 nmol/mg of protein), and low-affinity sites with an affinity constant of 1.3 X 10(3) M-1 (80 nmol/mg of protein). Both sites occur in hepatocytes with a relative enrichment of the low-affinity site. Nadh dehydrogenase preparations only apparently contain high-affinity binding sites. Only low-affinity sites occur in erythrocytes. 4. 2,4-Dichlorodiphenyleneiodonium competes with diphenylene[125I]iodonium for both low- and high-affinity sites, whereas tri-n-propyltin only competes for the low-affinity sites. 5. The high-affinity sites are apparently associated with NADH dehydrogenase and the low-affinity sites probably represent electrostatic binding of diphenylene[125I]iodonium to phospholipids. The high-affinity site does not appear to be associated with a rate-limiting stage of NADH oxidation.     

The FML (Fucose Mannose Ligand) of Leishmania donovani. a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with alpha-amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca2+. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.     

Morbidity study of extruder personnel with potential exposure to brominated dioxins and furans. II. Results of clinical laboratory studies

OBJECTIVE: To test whether dioxins affect liver and thyroid function, lipid metabolism and glucose or immunological variables, in workers exposed to brominated dioxins and furans. METHODS: 34 male production employees (29 were extruder operators) and eight technical support personnel were studied, all of whom were potentially exposed to polybrominated dibenzo-p-dioxins (PBDDs) and furans (PBDFs) during production of resins containing polybrominated diphenyl ethers (PBDEs). Controls were from a similar resin producing plant that did not use PBDEs. Blood samples were analysed for tetra, penta, and hexabrominated congeners, but 2,3,7,8-TBDD was the only exposure measure used in the regression analyses. Seven liver function indicators, five measures of blood lipids and glucose, four haematology and blood coagulation measures, and three measures of thyroid function were examined. RESULTS: None of the variables was statistically related to concentration of 2,3,7,8-TBDD in the regression analyses. Cigarette smoking was related to several outcomes at the 0.05 level: aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase (GLDH), erythrocyte sedimentation rate, and white blood cell count. Body mass index was also related to alanine aminotransferase, gamma-glutamyltranspeptidase, cholinesterase, GLDH, cholesterol, triglycerides, high density lipoprotein, low density lipoprotein, and glucose concentrations. No definitive associations between liver, blood lipid, thyroid, or immunological variables and exposure to brominated dioxins or blood lipid concentration of 2,3,7,8-TBDD were found. CONCLUSIONS: The study population was small and hence the findings must be interpreted with caution. Nevertheless, these results provide a base for interpreting the results of clinical studies in similarly exposed populations.     

Ontogeny of iodothyronine deiodinases in human liver

The role of the deiodinases D1, D2, and D3 in the tissue-specific and time-dependent regulation of thyroid hormone bioactivity during fetal development has been investigated in animals but little is known about the ontogeny of these enzymes in humans. We analyzed D1, D2, and D3 activities in liver microsomes from 10 fetuses of 15-20 weeks gestation and from 8 apparently healthy adult tissue transplant donors, and in liver homogenates from 2 fetuses (20 weeks gestation), 5 preterm infants (27-32 weeks gestation), and 13 term infants who survived up to 39 weeks postnatally. D1 activity was determined using 1 microM [3',5'-125I]rT3 as substrate and 10 mM dithiothreitol (DTT) as cofactor, D2 activity using 1 nM [3',5'-125I]T4 and 25 mM DTT in the presence of 1 mM 6-propyl-2-thiouracil (to block D1 activity) and 1 microM T3 (to block D3 activity), and D3 activity using 10 nM [3,5-125I]T3 and 50 mM DTT, by quantitation of the release of 125I. The assays were validated by high performance liquid chromatography of the products, and kinetic analysis [Michaelis-Menten constant (Km) of rT3 for D1: 0.5 microM; Km of T3 for D3: 2 nM]. In liver homogenates, D1 activity was not correlated with age, whereas D3 activity showed a strong negative correlation with age (r -0.84), with high D3 activities in preterm infants and (except in 1 infant of 35 weeks) absent D3 activity in full-term infants. In microsomes, D1 activities amounted to 4.3-60 pmol/min/mg protein in fetal livers and to 170-313 pmol/min/mg protein in adult livers, whereas microsomal D3 activities were 0.15-1.45 pmol/min/mg protein in fetuses and <0.1 pmol/min/mg protein in all but one adult. In the latter sample, D3 activity amounted to 0.36 pmol/min/mg protein. D2 activity was negligible in both fetal and adult livers. These findings indicate high D1 and D3 activities in fetal human liver, and high D1 and mostly absent D3 activities in adult human liver. Therefore, the low serum T3 levels in the human fetus appear to be caused by high hepatic (and placental) D3 activity rather than caused by low hepatic D1 activity. The occasional expression of D3 in adult human liver is intriguing and deserves further investigation.     

An in vitro study on methotrexate hydroxylation in rat and human liver

Methotrexate (MTX) was investigated for possible effect on the metabolism of ethoxyresorufin, pentoxyresorufin and ethoxycoumarin, the model substrates of cytochrome P450. The investigation was carried out in liver microsomes of rats pretreated with classical inducers of cytochrome P450 as well as in microsomes of two human livers. Furthermore, we measured the conversion of MTX (100microM) to its main metabolite, 7-hydroxymethotrexate (7-OHMTX), in microsomes and cytosolic fractions of rat and human livers. The inhibition of 7-OHMTX formation by menadion (inhibitor of aldehyde oxidase) and allopurinol (inhibitor of xanthine oxidase) was studied in the cytosol of rat and human livers. In both species, MTX in the concentration range 0.5-500 microM exerted no inhibitory effect on enzymatic activities associated with cytochrome P450. Moreover, we did not observe any measurable formation of 7-OHMTX in liver microsomes. MTX was metabolized at a similar rate in the cytosol of rat and human liver. Allopurinol (100 microM) reduced the rate of MTX hydroxylation by 31.5% in the cytosol of human livers but had no effect in the rat. Menadion (100 microM) decreased the rate of 7-OHMTX formation in the cytosol of human and rat liver by 69% and 94%, respectively. Our results confirmed that MTX is oxidized by a soluble enzymatic system in both the rat and human liver. In human tissues, both aldehyde oxidase and xanthine oxidase may play an important role in the metabolism of MTX. Depression of cytochrome P450 and related enzymatic activities observed in vivo cannot be explained by a direct inhibitory action of MTX on cytochrome P450.     

Changes in the profile of liver enzymes in newborn calves induced by experimental, subclinical acidosis in pregnant cows and osmotic diarrhoea

Experimental, subclinical acidosis was induced by oral administration of sacharose during the last 2 months of pregnancy in 15 cows. Seven cows and their newborn calves were used as a control group. The liver enzyme activities in the serum and the blood acid-base status were determined in the 15 calves from the cows in the experimental group. Mannitol was administered orally to 8 calves from the experimental group to induce osmotic diarrhoea. It was concluded that subclinical acidosis in pregnant cows alters the biochemical liver profile of their newborn calves, affecting the aspartate aminotransferase, alanine transaminase, beta glucuronidase, glutamate dehydrogenase and bilirubin activities in the serum, which are associated with oedematous changes to the hepatocytes. Diarrhoea was accompanied by an increase in the alkaline phosphatase and gammaglutamyl transferase activities and a decrease in the total protein concentration in the serum. These changes were apparently related to the numerous necrotic foci in the liver and the proliferation of the Kuppfer cells. It would appear from these results that the liver damage in the newborn calves was associated with the subclinical, metabolic acidosis in their dams and that osmotic diarrhoea occurring in the neonatal period additionally impaired the liver function.     

Nerve fiber formation and catecholamine content in adult rat adrenal medullary transplants after treatment with NGF, NT-3, NT-4/5, bFGF, CNTF, and GDNF

Forward (-->ATP) and reverse (-->CrP) fluxes through the creatine kinase reaction were determined in isolated rat and bovine heart mitochondria and with soluble MM-CK from rabbit skeletal muscle, using 31P-saturation transfer NMR. With soluble MM-CK forward and reverse fluxes were identical in the absence and presence of BSA or rat liver mitochondria. Addition of liver mitochondria decreased fluxes with increasing mitochondria concentration. The fluxf/Vmax(f) ratio was 0.006 with 10 mg BSA and 0.04 with 10 mg rat liver mitochondria, respectively. With heart mitochondria, fluxr was considerably higher than fluxf and the fluxf/Vmax(f) ratio was 1.7 for rat heart and 0.22 for bovine heart. It is concluded that in the presence of isolated mitochondria, the flux through the creatine kinase is driven by the mitochondrial ATP-ADP turnover. Therefore the fluxf/Vmax(f) ratio is highest for rat heart mitochondria with a high ATP-ADP turnover, intermediate for bovine heart mitochondria and low for MM-CK in the presence of liver mitochondria. It is lowest with MM-CK alone, where the creatine kinase reaction is at equilibrium and external ATP-ADP turnover is absent. The higher reverse than forward fluxes of mitochondrial creatine kinase determined at steady state by saturation transfer NMR, are caused mainly by a high ATP<-->Pi exchange in heart mitochondria preparations, having a high ATPase activity, compared to liver mitochondria.     

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells

The effect of 2-aminobicyclo[2.2.1]heptan-2-carboxylic acid (BCH), an L-leucine nonmetabolizable analogue and an allosteric activator of glutamate dehydrogenase, on glucose and glutamine synthesis was studied in rabbit renal tubules incubated with alanine, aspartate or proline in the presence of glycerol and octanoate, i.e. under conditions of efficient glucose formation. With alanine+glycerol+octanoate the addition of BCH resulted in a stimulation of alanine and glycerol consumption, accompanied by an increased glucose, lactate and glutamine synthesis. In contrast, when alanine was substituted by either aspartate or proline, BCH altered neither glucose formation nor glutamine and glutamate synthesis, while an accelerated glycerol utilization was accompanied by a small increase in lactate production. In view of the BCH-induced changes in intracellular metabolite levels the acceleration of gluconeogenesis by BCH in the presence of alanine+glycerol+octanoate is probably due to (i) increased uptake of alanine via alanine aminotransferase, (ii) stimulation of phosphoenolpyruvate carboxykinase, a key-enzyme of gluconeogenesis, (iii) rise of glucose-6-phosphatase activity, as well as (iv) activation of the malate-aspartate shuttle resulting in an augmented glycerol utilization for lactate and glucose synthesis.