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J V Passonneau

Publications and source records attributed to J V Passonneau.

At least 19 recordsLinked to original sources
PubMed1994

Control of glucose utilization in working perfused rat heart.

Metabolic control analyses of glucose utilization were performed for four groups of working rat hearts perfused with Krebs-Henseleit buffer containing 10 mM glucose only, or with the addition of 4 mM D-beta-hydroxybutyrate/1 mM acetoacetate, 100 nM insulin (0.05 unit/ml), or both. Net glycogen breakdown occurred in the glucose group only and was converted to net glycogen synthesis in the presence of all additions. The flux of [2-3H]glucose through P-glucoisomerase (EC 5.3.1.9) was reduced with ketones, elevated with insulin, and unchanged with the combination. Net glycolytic flux was reduced in the presence of ketones and the combination. The flux control coefficients were determined for the portion of the pathway involving glucose transport to the branches of glycogen synthesis and glycolysis. Major control was divided between the glucose transporter and hexokinase (EC 2.7.1.1) in the glucose group. The distribution of the control was slightly shifted to hexokinase with ketones, and control at the glucose transport step was abolished in the presence of insulin. Analysis of the pathway from 3-P-glycerate to pyruvate determined that the major control was shared by enolase (EC 4.2.1.1) and pyruvate kinase (EC 2.7.1.40) in the glucose group. Addition of ketones, insulin, or the combination shifted the control to P-glycerate mutase (EC 5.4.2.1) and pyruvate kinase. These results illustrate that the control of the metabolic flux in glucose metabolism of rat heart is not exerted by a single enzyme but variably distributed among enzymes depending upon substrate availability, hormonal stimulation, or other changes of conditions.

3-Hydroxybutyric Acid↗
PubMed1992

Regulation of the purine salvage pathway in rat liver.

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

Acetates↗
PubMed1991

Enzymatic determination of total CO2 in freeze-clamped animal tissues and plasma.

An enzymatic method for measuring total carbon dioxide content in freeze-clamped animal tissues is described. Total carbon dioxide content [TCO2] was defined as the sum of the dissolved CO2, the bicarbonate concentration, and the carbonate concentration. Tissue was extracted in 80% methanol, 20 mM 2-amino-2-methyl-1-propanol, pH 9.5 at 25 degrees C and homogenized in a 1.5-ml Sardstat screw-top test tube containing 0.5-mm glass beads and a minibead beater. Total CO2 was determined as bicarbonate/carbonate by monitoring the oxidation of NADH at 340 nm using the coupled assay of phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). In the coupled assay system, 1 mumol of bicarbonate/carbonate consumed is equivalent to the oxidation of 1 mumol NADH at 340 nm. The assay medium comprised 50 mM 2-amino-2-methyl-1-propanol, pH 9.0 at 25 degrees C, 5 mM phosphoenolpyruvate (PEP), 0.25 mM NADH, 5 mM MgCl2, 5 mM mercaptoethanol, 0.02% bovine serum albumin, 10 mM oxamate, PEP carboxylase (0.5 units/ml), and malate dehydrogenase (0.5 units/ml). The total CO2 content measured in freeze-clamped rat heart, liver, brain, and skeletal muscle was 20.53 +/- 0.64, 17.34 +/- 0.67, 17.00 +/- 0.48, 16.06 +/- 0.53 mumol/g wet wt tissue, respectively (n = 5). The total CO2 in the crusher muscle of the lobster was found to be 5.0 +/- 0.33 mumol/g wet wt. Total CO2 was also enzymatically measured in arterial plasma from four chronically cannulated male wistar rats and was 24.65 +/- 1.81 mumol/ml plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

In vivo portal-hepatic venous gradients of glycogenic precursors and incorporation of D-[3-3H]glucose into liver glycogen in the awake rat.

Male Wistar fed rats were chronically cannulated and fed ground chow for 2 h for 6 days. On the 7th post-operative day, blood was simultaneously drawn from the portal and hepatic veins over a 2-h feeding period. The position of the hepatic vein cannula was verified using a tritiated water washout technique. In separate experiments, 200 microCi of [3-3H]glucose was added to the food in order to determine the relative contribution of D-glucose and 3-C precursors to newly synthesized glycogen. The 22-h fasting plasma portal vein concentrations of D-glucose, L-lactate, and L-alanine were 4.8 +/- 0.03, 0.81 +/- 0.06, and 0.20 +/- 0.03 mM, respectively (n = 5). The fasting hepatic vein plasma concentrations were 5.1 +/- 0.2, 0.70 +/- 0.15 and 0.19 +/- 0.03 mM, respectively. The portal-hepatic vein gradients after 22 h were -0.24, +0.16, and +0.01 mM for D-glucose, L-lactate, and L-alanine, respectively. At 20 min after beginning the meal, the respective gradients were +2.2, +0.53, and +0.44 mM, indicating hepatic uptake of all glycogen precursors. Of the total carbon from the three major precursors entering the liver as C-6, D-glucose contributed 82%, while alanine and lactate contributed 18% at 20 min. As portal vein D-glucose and L-alanine levels exceeded 6.65 +/- 0.69 and 0.32 +/- 0.07 mM, respectively, the portal-hepatic venous gradient became positive and increased linearly with portal concentrations. The glycogen concentration in the liver increased from a 22-h fast value of 5 mumol of glucosyl units/g wet weight to 101 +/- 7 mumol/g 2 h after the meal. The mean specific activity of portal vein plasma of [3-3H]glucose was 11,490 +/- 1,180 dpm/mumol (+/- S.E.) and that in the glycogen isolated from liver was 8,175 +/- 785 dpm/mumol of glycosyl units 2 h after the meal. The specific activity of liver [3H]glycogen relative to glucose after the meal was 0.73 +/- 0.08. It was concluded that a minimum of 73% of the newly synthesized glycogen was formed from the uptake and direct phosphorylation of portal blood D-glucose by the liver without prior conversion of glucose to 3-C units.

Alanine↗
PubMed1990

Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.

Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.

Adenine Phosphoribosyltransferase↗
PubMed1988

Metabolic alterations in fiber layers of the CA 1 region of the gerbil hippocampus following short-term ischemia: high-energy phosphates, glucose-related metabolites, and amino acids.

The CA 1 neurons of the gerbil hippocampus die at 4 days following 5 min of bilateral ischemia. The fiber and somal layers of the CA 1 region of the gerbil hippocampus were analyzed for high-energy phosphates, glucose-related metabolites, and amino acids from 0.75 hr to 4 days of postischemia. The results were compared to those from two layers of the CA 3 region and the cerebral cortex. The metabolite changes in the fiber layers of the CA 1 region were qualitatively similar to those in the somal layer. The energy status of the tissues taken from the CA 1 region was never compromised for up to 2 days of recirculation, after which the ATP and P-creatine in the somal layer decreased to 43 and 56% of the control, respectively, whereas the average decreases in the CA 1 fiber layers were only 71 and 88% of the control, respectively. Thus, the high-energy phosphate response of the neuronal elements in the fiber layers was temporally similar to that found in the somal layer of the CA 1 region. The biphasic increases in glycogen, glucose, glucose-6-phosphate, and high-energy phosphates to values greater than the control indicated that the metabolic restoration following transient ischemia is a dynamic process which persists for up to 2 days of recirculation, even in resistant tissues. A similar pattern of delayed changes was observed in glutamate, gamma-aminobutyric acid (GABA), and glutamine, but the change in each amino acid appeared to be independent of the others despite their close metabolic relationship. The delayed decreases in GABA would favor a loss of inhibition to the CA 1 neurons and may be related to the phenomenon of delayed neuronal death.

Adenosine Triphosphate↗
PubMed1986

Effect of hyperglycemia on sorbitol and myo-inositol content of cultured rat conceptus: failure of aldose reductase inhibitors to modify myo-inositol depletion and dysmorphogenesis.

Growth retardation and dysmorphogenesis were seen in the rat conceptus following culture from day 9.5 to 11.5 of development in the presence of increased amounts of glucose. Sorbitol accumulated in the conceptus during the hyperglycemia whereas total protein, DNA, and free myo-inositol decreased. Similar glucose-related compositional changes were present in separated embryos and extra-embryonic membranes suggesting that all portions of the conceptus participate in the alterations. The aldose reductase inhibitors Sorbinil and Statil obtunded the rises in sorbitol but did not modify the increased incidence of malformations and the fall in DNA, protein and myo-inositol. Thus, the aldose reductase pathway may function in the early post-implantation rat conceptus but the resultant accumulation of sorbitol does not appear to contribute to the growth-retarding, dysmorphogenic, and myo-inositol-depleting effects of hyperglycemia.

Aldehyde Reductase↗
PubMed1986

Energy metabolism in delayed neuronal death of CA1 neurons of the hippocampus following transient ischemia in the gerbil.

The delayed death of CA1 neurons in the gerbil has been reported to occur at 4 days of reflow following 5 min of bilateral ischemia. Samples of the CA1 and CA3 somal region of the hippocampus, as well as of the parietal cortex, were dissected from frozen dried sections of gerbil brains frozen in situ between 1.5 and 96 hr of reflow following 5 min of bilateral ischemia and the concentrations of the adenylates, P-creatine, glucose, glycogen, and lactate were determined. The values for high-energy phosphates were restored by 1.5 hr of recirculation in all three regions and remained at or above control in the CA3 region and cortex for up to 96 hr. In contrast, the P-creatine and ATP decreased in the CA1 region at 48 and 96 hr of reflow, respectively. The total adenylates also decreased in the CA1 region at 96 h, but the normal energy charge in this area indicated that the surviving tissue was metabolically viable. A glucose overshoot was exhibited in the three regions at all time periods except 6 and 96 hr. At 6 hr of reflow, there was a transient return of glucose levels toward those of control. By 96 hr, the glucose in the CA3 region and cortex was not significantly different from control but was elevated in the CA1 region. The lactate levels were depressed from 1.5 to 12 hr of recirculation in all areas, but the decrease was significant only in the cerebral cortex. The concentration of glycogen was significantly elevated at 6 hr in all regions, then was restored by 24 to 48 hr, only to increase once again in the affected CA1 region. The results clearly indicate that metabolic perturbations persist for long periods of time after ischemic durations that are compatible with the survival of the animal but that the loss of the CA1 neurons cannot be attributed to a failure in energy metabolism.

Adenine Nucleotides↗
PubMed1986

In vivo cGMP levels in frog photoreceptor cells as a function of light exposure.

By employing a combination of highly sensitive radioimmunoassays and histochemical techniques, an in vivo time course of cGMP levels has been determined in the outer segment, photoreceptor cell and outer plexiform layers of frog retina. Frogs (Rana pipiens) were dark-adapted overnight and either frozen rapidly (approximately 3 sec) in liquid nitrogen or exposed to periods of light varying between 0.1 sec and 2 hr before freezing. Frozen retinal sections were cut, freeze-dried, and samples of individual layers dissected out and analysed for cGMP. In the outer plexiform layer, there was a 42% drop in cGMP concentration after 2 sec of light (250 ft candles) followed by a 34% rise after 2 min; a steep concentration gradient formed around the layer after the 2 min exposure. In both the outer-segment layer and photoreceptor-cell layer (which includes outer segments, inner segments and outer nuclear layers), cGMP levels declined from a dark value of 56 mumol kg-1 (dry) to 9 mumol kg-1 (dry) as a result of increasing exposure to several types of light source: levels appear to be primarily a function of total ft candle min. Cyclic GMP concentrations at the longest exposures (2 min with a fiber optic light source or 2 hr with fluorescent room light) reached identical minimum levels. In the outer segments, a 15% decrease in cGMP was observed after 0.1 sec of light exposure. Although the freezing time is too long to be able to say whether the 15% decrease in cGMP at the 0.1 sec exposure is involved in transduction, the low identical levels reached gradually after longer exposures appear to indicate that a light-induced biochemical adjustment in cGMP metabolism occurs over a relatively long time period separate from the msec time course of the transduction process.

Animals↗
PubMed1985

Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas.

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Adenine nucleotide and P-creatine levels in layers of frog retina as a function of dark and light adaptation.

Eight layers of frog retina were analyzed for ATP, P-creatine, ATP + ADP, and AMP under conditions of dark, 2 sec, 2 min, and in the case of AMP, 2 hr of light adaptation. Samples of each layer, usually ranging between 5 and 50 ng, were dissected from lyophilized frozen sections. After brief light exposure, ATP dropped while ADP rose sharply in the pigment epithelium, outer segments, and inner segments; ADP was too low to be measured accurately in the inner retina. The profile of ATP, P-creatine, and ATP + ADP concentrations showed peaks in the inner nuclear and ganglion layers. AMP, by contrast, was highest in the two plexiform layers. Levels in the inner retina dropped after only 2 sec of light but rose after 2 hr to levels that were higher than dark values in all retinal layers. AMP was often characterized by a non-uniform distribution: adjacent areas of a layer agreed very closely in value to each other but could vary several-fold from a different section of the same frog or from another frog exposed to the same conditions. This distribution produced clusters of values, particularly prominent in dark-adapted animals, something not observed with the other metabolites measured. The peaks of AMP in the plexiform layers suggest that AMP may be a by-product of dopamine-stimulated adenylate cyclase which also has peaks in these same layers.

Adenine Nucleotides↗
PubMed1985

Effect of nimodipine on cerebral metabolism during ischemia and recirculation in the mongolian gerbil.

The effect of nimodipine on cerebral metabolism during ischemia and reflow was studied in female mongolian gerbils. Animals were divided into three experimental groups. Group 1 received 1 mg/kg nimodipine i.p. 1 h prior to ischemia. Group 2 received an injection of the vehicle, 5% polyethylene glycol 400. Group 3 received an equal volume of normal saline. Cerebral ischemia was induced by bilateral common carotid artery occlusion for 1, 2, or 5 min. Recirculation was established for 0, 1, or 5 min. Sham-operated animals served as nonischemic controls. Gerbils were killed by microwave irradiation. Regional levels of ATP, phosphocreatine, glucose, glycogen, cyclic AMP, and cyclic GMP were measured in brain extracts using standard assay techniques. Levels of metabolites in sham-operated animals did not differ among Groups 1, 2, and 3. At 1 min of ischemia, cortical and striatal ATP levels were highest in Group 1 (p less than 0.05 and p less than 0.01, respectively). After 5 min of recirculation, cortical and striatal glucose levels were highest in Group 1 (p less than 0.005). Regional levels of the metabolites measured at other times did not differ significantly among the three groups. Pretreatment with nimodipine thus retards the fall in ATP and facilitates the recovery of glucose in mongolian gerbils subjected to common carotid artery occlusion. A regional variability of this effect was observed.

Adenosine Triphosphate↗
PubMed1985

Changes in brain energy metabolism and protein synthesis following transient bilateral ischemia in the gerbil.

The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1-2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.

Adenine Nucleotides↗
PubMed1985

Nimodipine levels in gerbil brain following parenteral drug administration.

Nimodipine binding to the particulate fraction of gerbil brain homogenate was characterized using tritiated (3H)-nimodipine as the radioactive ligand. Binding was monophasic and saturable, with the apparent affinity constant (KD) = 0.4 nM and the maximum number of binding sites (Bmax) = 12 nmol/kg wet wt. A competitive binding assay was validated for the measurement of nimodipine using gerbil brain as the source of receptors for the drug. Binding characteristics were sufficiently similar in specimens from different animals to allow the use of homogenates from individual animals as the source of both membrane-binding sites and competing ligand. Nimodipine could be detected in the brains of animals sacrificed soon after drug injection, and reached a peak level within 15 minutes. Brain drug level at a given time was a linear function of dose administered. One hour after a 1-mg/kg dose, the level of drug measured in brain was approximately 100 nmol/kg wet wt, more that 200 times the KD. Sufficient drug to mediate a maximal pharmacological effect accumulated in brain even after a dose of only 0.25 mg/kg. Thus, in this species, effective tissue nimodipine levels may be achieved at doses which minimize the risk of systemic hypotension.

Animals↗
PubMed1984

An examination of the efficiency of glucose and glutamine as energy sources for cultured chick pigment epithelial cells.

Embryonic chick pigment epithelial cells in culture require glucose as their major energy source for long-term growth, pigment formation, and colony organization. Cell number increases with glucose concentration at least up to 5.0 mM. Cells can be grown with glutamine as the major energy source but produce comparable cell numbers for only the first 3 days in culture, after which they cease growing. However, they are able to metabolize glutamine at a two to sixfold higher rate than cells grown in the presence of glucose as measured by CO2 release and by incorporation into protein. In cells grown in the presence of both glucose and glutamine, basal ATP levels were 31.1 nmoles/mg protein; P-creatine averaged 15.2 nmoles/mg protein and showed marked variability between experimental groups. During starvation, P-creatine levels fell while ATP levels remained relatively constant. Glucose was required for the recovery of P-creatine to prestarvation levels when measured 5 min after refeeding. Because of these marked changes in P-creatine concentration as a function of nutritional status, the ATP/P-creatine ratio becomes a useful measure of the energy state of the cell.

Adenosine Triphosphate↗
PubMed1984

An in vitro amino acid incorporation method for assessing the status of in vivo protein synthesis.

A quantitative in vitro amino acid incorporation assay is described which can be used to assess the status of in vivo protein synthesis. The preparation and incubation conditions employed result in constant precursor specific activity and limit amino acid incorporation to completion of nascent peptide chains. Results obtained with this method correlate well with measurements of polyribosome profiles using sucrose gradient centrifugation. The assay is easily applied to a large number of samples, and requires only a fraction of the time and tissue necessary for conventional measures of polysome aggregation. The method has been found suitable for studies of protein synthesis in mouse brain and liver, and in gerbil brain, but not in mouse kidney. Products of in vitro protein synthesis can be separated by standard electrophoretic techniques, allowing a characterization of proteins whose mRNAs are actively translated in vivo.

Amino Acids↗