Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

Aurintricarboxylic Acid Prevents NMDA-Mediated Excitotoxicity: Evidence for Its Action as an NMDA Receptor Antagonist

Abstract: The effect of the endonuclease inhibitor aurintricarboxylic acid (ATA) versus NMDA-mediated delayed cell death was examined in an ex vivo chick retinal preparation. Transient exposure to 100 μM NMDA for 60 min followed by a 24-h recovery period resulted in a sevenfold increase in lactate dehydrogenase (LDH) release into the medium. ATA at 100 μM significantly reduced NMDA-mediated LDH release by 60%. In clarifying the mechanism of protection versus NMDA, ATA was found to inhibit several acute NMDA-mediated effects: ATA attenuated NMDA-mediated GABA release in a dose-dependent manner (IC₅₀= 29.5 μM ), prevented NMDA-stimulated cyclic GMP formation, and blocked NMDA-mediated ²²Na⁺ influx. These acute inhibitory effects of ATA were overcome by increasing the NMDA concentration, which suggested a competitive interaction between NMDA and ATA. In a binding assay using membranes prepared from adult rat forebrain, ATA displaced the competitive NMDA receptor ligand [³H]CGS 19755 with an IC₅₀ of 26.9 μM. Maximal displacement was 88% with 100 μM ATA. These studies demonstrate that ATA protected neurons from NMDA-mediated cell death upstream of endonuclease inhibition, i.e., by antagonizing NMDA receptor activity in a manner consistent with competitive antagonism.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

N-Methyl-D-Aspartate Receptor Activation and Ca2+ Account for Poor Pyramidal Cell Structure in Hippocampal Slices

The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36–37°C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca²⁺ and much improved if it contains N-methyl-D-aspartate (NMDA) receptor antagonists or 0 mM Ca²⁺ and 10 mM Mg²⁺. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca²⁺, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Similar affinities of ADP and ATP for G-actin at physiological salt concentrations

The equilibrium constant for the exchange of ATP and ADP at G-actin was determined by fluorimetric titration of G-actin-bound ε-ATP by ATP or ADP. The affinity of ATP for G-actin was found to be only about 3-fold higher than the affinity of ADP for G-actin at 37°C, pH 7.5 and physiologically relevant salt concentrations (100 mmol K⁺/l, 0.8 mmol Mg²⁺/l, <0.01 mmol Ca²⁺/l).     

Regulation of rat brainstem tryptophan 5-monooxygenase. Calcium-dependent reversible activation by ATP and magnesium.

Tryptophan 5-monooxygenase in rat brainstem cytosol was activated about twofold by incubation with 0.5 mm ATP and 5 mm MgCl₂. The activation required micromolar concentrations of Ca²⁺ but was not dependent on either cyclic AMP or cyclic GMP. Rat brain cytosol was shown to possess an endogenous protein kinase which was markedly stimulated by the addition of Ca²⁺ using endogenous protein substrates. Following activation by ATP and Mg²⁺ in the presence of Ca²⁺, tryptophan 5-monooxygenase was reversibly deactivated to the original level by incubation at 30 °C after removal of Ca²⁺ by adding ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid and was then reactivated by incubation at 30 °C after subsequent addition of Ca²⁺ and ATP. The deactivation was markedly inhibited by the omission of Mg²⁺ or by the addition of NaF.     

Enhancement of NMDA-mediated responses by cyanide

The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg²⁺ resulted in a five-fold increase in NMDA-induced peak of [Ca²⁺]_i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg²⁺. Cyanide enhanced the immediate rise in [Ca²⁺]_i produced by NMDA, followed over a 1–5 min period by a gradual increase of [Ca²⁺]_i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg²⁺. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [³H]MK-801 to rat forebrain membranes in the presence of Mg²⁺, whereas in the absence of Mg²⁺, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca²⁺ influx and inward current by interacting with the Mg²⁺ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg²⁺ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca²⁺ influx resulting from cellular energy reserve depletion.Abbreviations NMDA N-Methyl-D-Aspartate - EAA excitatory amino acid - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohept-5,10-imine maleate     

Sorbitol Uptake in Plasma Membrane Vesicles Isolated from Immortalized Rabbit TALH Cells: Activation by a Ca2+/Calmodulin-dependent Protein Kinase

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg²⁺, Ca²⁺, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37°C to 4°C. A lower level of uptake was also observed in the presence of 100 μm quinidine and when Ca²⁺ or ATP were omitted from the medium. Membranes preincubated with Mg²⁺, Ca²⁺, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 μm trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca²⁺/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca²⁺/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells. Received: 31 March 1997/Revised: 11 June 1997     

Hypothermia preserves myocardial function and mitochondrial protein gene expression during hypoxia

Hypothermia before and/or during no-flow ischemia promotes cardiac functional recovery and maintains mRNA expression for stress proteins and mitochondrial membrane proteins (MMP) during reperfusion. Adaptation and protection may occur through cold-induced change in anaerobic metabolism. Accordingly, the principal objective of this study was to test the hypothesis that hypothermia preserves myocardial function during hypoxia and reoxygenation. Hypoxic conditions in these experiments were created by reducing O2 concentration in perfusate, thereby maintaining or elevating coronary flow (CF). Isolated Langendorff-perfused rabbit hearts were subjected to perfusate (Po2 = 38 mmHg) with glucose (11.5 mM) and perfusion pressure (90 mmHg). The control (C) group was at 37 degrees C for 30 min before and 45 min during hypoxia, whereas the hypothermia (H) group was at 29.5 degrees C for 30 min before and 45 min during hypoxia. Reoxygenation occurred at 37 degrees C for 45 min for both groups. CF increased during hypoxia. The H group markedly improved functional recovery during reoxygenation, including left ventricular developed pressure (DP), the product of DP and heart rate, dP/dtmax, and O2 consumption (MVo2) (P < 0.05 vs. control). MVo2 decreased during hypothermia. Lactate and CO2 gradients across the coronary bed were the same in C and H groups during hypoxia, implying similar anaerobic metabolic rates. Hypothermia preserved MMP betaF1-ATPase mRNA levels but did not alter adenine nucleotide translocator-1 or heat shock protein-70 mRNA levels. In conclusion, hypothermia preserves cardiac function after hypoxia in the hypoxic high-CF model. Thus hypothermic protection does not occur exclusively through cold-induced alterations in anaerobic metabolism.     

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2)

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31°C for 16 h), a conventional in vitro heat shock (43°C for 30 min) or control conditions (37°C), then harvested immediately or allowed to recover for 3 h at 37°C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.     

EFFECT OF HYPOTHERMIA UPON ORGANIC PHOSPHATES, GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS IN RAT CEREBRAL CORTEX

—The influence of hypothermia upon the metabolism of the brain was studied by reducing body temperature in N₂O-anaesthetized rats to 32, 27 or 22°C, with subsequent measurements of organic phosphates, glycolytic metabolites, citric acid cycle intermediates and associated amino acids. Hypothermia was maintained for either 1 or 2 h and the effect of anaesthesia was evaluated by maintaining unanaesthetized animals at 22°C. Hypothermia had no influence on the cerebral cortical concentrations of ATP, ADP or AMP and there was only a small increase in phosphocreatine. Since the tissue concentrations of glucose and glycogen were reduced, it is concluded that the well known resistance of the hypothermie brain to ischaemia is unrelated to increased energy stores. Hypothermia was accompanied by decreases in the tissue concentrations of fructose-1,6-diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, pyruvate, lactate, α-ketoglutarate, succinate and malate, but not of glucose-6-phosphate or citrate. These results indicate that metabolic flux is retarded mainly at the phosphofructokinase and isocitrate dehydrogenase steps. The largest relative reduction was seen in α-ketoglutarate, which was possibly secondary to accumulation of ammonia. There was no change in GABA, but a decrease in glutamate and increases in aspartate and alanine. These, changes are compatible with shifts in the aspartate and alanine aminotransferase reactions, possibly induced by the fall in α-ketoglutarate.     

Suppression of amber mutants in vitro induced by low temperature.

Amber mutations are efficiently and specifically suppressed during protein synthesis in vitro in an Su^? S-30 extract at 25 °C, but not at 37 °C. Eight different amber mutations in three different genes have been tested, and all are suppressed. The efficiencies of suppression range from 20 to 35%, when protein synthesis is at the Mg²⁺ concentration optimal for β-galactosidase synthesis at 25 °C. The suppression efficiency increases to approximately 60% at higher Mg²⁺ concentrations, and is reduced to less than 5% at very low concentrations. Ochre and UGA mutations are not suppressed at all under these conditions. The amber suppression is inhibited by addition of a purified protein synthesis release factor to the reaction, or when the protein synthesis reaction takes place in extracts derived from bacteria which are streptomycin-resistant.     

The sensitivity of ATPases from mouse tissues to DDT at different temperatures.

Oligmoycin-sensitive (O-S) Mg²⁺ ATPase from mouse brain has a higher sensitivity to DDT at a low temperature, 17°C than at 27° or 37°. The I₅₀ value for 17° was 0.24 μM DDT. The DDT sensitivity did not differ significantly at 27° and 37°C. This negative temperature correlation is similar to results in brain and muscle tissues of insects. Oligomycin-insensitive Mg²⁺ ATPase, also was inhibited by DTT more effectively at cooler temperatures. In contrast, O-S Mg²⁺ ATPase from mouse muscle showed no significant sensitivity difference to DDT at the 3 temperatures. Na⁺-K⁺ ATPase, inhibited to a lesser degree by DDT, was inhibited to a much greater extent (61%) at 37° than at 17° (23%). This positive temperature correlation is similar to findings in insect homogenates.     

Fast reversal of the initial reaction steps of the plasma membrane (Ca2+ + Mg2+)-ATPase

(1) Calmodulin-depleted red cell membranes catalyse a Ca²⁺, Mg²⁺-dependent ATP-[³H]ADP exchange at 37° C. The Ca²⁺, Mg²⁺-dependent exchange, measured at 20 μM CaCl₂, 1.5 mM MgCl₂, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca²⁺ + Mg²⁺)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca²⁺-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg²⁺-containing medium, while their Ca²⁺-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl₂ to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca²⁺-dependent ATP-ADP exchange, slightly less than it stimulates the Ca²⁺-dependent ATP hydrolysis. The effect of 1.5 mM MgCl₂ on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca²⁺ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca²⁺ pump should then be due to one or more reaction steps later than and including Ca²⁺ release and dephosphorylation.     

Characterization of the membrane bound Mg2+-ATPase of rat skeletal muscle

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg²⁺-ATPase (5–25 μmol P_i/mg min). The rate of ATP hydrolysis by the Mg²⁺-ATPase was nonlinear but decayed exponentially (first-order rate constant ≥0.2 s^?1 at 37°C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5′-adenylyl imidodiphosphate (AdoPP[NH]P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or AdoPP[NH]P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg²⁺-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg²⁺-ATPase to detergents. It is proposed that the regulation of the Mg²⁺-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.     

Regulation of the shape of unsealed erythrocyte membranes by Mg-ATP and Ca2+

In the presence of MgCl₂ and ATP, the specific viscosity of suspensions of unsealed freezethawed erythrocyte membranes decreased slowly with time at 37 °C. The decrease in viscosity was found to be an index of Mg-ATP-specific induced folding of these membranes. Mg-ATP-dependent shape or viscosity changes were found to be highly temperature dependent and the viscosity of these membranes did not decrease in the presence of 2 mm 5′-adenyl imidodiphosphate and MgCl₂. Cyclic AMP, NaCl, or KCl did not have any effect on the rate of Mg-ATP-induced viscosity decreases. The Mg-ATP-dependent viscosity decreases were inhibited 100% by 1 mm chlorpromazine or 1 mmN-ethylmaleimide. Mg-ATP-dependent viscosity decreases were half-maximally inhibited by 1 μm Ca²⁺ and completely inhibited by 3–5 μm Ca²⁺. Ca²⁺ (5 μm) also inhibited Mg²⁺-dependent phosphorylation 25 to 30% in these membranes. However, if these membranes were preincubated in the absence of Ca²⁺ for greater than 10 min at 37 °C, 5 μm Ca²⁺ no longer inhibited Mg-ATP-dependent viscosity decreases and only inhibited Mg²⁺-dependent phosphorylation 5% in these preincubated membranes. Preincubation of these membranes at 37 °C for 10 min in the absence of Ca²⁺ also resulted in the loss of approximately 40 to 50% of the high-Ca²⁺ affinity Ca + Mg-ATPase activity. The presence of 5 μm Ca²⁺ in the preincubation medium protected against the loss of the inhibitory effect of Ca²⁺ on Mg²⁺-dependent phosphorylation and Mg-ATP-dependent viscosity decreases. The presence of Ca²⁺ in the preincubation medium also protected against the loss of Ca + Mg-ATPase activity in these membranes. It is hypothesized that freeze-thawed erythrocyte membranes contain a Ca²⁺ phosphatase activity which is temperature labile in the absence of Ca²⁺ and that this Ca²⁺ phosphatase activity may be involved in the regulation of shape of these membranes. Also discussed is the possible relationship of this Ca²⁺ phosphatase with Ca + Mg-ATPase activity and the problems inherent in studying Ca²⁺-regulated functions in freeze-thawed erythrocyte membranes.     

Origins of the Extracellular Glutamate Released During Total Metabolic Blockade in the Immature Retina

Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time-dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [³H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing ³H-labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [³H]acetate plus [U-¹⁴C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [³H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5- and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [³H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, ¹⁴C-labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of ¹⁴C-labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na⁺-dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [³H]glutamate, constancy of the specific activity of extracellular [³H]glutamate, decrease in the specific activity of extracellular [¹⁴C]glutamate, and attenuation of release by prior loading with t-PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.     

Effects of hypothermia on canine kidney mitochondria

The effect of hypothermia on the function of isolated dog kidney cortex mitochondria was determined with an FAD- and NAD⁺-linked substrate. In dog kidney mitochondria, temperatures of 10 °C or less suppress ADP stimulation of respiration but have little or no effect upon uncoupler, Ca²⁺ or valinomycin-K⁺ stimulation of respiration. This suggests that the adenine nucleotide translocase which catalyses the transport of ADP into the mitochondria limits the rate of respiration and generation of ATP at 10 °C in kidneys undergoing preservation. The coupling of oxidation to phosphylation, as determined by measuring the amount of ATP formed at low temperatures, indicates, however, that mitochondria are fully coupled at both 10 and 5 °C. The respiratory control index at 15 °C is greater (with pyruvate plus malate) than at 30 or 10 °C and suggests that 15 °C may be the optimum perfusion temperature for maintaining adenine nucleotide levels in the perfused kidney.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.