ABSTRACT
Our previous studies have shown a local decrease in glutamate and aspartate levels during seizures, induced by picrotoxin microdialysis in the hippocampus of chronic freely moving rats. In this paper, we study the effect of continuous hippocampal microperfusion of the NMDA, AMPA and kainate glutamate receptor inhibitors 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801); 6,7-dinitroquinoxaline-2,3-dione (DNQX), and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). We also examine the action of L(-)-threo-3-hydroxyaspartic acid (THA), a glutamate and aspartate reuptake blocker, on the modification of extracellular glutamate and aspartate levels induced by picrotoxin, using the microdialysis method in freely moving rats. We found that changes in extracellular hippocampal concentrations in both amino acids are prevented by NMDA, AMPA and kainate receptor inhibitors. Seizures elicited under DNQX also induce a transient increase in aspartate extracellular levels coincident with seizure time. L(-)-threo-3-hydroxyaspartic acid increased the basal extracellular concentrations of both amino acids, but did not prevent the seizure-related decrease. Our results suggest that glutamate, the major neurotransmitter at the synaptic level, may also play an important role in non-synaptic transmission during seizures.
Subject(s)
Aspartic Acid/metabolism , Benzodiazepines , Excitatory Amino Acid Antagonists/pharmacology , Glutamic Acid/metabolism , Picrotoxin , Receptors, Glutamate/physiology , Seizures/metabolism , Animals , Anti-Anxiety Agents/pharmacology , Aspartic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Dizocilpine Maleate/pharmacology , Extracellular Space/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Male , Microdialysis , Quinoxalines/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Glutamate/drug effects , Seizures/chemically inducedABSTRACT
The relation between changes in the concentrations of some of the neuroactive extracellular amino acids (glutamate, aspartate, gamma-aminobutyric acid, glycine and taurine) and epileptic seizures has been tested in a new experimental model of seizures induced by picrotoxin microdialysis in chronic freely moving rats. During ictal discharges (paroxysmal electroencephalographic discharges associated with behavioral seizures), a significant decrease in the levels of extracellular aspartate and glutamate was observed. However, no changes were found during the interictal discharges (paroxysmal electroencephalographic discharges, without concomitant seizures). Our results suggest that modifications in extracellular aspartate and glutamate may be related to neuronal synchronization rather than to paroxysmal activity, supporting the neurophysiological differences between non-ictal and ictal paroxysmal activity.
Subject(s)
Amino Acids/metabolism , Extracellular Space/metabolism , Hippocampus/metabolism , Picrotoxin , Seizures/chemically induced , Animals , Aspartic Acid/metabolism , Electroencephalography , Glutamic Acid/metabolism , Glycine/metabolism , Hippocampus/drug effects , Male , Microdialysis , Rats , Rats, Sprague-Dawley , Taurine/metabolism , gamma-Aminobutyric Acid/metabolismABSTRACT
Phosphofructokinase purified from mantle tissue of the sea mussel Mytilus galloprovincialis, was phosphorylated "in vitro" by the catalytic subunit of cyclic AMP-dependent protein kinase. The incorporation of phosphate gave rise to an activation of the enzyme by increasing its affinity for fructose-6-phosphate, by decreasing its sensitivity to the inhibition by ATP and by enhancing the effect of allosteric activators (5'-AMP and fructose-2,6-bisphosphate). In addition, the effects of phosphorylation on the catalytic activity are pH-dependent.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Phosphofructokinase-1/isolation & purification , Animals , Bivalvia , Electrophoresis, Polyacrylamide Gel , Frozen Sections , Fructosephosphates/metabolism , Hydrogen-Ion Concentration , In Vitro Techniques , Kinetics , Phosphofructokinase-1/metabolism , Phosphorylation , Tissue PreservationABSTRACT
A critical part of natural freeze tolerance is the production of low molecular weight cryoprotectants; in freeze tolerant frogs this involves a freezing-induced activation of liver glycogenolysis that leads to the accumulation of glucose as the cryoprotectant, in amounts up to 300 mM, in all organs. The present study shows that the synthesis and maintenance of high organ glucose pools is facilitated by changes in the levels of fructose-2,6-bisphosphate (F2,6P2) and an inhibition of liver 6-phosphofructo-2-kinase (PFK-2) activity that blocks the catabolism of glucose by glycolysis. Freezing exposure (24 h at -2.5 degrees C) resulted in a sharp drop in F2,6P2 levels in four organs, to 23-75% of control values, but F2,6P2 rebounded when frogs were thawed. Freezing also stimulated changes in the properties of liver PFK-2 including a decrease in maximal velocity, a basic shift in pH optimum, a 10-fold increase in Km for fructose-6-phosphate, and increased I50 values for enzyme inhibitors. I50 values for glycerol-3-phosphate and phosphoenolpyruvate were 60- and 2.4-fold higher, respectively, for liver PFK-2 from frozen frogs compared with controls. Changes in liver PFK-2 properties are consistent with a freezing-induced phosphorylation of the enzyme to produce a less active enzyme form, resulting in reduced organ F2,6P2 levels and a decrease in 6-phosphofructo-1-kinase activity.
Subject(s)
Adaptation, Physiological , Cryoprotective Agents/metabolism , Freezing , Glucose/metabolism , Phosphotransferases/antagonists & inhibitors , Ranidae/physiology , Allosteric Regulation , Animals , Fructosediphosphates/metabolism , Male , Phosphofructokinase-2ABSTRACT
Fructose-2,6-bisphosphatase (FBPase-2) from the mantle tissue of the mussel Mytilus galloprovincialis shows a hyperbolic kinetic with a Km value (0.40 mM) for its substrate, that suggest that the "in vivo" Fru-2,6-P2 concentration is not a limiting factor for activity. The enzyme possesses an optimum pH for activity between 6 and 7 units, similar to the reached in mussel mantle during physiological hypoxia. The modulation of activity by the pH, and in addition, the positive effect of ATP are in keeping with the little decrease in concentration of the Fru-2,6-P2 that occurs during the first hour of hypoxia due to the valve closure.
Subject(s)
Bivalvia/metabolism , Fructosediphosphates/metabolism , Phosphoric Monoester Hydrolases/metabolism , Adenosine Triphosphate/pharmacology , Animals , Cell Hypoxia , Enzyme Activation , Hydrogen-Ion Concentration , Phosphofructokinase-2ABSTRACT
PKF-2 from mussel mantle was phosphorylated by cAMP-dependent protein kinase. The phosphorylation does not change the enzyme activity at neutral pH values, but at acid pH the activity of the phosphorylated form is higher than the native PFK-2. With respect to the native enzyme, the activation consisted of a reduction in the Km for Fru-6-P and a decrease in the inhibitory effect of PEP. These results are in keeping with the stabilized concentration of Fru-2,6-P2 found in the mussel mantle during the physiological hypoxia caused by the closure of the valves.
