In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Energy aspects of the growth of Escherichia coli synchronized by starvation

The quantitative determination of adenyl nucleotides based on the separation of their dansyl derivatives by thin layer chromatography has made it possible to study the dynamics of changes in the pool of ATP, ADP and AMP in Escherichia coli K-12 during its synchronous growth after glucose starvation. The energy parameters (the adenylate pool, energy charge, teh ATP/ADP ratio, the rates of oxygen uptake and ATP generation, the economic coefficients of oxygen and ATP utilization) were compared with changes in the growth characteristics (the rate of growth and biomass concentration). This comparison allowed the authors to draw the conclusion about the uncoupled constructive and energy metabolism and about the possible regulatory role of energy parameters in the synchronised culture growth.     

Cellular energy systems and reserpine ulcer in rats

Gastric ulcer was elicited in rats by reserpine (5 mg x kg-1 sc.) administration. Ulcer formation (number and severity) was measured 6, 12, 18 and 24 hr after reserpine administration. At the time of killing of the animals, tissue levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP) were measured enzymatically and by radioimmunoassay in the gastric fundal mucosa. The sum of ATP + ADP + AMP (adenylate pool) and the ratio of ATP x ADP-1 were calculated. It was found that (1) the tissue levels of ATP, AMP, cAMP, sum of ATP / ADP + AMP (adenylate pool) and ratio of ATP x ADP-1 increased significantly in the gastric fundal mucosa 6 hr after reserpine administration, thereafter these values decreased gradually and significantly; (2) the tissue level of ADP increased significantly in the gastric fundal mucosa 6 hr after reserpine administration, meanwhile its level increased significantly at 18 and 24 hr; (3) the value of energy charge (ATP + 0.5 ADP x ATP + ADP + AMP-1) remained unchanged; (4) the peaks of biochemical alterations in the gastric fundus mucosa preceded he appearance of ulcers. It was concluded that (1) reserpine ulcer appears after an active metabolic response in the rat gastric fundal mucosa; (2) hypoxaemic damage in the gastric fundal mucosa can be excluded as a possible underlying mechanism of ulcer formation produced by reserpine administration; (3) before the appearance of reserpine ulcer, significant changes in the feedback mechanism, system, i.e. between the ATP--membrane ATPase--ADP and the ATP--adenylate cyclase--cAMP energy systems, can be observed in the rat gastric fundal mucosa.     

Changes of gastric mucosal biochemistry in ethanol-treated rats with and without acute surgical vagotomy

The biochemical background of ethanol-(ETOH) induced gastric mucosal damage was studied in rats with intact vagus and after acute surgical vagotomy. Observations were carried out on Sprague-Dawley (CFY) strain rats of both sexes. Gastric mucosal lesions were produced by intragastric administration of 1 ml 96% ethanol. Bilateral truncal surgical vagotomy was carried out 30 min before ETOH administration. The number and severity of gastric mucosal lesions was noted 1 h after ETOH administration. Biochemical measurements (gastric mucosal level of ATP, ADP, AMP, cAMP and lactate) were carried out from the total homogenized gastric mucosa. The adenylate pool (ATP + ADP + AMP), energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) and ratio of ATP/ADP were calculated. It was found that: 1) ATP transformation into ADP increased, while ATP transformation in cAMP decreased in ethanol-treated animals with intact vagus nerve, while these transformations were quite the opposite in vagotomized animals; 2) no significant changes were found in the tissue level of lactate: and 3) the extent of biochemical changes was significantly less after surgical vagotomy. It is concluded that an intact vagus is basically necessary for the metabolic adaptation of gastric mucosa.     

Glucose Deprivation Converts Poly(ADP-ribose) Polymerase-1 Hyperactivation into a Transient Energy-producing Process

Massive poly(ADP-ribose) formation by poly(ADP-ribose) polymerase-1 (PARP-1) triggers NAD depletion and cell death. These events have been invariantly related to cellular energy failure due to ATP shortage. The latter occurs because of both ATP consumption for NAD resynthesis and impairment of mitochondrial ATP formation caused by an increase of the AMP/ADP ratio. ATP depletion is therefore thought to be an inevitable consequence of NAD loss and a hallmark of PARP-1 activation. Here, we challenge this scenario by showing that PARP-1 hyperactivation in cells cultured in the absence of glucose (Glu⁻ cells) is followed by NAD depletion and an unexpected PARP-1 activity-dependent ATP increase. We found increased ADP content in resting Glu⁻ cells, a condition that counteracts the increase of the AMP/ADP ratio during hyperpoly(ADP-ribosyl)ation and preserves mitochondrial coupling. We also show that the increase of ATP in Glu⁻ cells is due to adenylate kinase activity, transforming AMP into ADP which, in turn, is converted into ATP by coupled mitochondria. Interestingly, PARP-1-dependent mitochondrial release of apoptosis-inducing factor (AIF) and cytochrome complex (Cyt c) is reduced in Glu⁻ cells, even though cell death eventually occurs. Overall, the present study identifies basal ADP content and adenylate kinase as key determinants of bioenergetics during PARP-1 hyperactivation and unequivocally demonstrates that ATP loss is not metabolically related to NAD depletion.     

Effects of melatonin on 3-nitrotyrosine formation and energy charge ratio in guinea pig kidney in LPS-induced stress

The aim of this study was to evaluate the effects of Escherichia coli-derived lipopolysaccharide on guinea pig kidney by measuring the energy charge ratio and 3-nitrotyrosine levels. In addition the possible protective role of melatonin against lipopolysaccharide-mediated peroxynitrite formation and energy depletion of kidney was determined. Guinea pigs were either pretreated with melatonin or saline (for the control) followed by intraperitoneal administration of E. coli. Six hours after the administration of E. coli, guinea pig kidney ATP, ADP, AMP and 3-nitrotyrosine levels were measured by reverse-phase high performance liquid chromatography. There was a significant increase in the formation of 3-nitrotyrosine and decrease in energy charge in the endotoxin-induced group. However melatonin administration prevented 3-nitrotyrosine formation while failing to prevent or restore changes in the energy charge ratio of the kidney.     

Luminometric assays of ATP, phosphocreatine, and creatine for estimation of free ADP and free AMP.

We present methods to measure ATP, phosphocreatine, and total creatine (the sum of creatine and phosphocreatine) in alkaline cell extracts. Knowledge of these parameters, together with the known equilibrium constants for the creatine kinase and adenylate kinase-catalyzed reactions, allows one to estimate the levels of free ADP and free AMP inside cells. The enzymatic assays for the above-mentioned metabolites all lead up to the production of ATP, which is measured luminometrically with the ATP-dependent oxidation of luciferin catalyzed by firefly luciferase. To determine phosphocreatine, endogenous ATP is first destroyed, and phosphocreatine is then quantitatively reacted with exogenous ADP to form ATP. Total creatine is measured after quantitative conversion of creatine to phosphocreatine with a large excess of exogenous ATP, conversion of all ATP to ADP, and final reaction of phosphocreatine with ADP to form ATP. We used 5-microl samples in 0.5-ml microcentrifuge tubes and subsequent 5-microl additions of analytical reagents. We expect that the volumes can be changed easily. We tested the methods with glucagon- and insulin-secreting cells. Estimates of free ADP and AMP are expected to be useful in many different areas of research, such as cellular energy metabolism, purine nucleotide metabolism, adenine nucleotide gating of ion channels, and release of vasoactive or angiogenic factors.     

Control of chlorophyll synthesis in Chromatium vinosum

Summary The role of adenine nucleotides in the control of the energy metabolism of Chromatium has been studied through the measurement of the levels of ATP ADP, and AMP in growing cultures. Comparison of these levels with the cellular chlorophyll levels indicates that ATP concentration, and not those of ADP, AMP, or any function of concentration of these three nucleotides, is a controlling factor in chlorophyll synthesis. In addition, the relationship of cellular ATP and chlorophyll levels to sulfur metabolism furnishes further evidence for the existence of two photosystems, postulated previously. The effects of high levels of exogenous ATP and ADP are consistent with these findings.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.     

Interrelationships between the membrane-bound ATP-dependent energy systems of the gastric mucosa and the gastric cytoprotective effect of beta-carotene on the development of gastric mucosal damage produced by 0.6 M HCl in rats

The effects of different doses (0.01-0.1-1.0-10.0/mg/kg-1) of beta-carotene were studied on gastric secretory responses of 4 hr pylorus-ligated rats: development of gastric mucosal damage (as assessed by number and severity of lesions) produced by intragastric administration of 0.6 M HCl; tissue level of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenylate pool (ATP + ADP + AMP), ratio of ATP X ADP-1, "energy charge" (ATP + 0.5 ADP X X (ATP + ADP + AMP)-1) (during the development of gastric mucosal damage by 0.6 M HCl and of gastric cytoprotection by beta-carotene. It was found that beta-carotene did not decrease the gastric secretory responses of 4 hr pylorus-ligated rats; The development of gastric mucosal damage could be decreased dose-dependently by the administration of beta-carotene; the ATP transformation could be decreased by beta-carotene; the tissue levels of cAMP and AMP could be increased significantly and dose-dependently by beta-carotene; the ratio of ATP X ADP-1 could be increased significantly and dose-dependently by beta-carotene; the values of adenylate pool and "energy charge" remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

NTPDase and 5′-nucleotidase Activities of Synaptosomes from Hippocampus of Rats Subjected to Hyperargininemia

ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system. Since the ecto-nucleotidase cascade that hydrolyzes ATP to adenosine is involved in the control of brain functions and previous studies realized in our laboratory have recently reported that acute administration of Arg decreases the NTPDase and 5′-nucleotidase activities of rat blood serum, in the present study we investigated the effect of arginine administration on NTPDase and 5′-nucleotidase activities by synaptosomes from hippocampus of rats. First, sixty-days-old rats were treated with a single or a triple intraperitoneal injection of arginine (0.8 g/Kg) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. Second, rats received an intracerebroventricular injection of 1.5 mM arginine solution or saline (5 μL) and were killed 1 h later. We also tested the in vitro effect of arginine (0.1–1.5 mM) on nucleotide hydrolysis in synaptosomes from rat hippocampus. Results showed that intraperitoneal arginine administration did not alter nucleotide hydrolysis. On the other hand, arginine administered intracerebroventricularly reduced ATP (32%), ADP (30%) and AMP (21%) hydrolysis, respectively. In addition, arginine added to the incubation medium, provoked a decrease on ATP (19%), ADP (17%) and AMP (23%) hydrolysis, respectively. Furthermore, kinetic studies showed that the inhibitory effect of arginine was uncompetitive in relation to ATP, ADP and AMP. In conclusion, according to our results it seems reasonable to postulate that arginine alters the cascade involved in the extracellular degradation of ATP to adenosine.     

Effects of cimetidine administered in cytoprotective and antisecretory doses on the membrane-bound ATP-dependent energy systems in the gastric mucosal lesions induced by HCl in rats

CFY strain rats (both sexes, 180-210 g) were fasted for 24 hr. Different doses of cimetidine (2.5, 10 and 50 mg X kg-1 i.p.) were given 30 min prior to the gastric mucosal lesions induced by the intragastric application of 0.6 M HCl. Animals were sacrificed 1 hr after the administration of the necrotizing agent. The number of gastric lesions was determined and their severity scored. Samples from the gastric fundic mucosa were taken for biochemical analysis. The tissue levels of adenosine-5'-triphosphate (ATP), adenosine-5'-diphosphate (ADP), adenosine-5'-monophosphate (AMP) and L-(+)-lactate were determined enzymatically, while the tissue contents of cyclic 3',5' adenosine monophosphate were measured by radioimmunoassay. The values for adenylate pool (ATP + ADP + AMP)-1 were calculated. All biochemical results were computed for 1.0 mg mucosal protein. We found that (1) the levels of ADP and lactate rose significantly, while ATP, AMP, cAMP, ATP X ADP-1 and energy charge decreased during the development of gastric lesions induced by HCl: (2) cimetidine decreased dose-dependently the number and severity of lesions: (3) the levels of ATP, ADP X ADP-1, and energy charge were increased dose-dependently by cimetidine, while AMP and lactate were decreased: (4) the levels of ADP, adenylate pool and cAMP did not change significantly by cimetidine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Content of Adenosine Phosphate Compounds in a Long-Day Duckweed, Lemna gibba G3, under Different Light and Nutritional Conditions

The levels of ATP ADP and AMP as well as the energy charge were examined in the long-day duckweed. Lemna gibba G3, under different light and nutritional conditions. ATP and ADP content, but not AMP, decreased slightly when the plant was cultured with a medium depleted of sucrose or was exposed to continuous darkness. The energy charge was not affected either by changes in the light conditions or by the depletion of sucrose from the medium, although increase of fresh weight changed drastically under these conditions. The levels of ADP and AMP and the value of the energy charge remained nearly constant throughout a 1-day period when the plant was exposed to short-day, continuous light and continuous dark. ATP content decreased gradually during the light period of the short day and thereafter remained constant during the dark period. When the plant was exposed to continuous light. ATP content decreased until the 8th hour after beginning of the continuous light, only to recover rapidly to its original level during the phase coinciding with the preceding dark period of the short day. Under continuous dark conditions the ATP content remained constant throughout the day. It was concluded that the diurnal rhythm of physiological activities previously reported cannot be related to the energy charge.     

Enhanced hippocampal F2-isoprostane formation following kainate-induced seizures

We attempted to obtain evidence for the occurrence of oxidant injury following seizure activity by measuring hippocampal F2-isoprostanes (F2-IsoPs), a reliable marker of free radical-induced lipid peroxidation. Formation of F2-IsoPs esterified in hippocampal phospholipids was correlated with hippocampal neuronal loss and mitochondrial aconitase inactivation, a marker of superoxide production in the kainate model. F2-IsoPs were measured in microdissected hippocampal CA1, CA3 and dentate gyrus (DG) regions at various times following kainate administration. Kainate produced a large increase in F2-IsoP levels in the highly vulnerable CA3 region 16 h post injection. The CA1 region showed small, but statistically insignificant increases in F2-IsoP levels. Interestingly, the DG, a region resistant to kainate-induced neuronal death also showed marked (2.5-5-fold) increases in F2-IsoP levels 8, 16, and 24 h post injection. The increases in F2-Isop levels in CA3 and DG were accompanied by inactivation of mitochondrial aconitase in these regions. This marked subregion-specific increase in F2-Isop following kainate administration suggests that oxidative lipid damage results from seizure activity and may play an important role in seizure-induced death of vulnerable neurons.     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.     

Involvement of p38alpha in kainate-induced seizure and neuronal cell damage

We investigated how p38alpha mitogen-activated protein kinase (p38) is related to kainate-induced epilepsy and neuronal damages, by using the mice with a single copy disruption of the p38 alpha gene (p38alpha(+/-)). Mortality rate and seizure score of p38alpha(+/-) mice administered with kainate were significantly reduced compared with the case of wild-type (WT) mice. This was clearly supported by the electroencephalography data in which kainate-induced seizure duration and frequency in the brain of p38alpha(+/-) mice were significantly suppressed compared to those of WT mice. As a consequence of seizure, kainate induced delayed neuronal damages in parallel with astrocytic growth in the hippocampus and ectopic innervation of the mossy fibers into the stratum oriens in the CA3 region of hippocampus in WT mice, whose changes were moderate in p38alpha(+/-) mice. Likewise, kainate-induced phosphorylation of calcium/calmodulin-dependent kinase II in the hippocampus of p38alpha (+/-) mice was significantly decreased compared to that of WT mice. These results suggest that p38alpha signaling pathway plays an important role in epileptic seizure and excitotoxicity.     

A kinetic study of the rat liver adenosine kinase reverse reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP --> AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP --> ATP + inosine + NH(3). The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.