Library

Notes: The Lagrangian proposed by York et al. and the covariant first-order Lagrangian for general relativity are reviewed. They both deal with the (vacuum) gravitational field on a reference background and were conjectured to be equivalent. The two corresponding actions are compared and we show that the first one can in fact be obtained from the latter under suitable hypotheses. A conditioned correspondence among Nöther conserved quantities of covariant first-order Lagrangian, Brown–York quasilocal energy and the standard ADM Hamiltonian is also established. © 2001 American Institute of Physics.

Notes: Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [3H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC5017.2 μM) and Asp (EC50 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na+ dependent. Glu enhanced the release of [3H]GABA (EC50 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d-aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na+ sensitive. Similarly to Glu, D-Asp increased [3H]GABA release (EC50 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [3H]D-Asp outflow (EC50 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na+ dependent. Glu increased the release of [3H]- GABA from hippocampal synaptosomes (EC50 7.1 μM) in an N-methyl-d-aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na+ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.

Notes: Abstract: The release of cholecystokinin-like immunoreactivity (CCK-LI) from the frontal cortex of freely moving rats has been studied using a transcerebral microdialysis technique coupled to a radioimmunoassay procedure. Basal levels of CCK-LI in the dialysate were above detection limits (2.4 ± 0.7 pg/20 min; n = 8). High-K+ media evoked CCK-LI overflow in a concentration-dependent manner. The threshold concentration was 50 mM KCI. The peak overflow evoked by 100 mM K+ amounted to 42.7 ± 2.8 pg/20 min (n = 6); it was totally Ca2+ dependent but insensitive to 1 μM tetrodotoxin. Infusion of 4-aminopyridine (1 mM; 20 min) evoked an overflow of CCK-LI (32 ± 2.3 pg/ 20 min; n = 4), wnich was totally Ca2+ dependent and tetrodotoxin sensitive. Depolarization with 100 μg/ml of veratrine (20 min) provoked a CCK-LI overflow (62.2 ± 10 pg/20 min; n = 6), which was also blocked by tetrodotoxin or by the absence of Ca2+ ions. The CCK-LI material collected under basal conditions or during veratrine infusion consisted essentially of CCK octapeptide sulfate. The veratrine-induced CCK-LI overflow did not change significantly when the infusion time was prolonged to 100 min. A second 20-min stimulus with 100 μg/ml of veratrine applied 200 min after a first 20-min stimulus evoked a barely significant CCK-LI overflow. These data suggest that one single 20-min stimulus with 100 μg/ml of veratrine may be sufficient to deplete the CCK-LI releasable stores and that 〉200 min are required to replenish the depleted CCK-containing vesicles. Taken together the data allow us to conclude that the physiology and the pharmacology of CCK release can be adequately studied in vivo by brain microdialysis.

Notes: Abstract: The ability of γ-aminobutyric acid (GABA) and glycine (Gly) to modulate each other's release was studied in synaptosomes from rat spinal cord, cerebellum, cerebral cortex, or hippocampus, prelabeled with [3H]GABA or [3H]-Gly and exposed in superfusion to Gly or to GABA, respectively. GABA increased the spontaneous outflow of [3H]Gly (EC50, 20.8 μM) from spinal cord synaptosomes. Neither muscimol nor (-)-baclofen, up to 300 μM, mimicked the effect of GABA, which was not antagonized by either bicuculline or picrotoxin. However, the effect of GABA was counteracted by the GABA uptake inhibitors nipecotic acid and N-(4,4-diphenyl-3-butenyl)nipecotic acid. Moreover, the GABA-induced [3H]Gly release was Na+ dependent and disappeared when the medium contained 23 mM Na+. The effect of GABA was Ca2+ independent and tetrodotoxin insensitive. Conversely, Gly enhanced the outflow of [3H]-GABA from rat spinal cord synaptosomes (EC50, 100.9 μM). This effect was insensitive to both strychnine and 7-chlorokynurenic acid, antagonists at Gly receptors, but it was strongly Na+ dependent. Also, the Gly-evoked [3H]-GABA release was Ca2+ independent and tetrodotoxin insensitive. GABA increased the outflow of [3H]Gly (EC50, 11.1 μM) from cerebellar synaptosomes; the effect was not mimicked by either muscimol or (—)-baclofen nor was it prevented by bicuculline or picrotoxin. The GABA effect was, however, blocked by GABA uptake inhibitors and was Na+ dependent. Gly increased [3H]GABA release from cerebellar synaptosomes (EC50, 110.7 μM) in a strychnine- and 7-chlorokynurenic acid-insensitive manner. This effect was Na+ dependent. The effects of GABA on [3H]Gly release seen in spinal cord and cerebellum could be reproduced also with cerebrocortical synaptosomes. However, in cortex, the effect of Gly on [3H]GABA release was much lower than in spinal cord or cerebellum, although it was partly Na+ dependent. No changes of [3H]Gly release were observed in hippocampal synaptosomes exposed to GABA. It is suggested that transporters specific for GABA or Gly are colocalized on the same nerve terminal in rat spinal cord, cerebellum, and cerebral cortex, but not in hippocampus. Moreover, GABA uptake modulates Gly release and, at least in spinal cord and cerebellum, Gly uptake modulates GABA release. These conclusions are compatible with the reported coexistence of GABA and Gly in spinal and cerebellar neurons.

Notes: Rat cerebral cortex synaptosomes were exposed in superfusion to various depolarizing stimuli and the release of somatostatin-like immunoreactivity (SRIF-LI) was measured by means of a radioimmunoassay procedure. High KC1 (9-50 mM) concentration dependently evoked SRIF-LI release; the evoked overflow reached a plateau at 25 mM KC1 and was completely abolished when Ca2+ ions were omitted from the superfusion medium, independently of the concentration of KC1 used. The 15 mM K+-evoked release of SRIF- LI increased sharply as the Ca2+ concentration was raised to 0.8 mM, then leveled off and reached a plateau at 1.2 mM. The 15 mM K+-evoked overflow, but not the spontaneous outflow, was partially decreased (50%) by 1 μM tetrodotoxin. The presence in the superfusion fluid of a mixture of peptidase inhibitors did not improve the recovery of SRIF-LI both in the absence and in the presence of high K+. Exposure of synaptosomes to veratrine (1-50 μM) induced release of SRIF-LI in a concentration-dependent way. The effect of the alkaloid was strictly Ca2+ and tetrodotoxin sensitive. Replacement of extracellular Na+ by sucrose caused an acceleration of the spontaneous SRIF-LI outflow that was inversely correlated to the Na+ content in the superfusion medium. The release evoked by the sodium-deprived media did not exhibit any calcium dependence. HPLC analysis of the samples collected during superfusion showed that 〉90% of the SRIF-LI released either during the spontaneous outflow or by 15 mM KC1 was represented by SRIF-14 (SRIF-2814-28). These values reflected the ratio SRIF-14/SRIF-28 found in synaptosomes at the end of the experiments.

Notes: Abstract: Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [3H]acetylcholine and [3H]noradrenaline from rat hippocampal synaptosomes and of [3H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca2+ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [3H]noradrenaline, leaving unaffected that of [3H]acetylcholine or [3H]dopamine. 1,2-Bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [3H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [3H]dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [3H]noradrenaline but not that of [3H]acetylcholine or [3H]dopamine. It is concluded that (a) the release of [3H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca2+]-independent releases of [3H]acetylcholine and [3H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca2+, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca2+.

Notes: Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase-2 (COX-2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up-regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX-2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE2 and 8-epi-PGF2α following infusion of N-methyl-d-aspartate (NMDA). A 20-min application of 1 mm NMDA caused an immediate, MK-801-sensitive increase of both PGE2 and 8-epi-PGF2α basal levels. These effects were largely prevented by the specific cytosolic phospholipase A2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non- selective COX inhibitors indomethacin and flurbiprofen or by the COX-2 selective inhibitor NS-398, suggesting that the NMDA-evoked prostaglandin synthesis and free radical-mediated lipid peroxidation are largely dependent on COX-2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX-2 activity.