O. Barnabei

Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K+ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R‐phenylisopropyladenosine. All these effects were reversed by the selective adenosine A1 receptor antagonist 8‐cyclo‐pentyltheophylline. In the same synaptosomal preparation, K+ (30 mM) strongly stimulated the release of the preloaded [3H]adenosine in a partially Ca2+‐dependent and tetrodotoxin (TTX)‐sensitive manner. Moreover, in the same experimental conditions, both l‐glutamate and l‐aspartate enhanced the release of [3H]adenosine derivatives ([3H]ADD). The gluta‐mate‐evoked release was dose dependent and appeared to be Ca2+ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d‐glutamate and d‐aspartate were able to stimulate [3H]ADD release. In contrast, the specific glutamate agonists N‐methyl‐d‐aspartate, kainate, and quisqualate failed to stimulate [3H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg2+ and l‐glutamate‐evoked release was not inhibited by adding the specific antagonists 2‐amino‐5‐phosphonovaleric acid or 6–7‐dinitroquinoxaline‐2, 3‐dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ‐anunobutyric acid stimulated [3H]ADD release in a dose‐related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each others release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.

Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Abstract: Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome‐enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+‐dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40–80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D‐[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+‐stimulated release. Kainic acid at 0.1 mM had no clear‐cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier‐mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high‐affinity uptake of L‐glutamate and D‐aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.

Role of chromatin phospholipids on template availability and ultrastructure of isolated nuclei.

The influence of phospholipid vesicles has been tested on isolated nuclei by evaluating the endogenous DNA-dependent RNA polymerase and the chromatin ultrastructure. Negatively charged phosphatidylserine liposomes have a stimulating effect on RNA synthesis, while the vesicles obtained with the neutral sphingomyelin, phosphatidylethanolamine and phosphatidylcholine, and the acidic phosphatidylinositol, are inhibitory. The enhancement of transcription by phosphatidylserine seems to be due to both elongation and initiation of RNA transcripts and very likely depends on interactions of the lipid with the template rather than with the enzymes. The morphological analysis indicates deep changes of the chromatin organization, mainly concerning the size and distribution of the fibers, without any variation of the nuclear volume. The rearrangement of the chromatin could account for the variations of the template availability for RNA synthesis induced by the vesicles and might be associated with changes of the nuclear matrix.

Protection from kainic acid neuropathological syndrome by NMDA receptor antagonists: Effect of MK-801 and CGP 39551 on neurotransmitter and glial markers

Systemic administration of kainic acid results in the development of a characteristic convulsive syndrome, accompanied by neuropathological alterations and loss of transmitter markers in some forebrain regions. Since some of these effects appear to involve the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors, the protection given by a non-competitive (MK-801) and a competitive (CGP 39551) NMDA receptor antagonist against the loss of glutamatergic and gamma-amino butyric acid (GABAergic) neurochemical markers was compared. Appropriate doses of both compounds (1 mg/kg MK-801 and 25 mg/kg CGP 39551) completely reversed the decrease of high affinity uptake of glutamate and activity of glutamate decarboxylase in the olfactory cortex, amygdala, hippocampus and lateral septum. In addition, they also essentially counteracted the increase of a glial marker, the enzyme glutamine synthetase, consequent to neuronal degeneration. The results confirmed that involvement of NMDA receptors is essential for the full expression of neuropathological effects of kainic acid. They also support the use of a competitive antagonist of the NMDA receptor, such as CGP 39551, to afford substantial protection against the excitotoxic damage, whilst giving fewer side effects and motor disturbances than MK-801.

Temporal, regional and cellular selectivity of neonatal alteration of the thyroid state on neurochemical maturation in the rat

SummaryThe effects of alteration of thyroid state on neurochemical maturation have been studied in rats made hypothyroid by daily injections of methimazole or hyperthyroid by daily supplementation with thyroid hormone (T3) from postnatal days 1 to 27. Biochemical assays on seven brain regions plus the spinal cord were carried out on 14 and 28 day-old rats as well as in adult rats after at least 40 days of recovery. 2′,3′cyclic nucleotide phosphohydrolase (CNPase), a specific marker for oligodendrocytes and myelination was significantly decreased in all regions except the spinal cord of hypothyroid rats. The astrocytic marker glutamine synthetase (GS) was slightly increased in the hippocampus of hypothyroid rats. Choline acetyltransferase (ChAT), a specific marker for cholinergic neurons, was decreased in the prefrontal and visual cortices, the striatum and the superior colliculus and increased in the cerebellum of hypothyroid rats; in addition, the enzyme activity was increased in the prefrontal cortex and striatum and decreased in the cerebellum of hyperthyroid rats. Acetyl-cholinesterase (AChE) activity was decreased in the prefrontal cortex and in the striatum of hypothyroid rats while 3H-quinuclidynil benzilate (QNB) muscarinic binding was decreased in all cortical areas and in the hippocampus of hypothyroid rats. Glutamate decarboxylase (GAD), a specific marker for GABAergic neurons, was decreased in the cortical areas of hypothyroid rats. Aromatic amino acid decarboxylase (AAD), a general marker for monoaminergic neurons, was unaffected. Alteration of neurochemical parameters was never observed in the spinal cord. Under our experimental conditions, the effects of alteration of thyroid state appeared graded and selective with respect to temporal, regional and cellular parameters.

Chromatin lipids and their possible role in gene expression. A study in normal and neoplastic cells

Abstract Phospholipids have been demonstrated to affect the thermal stability, the ultrastructure and the template activity both of DNA and nucleohistones in vitro. A relationship has been observed between the relative composition of the phospholipid fraction bound to NHCP and the metabolic condition of normal and neoplastic cells. These findings may suggest that gene expression may involve other regulatory molecules besides chromosomal proteins.

Conformational changes of nuclear chromatin related to phospholipid induced modifications of the template availability.

The phospholipid involvement in the regulation of the functional and structural properties of isolated nuclei has been studied by analyzing the composition and the possible function of the nuclear matrix bound phospholipids in rat liver and murine erythroleukemic cells. The digestion of the matrix phospholipids with phospholipases results in the release of essentially all the newly replicated matrix DNA. The exogenous addition of liposomal phosphatidylserine to rat liver nuclei induces chromatin structural changes consisting in a disaggregation of the heterochromatin, probably mediated by the matrix remodeling, and in a transition from the solenoid fiber to the nucleosome filament, due to the removal of the histone H1. These effects occur through a direct interaction of phospholipid molecules with the inner nuclear components, as demonstrated by carboxyfluorescein transfer and electron microscope autoradiography.

Regional maturation of neurotransmitter-related and glial markers during postnatal development in the rat

Neurotransmitter‐related (choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase, l‐glutamate and GABA high affinity uptake) and glial neurochemical markers (glutamine synthetase, β‐alanine uptake and 2′,3′ cyclic nucleotide phosphohydrolase) have been quantitatively assayed in various regions of the rat CNS during normal postnatal development: spinal cord, cerebellum, superior colliculus, hippocampus, striatum, visual cortex, frontal sensory‐motor cortex and prefrontal cortex. In general, neurochemical markers show an obvious trend toward increasing levels in parallel with brain maturation. However, some relevant exceptions have been observed and discussed. Detailed knowledge of regional neurochemical brain maturation is important since it gives us information concerning some key events of brain development. In addition, this knowledge is the essential pre‐requisite for studies aimed at the alteration of specific regional and temporal parameters through experimental manipulation.

Nuclear polyphosphoinositides during cell growth and differentiation

When highly purified nuclei of Swiss mouse 3T3 cells are incubated with gamma-32P-ATP, radioactivity is incorporated into phosphatidic acid and the two polyphosphoinositol lipids, phosphatidylinositol(4)P and (4,5)P2. If the cells are pre-treated with IGF-I, the incorporation into the polyphosphoinositides is decreased. This effect is maximal by 2 min, is transient in that it disappeared by 1 hr, and is increased markedly by the co-addition of bombesin, even though bombesin alone has no effect. Friend cells exhibit a related phenomenon in that the labelling of PIP2 in isolated nuclei is increased by conditions which cause erythroid differentiation (DMSO addition). We suggest that some aspect of nuclear polyphosphoinositide metabolism is modified when the nucleus is induced to divide or to differentiate, and that this change in inositide metabolism is a very early event in the sequence leading to cell division or differentiation.

Lipid-dependent nuclear signalling: Morphological and functional features

Enzymes involved in lipid metabolism exist within the nucleus and are responsive to external stimuli. In particular, the kinases which phosphorylate phosphatidylinositol and phosphatidylinositol-4-monophosphate have been demonstrated in nuclei of both undifferentiated and differentiated Friend cells and of quiescent Swiss 3T3 cells as well as of those exposed to insulin-like growth factor I. Besides the lipid kinases, also the phosphoinositidases C (PIC) are active inside the nucleus. In Swiss 3T3 cells the nuclear PIC beta 1 is activated and its activation by IGF-I temporally precedes the translocation to the nucleus of protein kinase C. In Friend cell nuclei, on the other hand, when erythroid differentiation is induced, the PIC beta 1 activity is reduced. Another aspect of the nuclear signalling transduction system which appears quite interesting is its actual localization at subcellular level. By using electron microscope immunogold labelling, the nuclear PIC isoforms (the beta 1 isoform in Swiss 3T3 cells, the beta 1 and gamma 1 in Friend cells) are localized mainly in the interchromatin domains. This localization has been further confirmed on in situ matrix preparations of 3T3 cells in which PIC beta 1 is associated with the inner nuclear matrix but not with the nuclear pore-lamina complex. Colocalization experiments indicate that nuclear PIC beta 1 is present in sites in which both nuclear phospholipids and PKC can be detected, while the cytoplasmic PIC gamma 1 can be identified in close association with cytoskeletal filaments identified by anti-actin antibodies. The precise localization of the different PIC isoforms strongly indicates that the signal transduction system operating at the nuclear level may be part of a cross-talk between the cytoplasm and the nucleus controlling either cell proliferation or differentiation.