Alessandro Poli

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.

PLC and PI3K/Akt/mTOR signalling in disease and cancer

Cancer cell metabolism is deregulated, and signalling pathways can be involved. For instance, PI3K/Akt/mTOR is associated with normal proliferation and differentiation, and its alteration is detectable in cancer cells, that exploit the normal mechanisms to overcome apoptosis. On the other hand, also the family of Phospholipase C (PLC) enzymes play a critical role in cell growth, and any change concerning these enzymes or their downstream targets can be associated with neoplastic transformation. Here, we review the role of PLC and PI3K/Akt/mTOR signal transduction pathways in pathophysiology.

Regional distribution of nitric oxide synthase and NADPH-diaphorase activities in the central nervous system of teleosts.

Neuronal nitric oxide synthase (nNOS) and NADPH-diaphorase activities were investigated in discrete areas of the central nervous system of goldfish and brown trout. Both species showed a similar distribution pattern of nNOS activity with regional differences in all examined areas. Telencephalon and hypothalamus showed the highest nNOS values, while in the goldfish cerebellum and its valvula nNOS was not detectable. In both species, NADPH-diaphorase activity showed a lower regional variability, compared to nNOS. The highest activity was measured in the olfactory bulbs where, conversely, low levels of nNOS activity were present. The non close correspondence between NOS and NADPH-diaphorase activities confirms the discrepancies indicated by morphological data. Western blot analysis revealed the presence of a nNOS isoform of about 150 kDa mol. wt. corresponding to that of mammals. The pattern of nNOS expression in the considered brain regions of the goldfish and trout was comparable to the levels of the nNOS activity.

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in goldfish brain

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which selectively damages dopaminergic neurons in mammals, caused a marked depletion of tyrosine hydroxylase (TH) immunoreactivity in the goldfish brain. The concomitant ultrastructural observations showed the neurotoxic effect of MPTP on telencephalic, diencephalic and medullar neurons. The affected neurons revealed darkening of the cytoplasm and swelling of the mitochondria and the endoplasmic reticulum. Concomitant significant decreases in dopamine (DA) and noradrenaline (NA) levels were determined in the brain areas where morphological observations were performed. The loss of catecholamine levels was completely prevented by the treatment with the monoamine oxidase (MAO) inhibitor pargyline to prevent MPTP oxidation. The results indicate that in goldfish brain, acute MPTP administration causes selective catecholamine depletion, without altering the serotoninergic system.

Protein kinase C involvement in cell cycle modulation

Protein kinases C (PKCs) are a family of serine/threonine kinases which act as key regulators in cell cycle progression and differentiation. Studies of the involvement of PKCs in cell proliferation showed that their role is dependent on cell models, cell cycle phases, timing of activation and localization. Indeed, PKCs can positively and negatively act on it, regulating entry, progression and exit from the cell cycle. In particular, the targets of PKCs resulted to be some of the key proteins involved in the cell cycle including cyclins, cyclin-dependent kinases (Cdks), Cip/Kip inhibitors and lamins. Several findings described roles for PKCs in the regulation of G₁/S and G₂/M checkpoints. As a matter of fact, data from independent laboratories demonstrated PKC-related modulations of cyclins D, leading to effects on the G₁/S transition and differentiation of different cell lines. Moreover, interesting data were published on PKC-mediated phosphorylation of lamins. In addition, PKC isoenzymes can accumulate in the nuclei, attracted by different stimuli including diacylglycerol (DAG) fluctuations during cell cycle progression, and target lamins, leading to their disassembly at mitosis. In the present paper, we briefly review how PKCs could regulate cell proliferation and differentiation affecting different molecules related to cell cycle progression.

Neurotoxic effect of kainic acid on ultrastructure and gabaergic parameters in the goldfish cerebellum

Kainic acid administration into the cerebellar dorsal lobe of the goldfish causes selective degeneration of some neuronal types. Stellate and Golgi neurons are very sensitive to the neurotoxin and undergo rapid degeneration. On the basis of their differential responses to kainic acid, Purkinje cells can be divided in two distinct sub-populations (i.e. sensitive and insensitive neurons). The degenerative changes of the Purkinje neurons are in addition remarkably slow in comparison with the same cells in mammals or with stellate and Golgi neurons in the goldfish. Granule cells, as well as the cerebellar afferent fiber system, are not significantly affected. Six days after kainic acid administration, the level of glutamate decarboxylase in the cerebellar dorsal lobe drops to about 40% of the control value. This result suggests that the neurons sensitive to kainic acid neurotoxicity are, at least in part, GABAergic. Light- and electron-microscopic autoradiography of cerebellar elements selectively accumulating [3H]GABA, supports this idea. Moderate decreases of acetylcholinesterase and protein content were also noticed in the kainic acid-treated cerebellar dorsal lobe.

Evidence for a neurotransmitter role of aspartate and/or glutamate in the projection from the torus longitudinalis to the optic tectum of the goldfish

Different experimental approaches have been used to demonstrate that aspartate and/or glutamate is a transmitter(s) in the projection from the torus longitudinalis to the marginal layer of the optic tectum in the goldfish. Slices of the optic tectum incubated in vitro in the presence of D-[3H]aspartate and processed for light microscopic autoradiography, demonstrated a preferential accumulation of the labeled compound in the marginal layer. Under the same experimental conditions several neurons in the central part of the torus longitudinalis selectively accumulated D-[3H]aspartate. Synaptosome-enriched preparations from the optic tectum showed high-affinity uptake for D-[3H]aspartate and the rate of the uptake was significantly decreased after disconnection from the ipsilateral torus longitudinalis. The same subcellular preparations showed Ca2+-dependent release of previously accumulated D-[3H]aspartate under high potassium stimulation. This release was significantly reduced in preparations from optic tecta 5 days after cutting their connection with the ipsilateral torus longitudinalis. Finally, D-[3H]aspartate injected in the optic tectum retrogradely labeled the fiber systems connecting the marginal layer with the ipsilateral torus longitudinalis as well as neuronal cell bodies in the torus longitudinalis itself. From autoradiographic experiments it was, in addition, noticed that several tectal neurons selectively accumulated D-[3H]aspartate in the cell bodies as well as in main dendritic trunks. This observation suggests tht aspartate and/or glutamate may be a transmitter(s) in some intrinsic circuits and extrinsic projections of the optic tectum.

Distribution and expression of A1 adenosine receptors, adenosine deaminase and adenosine deaminase-binding protein (CD26) in goldfish brain

The expression patterns of adenosine A(1) receptors (A(1)Rs), adenosine deaminase (ADA) and ADA binding protein (CD26) were studied in goldfish brain using mammalian monoclonal antibody against A(1)R and polyclonal antibodies against ADA and CD26. Western blot analysis revealed the presence of a band of 35 kDa for A(1)R in membrane preparations and a band of 43 kDa for ADA in both cytosol and membranes. Immunohistochemistry on goldfish brain slices showed that A(1) receptors were present in several neuronal cell bodies diffused in the telencephalon, cerebellum, optic tectum. In the rhombencephalon, large and medium sized neurons of the raphe nucleus showed a strong immunopositivity. A(1)R immunoreactivity was also present in the glial cells of the rhombencephalon and optic tectum. An analogous distribution was observed for ADA immunoreactivity. Tests for the presence of CD26 gave positive labelling in several populations of neurons in the rhombencephalon as well as in the radial glia of optic tectum, where immunostaining for ADA and A(1)R was observed. In goldfish astrocyte cultures the immunohistochemical staining of A(1)R, ADA and CD26, performed on the same cell population, displayed a complete overlapping distribution of the three antibodies. The parallel immunopositivity, at least in some discrete neuronal areas, for A(1)Rs, ADA and CD26 led us to hypothesize that a co-localization among A(1)R, ecto-ADA and CD26 also exists in the neurons of goldfish since it has been established to exist in the neurons of mammals. Moreover, we have demonstrated for the first time, that A(1)R, ecto-ADA and CD26 co-localization is present on the astroglial component of the goldfish brain. This raises the possibility that a similar situation is also shown in the glia of the mammalian brain.

Effect of kainic acid on ultrastructure and γ-aminobutyrate-related circuits in the optic tectum of the goldfish

Abstract Following unilateral microelectrophoretic delivery of kainic acid in the optic tectum of the goldfish, an ultrastructural and biochemical study was carried out. Kainic acid exerted a powerful neurotoxic effect against several types of tectal neurons, noticeably the periventricular neurons and the pyramidal and fusiform neurons of the stratum fibrosum et griseum superficiale. The neurotoxic effect of kainic acid was found to be highly selective. In fact, only some of the different neuronal populations underwent degenerative changes, while other neurons of the same type, and often in very close vicinity, were completely unaffected. Kainic acid neurotoxicity allows us therefore to discriminate between apparently homogeneous neuronal populations, probably on the basis of different neurochemical characteristics possessed by neurons of the same morphological type. The lack of neurotoxic effect against afferent fibres and axon terminals was assessed. Long-term observations of the affected optic tectum after kainic acid treatment demonstrated a remarkable level of structural rearrangement. A sharp decrease in the level of glutamate decarboxylase activity was noticed during the first six days after kainic acid treatment. This was followed by a partial recovery of enzyme activity, which, however, did not progress from 15 days to 2 months after operation. On the other hand no decrease of glutamate decarboxylase activity occurred in the left optic tectum six days and one month after surgical ablation of the right eye. These results suggest the presence of intrinsic γ-aminobutyrate-containing systems in the goldfish optic tectum. The existence of intrinsic neurons that take up γ-aminobutyrate was confirmed by light-microscopic autoradiography of the optic tectum of normal goldfish after local injection of [3H]γ-aminobutyrate.