Convulsant bicuculline modifies CNS muscarinic receptor affinity

BACKGROUND: Previous work from this laboratory has shown that the administration of the convulsant drug 3-mercaptopropionic acid (MP), a GAD inhibitor, modifies not only GABA synthesis but also binding of the antagonist [(3)H]-quinuclidinyl benzilate ([(3)H]-QNB) to central muscarinic receptors, an effect due to an increase in affinity without modifications in binding site number. The cholinergic system has been implicated in several experimental epilepsy models and the ability of acetylcholine to regulate neuronal excitability in the neocortex is well known. To study the potential relationship between GABAergic and cholinergic systems with seizure activity, we analyzed the muscarinic receptor after inducing seizure by bicuculline (BIC), known to antagonize the GABA-A postsynaptic receptor subtype. RESULTS: We analyzed binding of muscarinic antagonist [(3)H]-QNB to rat CNS membranes after i.p. administration of BIC at subconvulsant (1.0 mg/kg) and convulsant (7.5 mg/kg) doses. Subconvulsant BIC dose failed to develop seizures but produced binding alteration in the cerebellum and hippocampus with roughly 40% increase and 10% decrease, respectively. After convulsant BIC dose, which invariably led to generalized tonic-clonic seizures, binding increased 36% and 15% to cerebellar and striatal membranes respectively, but decreased 12% to hippocampal membranes. Kd value was accordingly modified: with the subconvulsant dose it decreased 27% in cerebellum whereas it increased 61% in hippocampus; with the convulsant dose, Kd value decreased 33% in cerebellum but increased 85% in hippocampus. No change in receptor number site was found, and Hill number was invariably close to unity. CONCLUSION: Results indicate dissimilar central nervous system area susceptibility of muscarinic receptor to BIC. Ligand binding was modified not only by a convulsant BIC dose but also by a subconvulsant dose, indicating that changes are not attributable to the seizure process itself. Findings support the notion that the muscarinic receptors play a major role in experimental epilepsy and provide a new example of differential neuronal plasticity

An endogenous Na+, K+-ATPase inhibitor enhances phosphoinositide hydrolysis in neonatal but not in adult rat brain cortex

The effect of an endogenous Na+, K+-ATPase inhibitor, termed endobain E, on phosphoinositide hydrolysis was studied in rat brain cortical prisms and compared with that of ouabain. As already shown for ouabain, a transient effect was obtained with endobain E; maximal accumulation of inositol phosphates induced by endobain E was 604 ± 138% and 186 ± 48% of basal values in neonatal and adult rats, respectively. The concentration-response plot for the interaction between endobain E and phosphoinositide turnover differed from that of ouabain, thus suggesting the involvement of distinct mechanisms. In the presence of endobain E plus ouabain at saturating concentrations, no additive effect was recorded, suggesting that both substances share at least a common step in their activation mechanism of inositol phosphates metabolism or that they enhance phosphatidylinositol 4,5-biphosphate breakdown from the same membrane precursor pool, until its exhaustion. Experiments with benzamil, a potent blocker of Na+/Ca2+ exchanger, showed that it partially and dose-dependently inhibited endobain E effect. These results indicate that the endogenous Na+, K+-ATPase inhibitor endobain E, like ouabain, is able to stimulate phosphoinositide turnover transiently during postnatal brain development.Fil: Calviño, Maria Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Peña, Carina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: De Lores Arnaiz, G. Rodríguez. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Brain mitochondria bioenergetics, nitrergic and neurotensinergic systems

Mitochondria display a central role in cellular activity, being essential for energetic metabolism, Ca2+ homeostasis and cell signalling. The brain is one of the organs with the highest oxygen consumption of the human body, which is employed, in great proportion, to maintain and restore ionic equilibria through neuronal membranes in resting conditions and after the passage of the nervous impulse. Furthermore, neurons rely almost exclusively on ATP synthesis from the mitochondrial respiratory chain and oxidative phosphorylation to fulfill their energy requirements for neurotransmitter synthesis, release and reuptake. Differences between the characteristics of astrocyte and neuron mitochondria are evident such as for instance the distribution and organization of supercomplex respiratory chain components. In addition, some differences between oligodendrocytes and astrocytes bioenergetics have been reported.According to their distribution within the neurons, mitochondria are termed synaptic, when located within the nerve endings and non-synaptic ones, when present in neuronal soma. Environmental and energy demand differences for synaptic mitochondria versus non-synaptic mitochondria are apparent. Nitric oxide (NO) is a free radical that acts as an intercellular messenger in physiological processes such as neurotransmission, among other functions. Nitric oxide is generated by the activity of three NO synthase (NOS) enzyme isoforms. One of them, the neuronal NOS (nNOS) predominates in neuronal tissue. At central nervous system, besides the so-called classic neurotransmitters, a constellation of peptides have been identified. They present pharmacological characteristics and are colocalized and coreleased together with the classic neurotransmitters.Neurotensin is a tridecapeptide which at both central and peripheral nervous systems exerts a wide spectrum of effects. Neurotensin acts as a neuromodulator or as a neurotransmitter through its binding to specific receptors. The most important receptors are coupled to G protein, and are termed NTS1 and NTS2, which bind respectively the peptide with high and low affinity.When neurotensin binds to NTS2 receptor, it exerts either agonist or antagonist activity, according to this receptor-mediated pathway. The blockade of NTS2 receptor by levocabastine leads to a decrease in NOS activity accompanied by an enhancement of nNOS expression in brain mitochondria. At the same time, the activity of mitochondrial enzymatic complexes is markedly diminished. It is suggested an interrelationship between the neurotensinergic and the nitrergic systems. However, the effects of NTS2 activity on mitochondrial bioenergetics are likely to be independent of the regulation of NO synthesis.Fil: Rodríguez de Lores Arnaiz, Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Lores Arnaiz, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentin

Changes in synaptic proteins of the complex PSD-95/NMDA receptor/nNOS and mitochondrial dysfunction after levocabastine treatment

Nitric oxide generation is related to the activity of certain proteins located at synaptic sites. Previous findings show that NOS activity, nNOS protein expression, respiratory parameters and mitochondrial complex activities are altered in rat cerebral cortex by administration of levocabastine, an antagonist of histamine H1 and neurotensin NTS2 receptors. ATP provision by mitochondria may play an important role in the functional interaction between synaptic proteins NMDA receptor and PSD-95 with NO synthesis. In this context, our purpose was to evaluate the effect of levocabastine administration on protein expression of PSD-95, GluN2B and iNOS, as well as on mitochondrial ATP production. Male Wistar rats received a single (i.p.) dose of levocabastine (50 μg/kg) or saline solution (controls) and were decapitated 18 h later. Mitochondrial and synaptosomal membrane fractions were isolated from cerebral cortex by differential and sucrose gradient centrifugation. Expression of synaptic proteins was evaluated by Western blot assays in synaptosomal membrane fractions. Oxygen consumption, mitochondrial membrane potential and ATP production rate were determined in fresh crude mitochondrial fractions. After levocabastine treatment, protein expression of PSD-95, GluN2B and β-actin decreased 97, 45 and 55%, respectively, whereas that of iNOS enhanced 3.5-fold versus controls. In crude mitochondrial fractions levocabastine administration reduced roughly 15% respiratory control rate as assayed with malate-glutamate or succinate as substrates, decreased mitochondrial membrane potential (21%), and ATP production rates (57%). Results suggested that levocabastine administration induces alterations in synaptic proteins of the protein complex PSD-95/NMDA receptor/nNOS and in neuron cytoskeleton. Mitochondrial bioenergetics impairment may play a role in the functional link between synaptic proteins and NO synthesis.Fil: Lores Arnaiz, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Karadayian, Analia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Gutnisky, Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Miranda, Jennifer Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Rodríguez de Lores Arnaiz, Georgina. Universidad de Buenos Aires; Argentin