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A Cupello

Publications and source records attributed to A Cupello.

At least 55 records · Page 3Linked to original sources
PubMed1997

Modulation by calcium of Deiters' neuron GABAA receptors in the rabbit.

The function of classical GABAA receptors of the rabbit Deiters' neurons has been studied at the single membrane level by a biochemical micromethod involving the study of labelled chloride permeation. In particular, labelled chloride permeation across microdissected fresh single membranes was studied in a microchamber system. The stimulation of 36Cl- out-->in permeation by "extracellular" GABA was determined under different conditions in the respect of Ca++. When the conditions were such that "intracellular" Ca++ was 0.02 microM there appeared to be an optimal effect by GABA on chloride passage. Conditions presumably resulting in an increase of [Ca++]i beyond the level reported above led to a decreased GABA effect, especially at the highest GABA concentrations used (> or = 10(-4)M). However, complete removal of Ca++ by a high (12 mM) intracellular EGTA concentration erased completely the GABA effect. These results indicate that in these neurons an optimal GABAA receptor function requires [Ca++]i levels well below micromolar. The high [EGTA]i effect seems to imply that too low a [Ca++]i is also harmful to the proper function of these GABAA receptors. However, an alternative explanation is possible.

Animals↗
PubMed1996

Nitric oxide and GABAA receptor function in the rat cerebral cortex and cerebellar granule cells.

The aim of the present work was to investigate the mechanism by which the diffusible factor nitric oxide regulates GABAA receptor function in the brain. The effect of nitric oxide on GABAA receptor function has been studied in two different neuronal preparations: rat cerebral cortex microsacs and rat cerebellum granule cells in culture. In the first case, GABA-stimulated 36Cl-accumulation was studied as an index of GABAA receptor function. The maximal rate of GABA-stimulated 36Cl- accumulation (Vmax) was reduced by treatment of microsacs with nitric oxide chemical donors such as sodium nitroprusside (-26%) and S-nitroso-acetyl-penicillamine (-11%). The greater effect of the former agent is due to an additional interference by its breakdown products. The biochemical precursor L-arginine (1 mM) produced the same Vmax decrease as S-nitroso-acetyl-penicillamine. This effect was reversed by a nitric oxide synthase blocker and appears truly nitric oxide mediated. The action of nitric oxide in this system does not seem to imply cyclic GMP formation. GABAA receptor function was studied by whole-cell patch-clamp in rat cerebellum granule cells in culture. In this case, L-arginine (100 microM) profoundly reduced the Cl- current elicited by 10 microM GABA and its effect subsided following washing out. The effect of L-arginine was observed almost exclusively on the rapidly desensitizing component of the GABA-activated current. The action of L-arginine was blocked by a protein kinase G inhibitor and mimicked by its activators. Thus, it appears that this effect in these cells involves nitric oxide formation, cyclic GMP accumulation and protein kinase G-catalysed phosphorylation of GABAA receptor.

Animals↗
PubMed1995

Screening of the macromolecular component of CSF from various pathologies for its interference with GABAA receptor function.

Cerebrospinal fluid (CSF) samples from patients undergoing lumbar puncture for suspected neurological diseases were fractioned into macromolecular (MW > 3000 Daltons) and small molecule components (MW < 3000 Daltons). The macromolecular component was tested for its interference with GABA stimulation of 36Cl- accumulation in rat cerebral cortex microsacs. For many pathologies, no interference was apparent; an exception was two patients with Parkinson's disease in whom there were clear signs of stimulated GABA activity. This last result seems worthy of further investigation.

Adult↗
PubMed1994

Modulation by nitric oxide of rat brain GABAA receptors.

The effect of nitric oxide (NO) on the function of GABAA receptors was studied in two different rat brain neuron populations. Cerebral cortex neuronal GABAA receptors were studied by preparing microsacs and evaluating 36Cl- accumulation. Whether nitric oxide was provided by sodium nitroprusside (SNP) or by the metabolic precursor precursor arginine there was a 15-25% reduction in the Vmax for GABA-stimulated 36Cl- accumulation. The arginine effect could be reversed by the NO synthase (NOS) inhibitor N omega-nitro-L-arginine. GABAA receptor mediated Cl- currents were studied in rat cerebellar granule cells by whole-cell patch clamp. S-Nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside and L-arginine reduced the Cl- current elicited by 10 microM GABA. The L-arginine effect was reversible upon its washing out. This circumstance indicates that NO produced by endogenous NOS can inhibit GABAA receptor function in cerebellar granule cells.

Amino Acid Oxidoreductases↗
PubMed1994

Modulation by extracellular pH of the activity of GABAA receptors on rat cerebellum granule cells.

The Cl- currents activated by GABA via GABAA receptors in rat cerebellum granule cells in culture were studied by whole-cell patch-clamp. These currents were measured at various extracellular pH. The currents activated by 100 microM GABA, both the peak and the steady-state component, increase at acidic pH's and decrease at basic pH's. The transition point being at around 7.7. Interestingly, passing from pH 7.4 to 6.4 the GABA dose-response curve indicates that the increases in the peak current are related to an augmented maximal current. The increases in the steady-state component are mainly due to a higher affinity of the receptors for the neurotransmitter and disappear at saturating [GABA]. The study of the I-V curves for the GABA activated peak Cl- currents at pH 6.4, 7.4 and 8.4 reveals linearity in the latter instance. However, an outward rectification is present at the two more acidic pH's. This fact suggests that the protonation of basic amino acids at the acid pH does involve rectification of Cl- channel conductance. Overall, the data indicate that slight changes in in situ extracellular pH may have profound influences on GABAA receptor function.

Animals↗
PubMed1993

The increase in Cl- permeation across the Deiters' neuron membrane by GABA on its cytoplasmic side is abolished by protein kinase C (PKC) activators.

1. Cl- ion outward permeation across microdissected Deiters' neuron plasma membranes is augmented by GABA on the membrane cytoplasmic side. When these neurons are preincubated with a PKC activator, phorbol-12,13-dibutyrate (PdBu), there is a complex pattern of effects on basal and GABA-activated 36Cl- in-->out permeation. A distinct fact is an increase in basal Cl- passage and a disappearance of the 10(-6) M GABA effect at [PdBu] = 0.1 microM. 2. Likewise, 0.1 microM oleylacetylglycerol (OAG) treatment erases the effect completely, further supporting a role for PKC in modulating GABA-stimulated Cl- in-->out permeation. 3. The inactive ester, phorbol-12,13-didecanoate (Pdd), at 0.1 microM, does not affect GABA stimulation of Cl- passage. 4. High concentration (15-20 microM) of OAG and PdBu block the "intracellular" GABA efefct. However, the 20 microM PdBu effect is reversed by 30 microM H7. 5. These results indicate a role of endogenous PKC in Cl- extrusion by GABAA receptors on the cytoplasmic side of the Deiters' neuron membrane.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Electric shock convulsions in the rabbit and brain cortex GABAA receptor function.

The effect of electric shock convulsions (ESC) on the function of brain cortex GABAA receptors has been studied in the rabbit. Three single electroconvulsive shocks (ECS) were given at intervals of 48 hours and the brain cortex was sampled 36 hours after the last shock. The dose-response curve was determined for GABA-stimulated 36Cl-accumulation into brain cortex microsacs. The parameters of the curve (maximal accumulation rate, Ka and Hill coefficient, n) were constant when determined in two different series of experiences. Animals handled in the same way as the animals from the electric shock group but which did not receive the ECSs (sham ECS group) showed similar maximal accumulation rate and Ka. However, the average n coefficient was significantly higher in the electric shock group. Naive animals, taken from their cages just before the sacrifice, showed dose-response curves which varied from one experimental series to another. This last result (confirming previous observations) shows modifications and inconsistencies in the evaluation of GABAA receptor function in stressed handling-naive animals.

Animals↗
PubMed1993

Regulation of GABAA receptor in cerebellar granule cells in culture: differential involvement of kinase activities.

GABAA receptor function was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. The data show that GABA activates Cl- currents in these neurons which reverse at the appropriate membrane potential and are blocked by picrotoxin. The GABA-activated currents desensitize with time of application of the neurotransmitter at concentrations > or = 10(-6) M. The dose-response curve for the peak Cl- current gives a Ka value of 2.3 microM with a Hill coefficient of 1.2. The peak Cl- current elicited by GABA decreases with time of cell registration, with a time-constant of 7.3 min. Residual responsiveness though is maintained thereafter. This "run-down" phenomenon can be completely prevented by adding adenosine-5'-triphosphate + Mg2+ in the pipette solution. Treatments which directly (8-bromoadenosine-3',5'-cyclic-monophosphate; adenosine-3', 5'-cyclic-monophosphate) or indirectly (forskolin, isobutylmethylxanthine) increase the adenosine-3',5'-cyclic-monophosphate intracellular content reduce the GABA-induced Cl- current. Conversely, treatment with the protein kinase A and C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine potentiates the effect of GABA. On the whole, the data indicate that different protein kinase activities modulate the functional state of the GABAA receptors on granule cells from the rat cerebellum.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Modulation by intracellular Ca++ of GABA activated Cl- extrusion from Deiters' neuron.

The increase in 36Cl- in --> out permeation across plasma membranes from the Deiters' neurons by GABA on the cytoplasmic side is modulated by Ca++. The GABA stimulation was maximal with Ca++ on the intracellular side at the physiologically likely concentration of 3 x 10(-8) M. A lower effect was found in the 10(-7) - 10(-5) M [Ca++]i range with a total disappearance of it at [Ca++] of .2 mM. This Cl- extrusion mechanism is likely to be involved in the establishment of a Cl- out --> in electrochemical gradient. Modulation of it by intracellular Ca++ transients may be of physiological importance.

Animals↗
PubMed1992

Further evidence for the presence of gamma-aminobutyric acidA (GABAA) receptors on the cytoplasmic side of Deiters' membrane.

1. The permeation of labeled Cl- across single nerve membranes microdissected from rabbit Deiters' neurons was studied in a microchamber system. The in----out permeation of the ions was evaluated under control conditions and in the presence of either 10(-6) M GABA or 10(-6) M GABA plus 10(-5) M bicuculline methiodide (BMI) on the membrane cytoplasmic side. 2. In 32 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied with the addition of GABA. Within each experiment all the membranes were obtained from the same animal. 3. In an additional 10 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied in the presence of GABA plus bicuculline methiodide on the membrane cytoplasmic side. 4. The data show that 10(-6) M GABA on the Deiters' membrane cytoplasmic side stimulates Cl- permeation in----out by 42% (P = 0.0000001). When 10(-5) M BMI was present together with GABA, no stimulation of Cl- in----out permeation occurred.

Animals↗
PubMed1992

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection.

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5-30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that compartment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.

Animals↗
PubMed1992

Evaluation of the mechanisms by which gamma-amino-butyric acid in association with phosphatidylserine exerts an antiepileptic effect in the rat.

The i.p. injection in rats of GABA (740 mg/Kg) after sonication with an equal amount of phosphatidylserine (PS) has an antiepileptic effect. The injection of plain GABA has no such an effect. Blood, brain and synaptosomal accumulation of exogenous labeled GABA under the two circumstances are evaluated. In the case of GABA/PS injection there is a higher passage of the exogenous labeled neurotransmitter into the blood and brain nerve endings (synaptosomes). A higher synaptosomal accumulation of the exogenous labeled neurotransmitter is found even when GABA and PS are injected separately. Since these accumulation increases occur at a time when there is the antiepileptic effect, they seem relevant to it. Our interpretation of the chain of the events resulting in the antiepileptic action is that the phospholipid facilitates from the beginning the first passage of the exogenous neurotransmitter form the peritoneum to the blood. Then a higher passage to the brain tissue and eventually to the GABA-ergic nerve endings ensues. The brisker accumulation of the exogenous neurotransmitter in the nerve endings could be at the basis of a more efficient GABA-ergic inhibitory control in the brain.

Animals↗
PubMed1992

Components of basal and GABA activated 36Cl- influx in rat cerebral cortex microsacs.

The basal and GABA activated accumulation of labelled chloride in rat cerebral cortex microsacs has been studied as a function of incubation time. Basal accumulation has biphasic kinetics within 2 minutes of incubation with two components clearly visible. GABA activated stimulation has two phases as well, starting from (GABA) = 10(-5) M, one appearing within seconds of incubation range and the other one within tens of seconds. Both GABA-activated components are blocked by bicuculline methiodide (BMI). Practically no effect by 10(-3) M nipecotic acid was found on either the comparatively rapid or the slower phase. The two GABA activated components may correspond to two different populations of sealed vesicles with different receptor concentration per internal volume.

Animals↗
PubMed1991

Can Cl- ions be extruded from a gamma-aminobutyric (GABA)-acceptive nerve cell via GABAA receptors on the plasma membrane cytoplasmic side?

1. In this commentary we discuss results obtained by a micromethod for the study of Cl- permeability across single nerve membranes from rabbit Deiters' neurons. 2. These results showed the presence of GABAA receptors on the nerve cell membrane cytoplasmic side. 3. We could show that these receptor complexes have a higher affinity for GABA than their extracellularly facing counterparts. Moreover, they present a phenomenon of desensitization. Another distinct property is that upon activation by GABA, they expose positive charges at their cytoplasmic mouths. 4. We propose that these receptor complexes could function in situ as a device for extruding Cl- anions from the nerve cell interior. This phenomenon would create an electrochemical gradient for Cl- penetration into the cell upon the action of extracellular GABA, after its presynaptic release.

Animals↗
PubMed1991

Differential scanning calorimetry characterization of rabbit brain membrane fractions.

Various membrane fractions have been prepared from rabbit brain synaptosomes by centrifugation on discontinuous sucrose gradients after osmotic shock. These fractions were characterized by electron microscopy (E.M.), SDS-PAGE and GABA binding. The fractions were then studied by differential scanning calorimetry (DSC). The calorimetric results indicate that all the fractions studied show thermal transitions at around 60 degrees C which correspond to the "melting" of membrane structures. An additional transition at higher temperature (82 degrees C) seems to be associated with an enrichment in central myelin fragments.

Animals↗
PubMed1990

Effect of phosphatidylserine on the basal and GABA-activated Cl- permeation across single nerve membranes from rabbit Deiters' neurons.

The permeation of labeled Cl- ions across single plasma membranes from Deiters' neurons has been studied in the presence of various concentrations of phosphatidylserine (PS) on their extracellular side. PS reduces significantly basal Cl- permeation only at 10(-5) M on the membrane exterior. No effect was found at other concentrations. GABA activable 36Cl- permeation is heavily reduced and almost abolished at 10(-11) - 10(-5) M phosphatidylserine. This exogenous phosphatidylserine effect is difficult to interpret in relation to the function of the endogenous phospholipid. However, it may be involved in the epileptogenic effect in vivo of exogenous phosphatidylserine administration to rats.

Animals↗
PubMed1990

Stimulation of 36Cl- permeation in the in----out direction across the Dieters' neuron membrane by GABA on its cytoplasmic side: effect of different ionic conditions.

The permeation of labelled Cl- ions across single plasma membranes microdissected from rabbit Deiters' neuron was studied in a microchamber system. In particular, we studied 36Cl- in----out permeation and its stimulation by 10(-6) M GABA on the cytoplasmic side under different ionic conditions on both sides of the membrane. Three main results were found: a) The GABA effect turns up at a cytoplasmic side [Cl-], 21 mM, in the range of the normal steady state intracellular Cl- concentration. It disappears both at tracer level of Cl- on the cytoplasmic side and when [Cl-] is high there (140 mM). b) The increase in ionic strength due to the equinormal substitution of monovalent anions (Cl- and acetate) with the trivalent impermeant anion citrate on both sides of the membrane erases the GABA effect even if Cl- is at the optimal cytoplasmic side concentration, 21 mM. c) Citrate ions reduce to the limit of significance the GABA effect even when they are only on the membrane extracellular side and [Cl-] is at the optimal level on the other side. These results confirm that GABA stimulated Cl- permeation in the in----out direction is via Cl- channels exposing positive charges at their cytoplasmic mouth. In addition, they point out that such GABA activated channels are endowed with a partial electrical positivity at their extracellular exit.

Animals↗