Biomedical subjects

A Cupello

Publications and source records attributed to A Cupello.

At least 37 records · Page 2Linked to original sources

PubMed2001

Subfractionation of ficoll gradient purified synaptosomes: GABA uptake by the subfractions.

The classical synaptosomes preparation purified on discontinuous Ficoll gradient has been sufractionated into three further subfractions by introducing more Ficoll density layers. Among the three subfractions, the lightest one was the one which was more difficult to obtain in reproducible amounts and was only partially characterized in terms of labelled GABA uptake. The heaviest one most probably is largely made up of partially damaged nerve endings. The central one was the most reproducible in terms of yield and labelled GABA uptake and actually was the one we studied more thoroughly in terms of morphology, labelled GABA uptake and its pharmacology. A comparison has been made with the classical "total" purified synaptosomes fraction. An interesting result of these experiments is a paradoxical effect of the glial uptake inhibitor beta-alanine. This substance appears to favor, in both "total" and "central" fraction synaptosomes, a redistribution of taken up GABA from contaminating glia to actual nerve endings.

Animals↗

PubMed2001

GABA uptake by rabbit restiform body homogenates.

Restiform body (inferior cerebellar peduncle) preparations were obtained from rabbit brain stem slices and homogenized. When challenged with labelled GABA, these homogenates took it up briskly. We have characterized pharmacologically this uptake which resulted almost equally neuronal and glial. The neuronal component of the GABA uptake might be due to the adjacent cochlear nuclei coming along in the preparations, whereas the glial component probably belongs to the restiform body proper. Another possibility is that actually both components are due to the myelinated fibers and glia which make up the restiform body.

Acetanilides↗

PubMed2000

Unraveling of important neurobiological mechanisms by the use of pure, fully differentiated neurons obtained from adult animals.

An important, and often overlooked, problem in the neurochemical approach to neurobiological problems is that analysis of tissue involves almost always a heterogeneous population of cells (neurons, glia and other types of tissue cells). The use of cell cultures has obvious limitations such as that they derive from embryonic or immediately postnatal animals; in addition, the cell culture conditions most certainly are quite different from the real tissue environment for the nerve cells. We underline here an alternative strategy, which is not new, but which, in our view, has already given formidable contributions to neurobiological studies and still is giving results of great importance. This is the technique proposed and used since the late fifties and early sixties by the senior author (H. Hydén). The method involves the isolation of the big vestibular neurons from the adult rabbit vestibular nucleus. The neurons, fully differentiated and performing a precisely defined function, are obtained rapidly and completely free from surrounding glial cells. The separate microbiochemical study of these cells and their surrounding glia has yielded already in 1962, the information that modifications in gene expression are associated with plastic modifications of the function of the relevant neurons, which take place in the behavioral event of learning. Another important concept was formulated in the same time period following determination of the activities of energy metabolism related enzymes separately in vestibular neurons and their glia under vestibular stimulation. This is the concept that, under increased functional activity glia increases its anaerobic metabolism and passes then on the resulting metabolites to the neurons for aerobic metabolism. Both these concepts (RNA and memory; metabolic cooperation between glia and neurons) are nowadays widely accepted. In addition, this approach with pure big nerve cells has allowed in recent years the discovery of a novel mechanism for chloride extrusion in these cells. This mechanism utilizes structures similar to GABA activated chloride channels in cyclic modifications resulting in the final extrusion of chloride ions. The energy for the process is provided by a protein phosphorylation step. Future approaches are warranted such as the possibility of recognizing by RT-PCR specific neuronal mRNAs and their modification in expression in relation to function and plastic modifications (learning). Another possible interesting application appears to be the recognition of the mRNAs for GABA(A) receptor subunits expressed here in these neurons in relation to the physiological and pharmacological characteristics of these native neuronal GABA(A) receptors.

Animals↗

PubMed2000

Antiserum against S-100 protein prevents long term potentiation through a cAMP-related mechanism.

Long term potentiation (LTP) was induced in the CA1 region of rat hippocampal slices by tetanization of the Schaffer collaterals. Local pretreatment of CA1 with serum of rabbits immunized against S-100 prevented the potentiation. However, treatment of the slices with a membrane permeant cAMP analogue, such as 8-Br-cAMP, could protect against the blocking effect of anti S-100 serum. We suggest that in the rat endogenous S-100b is involved in transduction mechanisms during LTP induction, via its ability to stimulate adenylate cyclase. Possible mechanisms of this action are discussed.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2000

GABA(A) receptor modulation in rat cerebellum granule cells.

The inhibitory GABA(A) receptor is a key element in determining the pattern of nerve cell electrical activity. Thus, modulation of its function is of paramount impact in shaping neuronal functional activity under physiological and pathological conditions. This applies to cerebellar granule neurons as to all the other neurons in the brain. The culture of cerebellar granules from newborn rats is a convenient means by which to approach these cells for electrophysiological studies provided that they maintain, as far as GABA(A) receptors are concerned, the same characteristics as in situ. Thus, the regulation of GABA(A) receptor activity in these neurons has been studied by the patch-clamp technique, both in the whole-cell and outside-out configuration. An obvious first level of control of such receptors' activity is their desensitization under continued agonist application, with biphasic kinetics. The data do not allow one to conclude whether one is dealing with two different populations of receptors or with a single population with two desensitization phases; although the presence of two GABA(A) receptor populations is suggested by a host of observations. The granule cell GABA(A) receptors are modulated by changes in extracellular pH with lower pH resulting in an enhanced receptor activity. They display, under the conditions of whole-cell recording, a run-down phenomenon which is most probably due to a tyrosine phosphatase activity which is in turn under control by a protein serine kinase. Thus, in situ tyrosine phosphorylation is a key element in determining the efficiency of GABA mediated inhibition. Activation of protein kinase A or protein kinase G (PKG) down-regulates GABA(A) receptors' activity. This last event is involved in the depression of those receptors' activity by L-arginine via the production of nitric oxide. In addition, the activity of calmodulin-activated adenylate cyclase I is controlled by GABA(B) receptors. Dendritic GABA(A) receptor activity is partially blocked by previous activation of N-methyl-D-aspartate (NMDA) receptors via calcineurin mediated dephosphorylation/activation of protein tyrosine phosphatase and concomitant production of nitric oxide and PKG activation. The site phosphorylated by PKG is evidently not available for calcineurin-mediated serine dephosphorylation, due to calcineurin-specific membrane localization in respect of the GABA(A) receptor. Overall, a complex network of biochemical signals appear to keep granule cells GABA(A) receptors under a fine balance between up- and down-regulatory mechanisms. The overall data appear also to indicate the presence of two GABA(A) receptor populations: a dendritic one which can be modulated by Ca++ entering via NMDA receptors and a cell body one. The two populations are probably different in terms of desensitization kinetics and benzodiazepine sensitivity.

Animals↗

PubMed1999

Evidence of two populations of GABA(A) receptors in cerebellar granule cells in culture: different desensitization kinetics, pharmacology, serine/threonine kinase sensitivity, and localization.

GABA(A) receptors of rat cerebellar granule cells in culture have been studied by the whole cell patch clamp technique. The biphasic desensitization kinetic observed could be due either to different desensitization mechanisms of a single receptor population or to different receptor populations. The overall data indicate that the latter hypothesis is most probably the correct one. In fact, the fast desensitizing component was selectively potentiated by a benzodiazepine agonist and preferentially down-regulated by activation of the protein serine/threonine kinases A and G, as a consequence of the latter characteristic that receptor population was preferentially down-regulated by previous activation of N-methyl-d-aspartate glutamate receptors, via production of nitric oxide and PKG activation, most probably in dendrites. The other population is benzodiazepine insensitive and not influenced by activation of PKA or PKG. This slowly desensitizing population may correspond to the extrasynaptic delta subunit containing GABA(A) receptors described by other authors. Instead, the rapidly desensitizing population appears to represent dendritic synaptic GABA(A) receptors.

Animals↗

PubMed1999

Involvement of S-100 protein in anoxic long-term potentiation.

In in vitro rat hippocampal slices a short period (2 min) of hypoxia resulted in lasting potentiation of the population spike transynaptically evoked in CA1 by stimulation of Schaffer collaterals ("anoxic LTP"). Pretreatment of slices with antiserum against S-100 protein fully prevented this anoxic LTP. Since also "classical" (i.e., induced by high-frequency electrical stimulation) long-term potentiation is prevented by anti S-100 serum, this represents one more important similarity between these events.

Action Potentials↗

PubMed1999

Chloride permeation across the Deiters' neuron plasma membrane: activation by GABA on the membrane cytoplasmic side.

Single plasma membranes were microdissected from Deiters' neurons freshly obtained from the lateral vestibular nucleus of the rabbit and their chloride permeability was studied in a microchamber system. The basal in-->out 36Cl- permeation initially found was brought to zero by Zn2+, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid and iodide. GABA on the membrane cytoplasmic side resulted in a measurable in-->out 36Cl- passage, which was blocked by the GABA(A) antagonists bicuculline and picrotoxin. This effect peaked at 1 microM GABA on the inner side of the membrane. At higher GABA concentrations, a strong desensitization of the effect was found. Stimulation of Cl- permeability by GABA on the extracellular side of the membrane peaked at much higher GABA concentrations, 10-100 microM. This excludes an effect due to passage of the neurotransmitter from the inner to the outer compartment in our microchamber device. Moreover, this possibility is also dismissed by the fact that 1 microM GABA on the membrane outside did not evoke any 36Cl- in-->out permeation. In addition, pentobarbitone by itself could also stimulate 36Cl- in-->out permeation when added on the cytoplasmic side of Deiters' membrane. On these bases and in agreement with our previous reports, we propose that structures behaving pharmacologically as GABA(A) receptors respond to low levels of GABA on the cytoplasmic side of these neurons' membranes. We suggest that these structures are devices that, at the expense of ATP consumed in their phosphorylation, extrude Cl- after postsynaptic GABA uptake into the Deiters' neuron.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

GABA(B) receptor activation protects GABA(A) receptor from cyclic AMP-dependent down-regulation in rat cerebellar granule cells.

Interaction between GABAA and GABA(B) receptors was studied in rat cerebellar granule cells in culture, by the whole-cell patch-clamp approach. Our data show that the GABA(B) agonist (-)baclofen is not able, per se, to significantly change the muscimol-activated chloride current. However, (-)baclofen dose-dependently prevents the reduction of GABA(A) receptor function by forskolin, an activator of adenylate cyclase. The effect of baclofen is mediated by a pertussis toxin-sensitive G protein. In fact, in cells treated with pertussis toxin, baclofen and forskolin, the toxin is able to block baclofen action, allowing forskolin to act fully. The protective effect by GABA(B) receptor activation under these circumstances is most probably related to the prevention of cyclic AMP increases after forskolin treatment. In fact, in these neurons cyclic AMP and protein kinase A activation result in a down-regulation of GABA(A) receptor function. On the whole, the data indicate the presence of complex modulation of GABA(A) receptors by GABA(B) receptor types in cerebellum granule cells.

Adenylate Cyclase Toxin↗

PubMed1999

An electrogenic ionic pump derived from an ionotropic receptor: assessment of a candidate.

1. Data obtained studying permeability characteristics of single Deiters' membranes in a microchamber system show that intracellular GABA can activate chloride in-->out passage with a GABAA pharmacology. 2. The overall data suggest the presence of a chloride extrusion pump in these neurons based on intracellular GABA activated chloride channels. 3. This conclusion takes up a previous theoretical suggestion that ionic channels could work as ionic pumps provided an energy input modifies the energy profile along the permeation path. 4. According to our quantitative evaluation, this pumping mechanism works with a low yield and along a cycle with a strongly asymmetric behavior, being far from equilibrium due to powerful "leakage" pathways for chloride in these neurons.

Adenosine Triphosphate↗

PubMed1999

Stimulation of chloride in-->out permeation across the Deiters' neuron membrane by pentobarbital on the cytoplasmic side: additional evidence of GABA(A) receptors acting as chloride extrusion pumps.

Pentobarbital stimulates 36Cl- permeation across single Deiters' membranes in a microchamber system, acting on classical, extracellularly facing, GABA(A) receptors. However, when applied on the membrane cytoplasmic side it activates per se labeled chloride in-->out permeation. No effect was found on chloride out-->in permeation. Similarly, at lower concentrations it facilitates the increase of 36Cl- in-->out permeation by application of GABA on the membrane inside, again via asymmetric chloride channels allowing in-->out but not out-->in passage. These data confirm that on the Deiters' membrane cytoplasmic side there are structures behaving pharmacologically as GABA(A) receptors whose function is that of a Cl- extrusion pump. This mechanism involves a cycle of activation-phosphorylation/desensitization-reactivation of the receptor complexes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Intracellular GABA-activated in-->out permeation of chloride across the Deiters' neuron membrane: modulation by phosphorylating activities.

The modulation of intracellular GABA activated 36Cl- in-->out permeation across single Deiters' neuron membranes has been studied in a microchamber system. Addition of Mg2+/ATP on the membrane cytoplasmic side reduces strongly the GABA effect as does ATP alone. However, the greatest inhibition of the GABA effect is given by the addition of Mg2+ to the intracellular side buffer: a complete block of the stimulation by GABA of 36Cl- in-->out permeation. This is interpreted as due to the presence in this case of a constant concentration of exogenous Mg2+ acting together with endogenous ATP in the small cytoplasmic layer on the membrane inner side. The addition of ADP to Mg2+/ATP increases the inhibitory effect of the latter. This is presumably due to an extra increase of ATP, locally under the membrane, due to phosphorylation of ADP by endogenous phosphocreatine. Overall, the data confirm that phosphorylating conditions impair the intracellular GABA action on 36Cl- in-->out permeation.

Animals↗

PubMed1998

Cerebellar granule cell GABA(A) receptors studied at the single-channel level: modulation by protein kinase G.

Rat cerebellar granule cells GABA(A) receptors were studied at the single-channel level in outside-out patches. Three conductance levels were detected as activated by 0.1 microM GABA: 11, 20 and 30 pS. Single-channel I-V relationships were linear. The probability of opening did not vary over time within single patches. Kinetic analysis brought to a mean open time constant of 3.2, 2.9 and 2.8 ms respectively for each conductance level and a closed time histogram fitted by the sum of two exponential functions (tau c1 = 2.1 ms, 43%; tau c2 = 18.2 ms, 57%). Protein kinase G (PKG) activation did not affect single-channel conductances, but resulted in a reduction over time of single-channel open probability for all the conductance levels. Kinetically, protein kinase G modified the mean open time constants and the relative areas of the two components of the closed state distribution whereas the mean closed time constants remained unaffected. These results confirm and add details about cerebellar granule GABA(A) receptors down regulation by PKG.

Animals↗

PubMed1998

Measurements of (Na+,K+)ATPase after in vitro hypoxia and reoxygenation are affected by methods of membrane preparation.

(Na+,K+ )ATPase activity was evaluated in membranes from rat hippocampal slices after in vitro hypoxia and reoxygenation. Membranes were prepared with two different methods, one using an isotonic medium and another using a hypotonic one. The changes that were found after hypoxia went into opposite directions in the two cases. Membranes prepared in a hypotonic medium are probably more suitable for these measurements. Using these membranes, hypoxia results in a slight decrease of (Na+,K+)ATPase activity and in a further decrease after reoxygenation. We also found that expressing (Na+,K+)ATPase activity as a percent of total ATPase activity is appropriate for membranes prepared under hypotonic conditions and can unveil (by reducing variability between experiments) significant changes that may be masked in small samples like ours.

Animals↗

PubMed1998

Involvement of phosphatase activities in the run-down of GABA(A) receptor function in rat cerebellar granule cells in culture.

Run-down of GABA activated Cl- currents was found when rat cerebellar granule cells in culture were studied by the whole-cell patch-clamp technique in the absence of ATP in the pipette medium. This event could be prevented, even in the absence of ATP, by using the perforated-patch technique or by adding to the pipette medium either a blocker of protein tyrosine phosphatase, sodium vanadate, or deltamethrin, a blocker of the protein serine/threonine phosphatase calcineurin. Conversely, run-down could be partially induced, even in the presence of ATP, by blockers of tyrosine kinases. A reduction of GABA(A) receptor activity was also found in outside-out membrane patches when ATP was not on the membrane inside. The run-down phenomenon involved all three conductance levels found in these patches: 11, 20 and 30 pS. In all three cases it was due to a reduction of channels' open probability. The single-channel experiments showed that also in this case run-down was prevented by either sodium vanadate or deltamethrin on the membrane cytoplasmic side. Overall, through relatively unphysiological conditions (cells in culture and patch-clamp techniques), the study of the run-down phenomenon shows that the tyrosine phosphorylation state of GABA(A) receptors is of importance in maintaining it in a proper functional state. The data also show that tyrosine phosphorylation state is controlled by a protein tyrosine phosphatase, whose activity in turn is blocked via serine/threonine phosphorylation.

Adenosine Triphosphate↗

PubMed1997

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells.

The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg+2 containing medium. The NMDA effect was also absent when extracellular Ca+2 was replaced by Ba+2 and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca+2 channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca+2 influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5'-O-3-thiophosphate (ATP-gamma-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N omega-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-gamma-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca+2 influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca+2 influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity.

Animals↗

PubMed1997

Effect of nitric oxide donors on GABA uptake by rat brain synaptosomes.

The effect of nitric oxide donors and L-arginine on the uptake of GABA was studied in synaptosomes purified from rat brain. The neurotransmitter uptake was significantly reduced by S-nitrosoacetylpenicillamine and by sodium nitroprusside, although in this case to a lesser extent. A slight inhibitory effect was found preincubating rat brain synaptosomes with 1 mM L-arginine as well. The S-nitrosoacetylpenicillamine effect gradually disappeared with decomposition of the substance by exposure to light. The nitric oxide effect appears to be mainly due to a decrease in the V for synaptosomal GABA uptake and seems to be related to a partial collapse of nerve endings ionic gradients. Functionally, it could result over time in a reduced availability of GABA at the synapses involved.

Animals↗

PubMed1997

Ca(2+)-ATPase pump forms and an endogenous inhibitor in bovine brain synaptosomes.

Two forms of Ca(2+)-pump were identified in bovine brain synaptic membranes as aspartylphosphate intermediates and were characterized. The 140 kDa and 97 kDa phosphoproteins were digested by calpain, producing two phosphorylated fragments, of M.W. 124 and 80 kDa respectively, not inhibited by thapsigargin, and displayed a trypsin digestion pattern with the formation of one phosphorylatable fragment of about 80 kDa. These results suggest that both pumps belong to the Plasma Membrane-type of Ca2+ ATPases, differing from the Sarco- or Endoplasmic Reticulum kind. A plasma membrane Ca(2+)-ATPase proteinaceous inhibitor with molecular weight between 6,000 and 10,000 Da was resolved from synaptic terminal cytosol, where it is enriched by fourfold with respect to frontal cortex brain cytosol. Such enrichment is already evident in the correspondent crude fractions. The presence of calcium pump and its proteinaceous inhibitor inside the synaptic terminals from bovine brain is discussed in terms of free calcium level regulation in neuron synaptoplasm.

Animals↗