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Biomedical subjects

A Cupello

Publications and source records attributed to A Cupello.

At least 19 recordsLinked to original sources

The creatine transporter mediates the uptake of creatine by brain tissue, but not the uptake of two creatine-derived compounds.

Hereditary creatine transporter deficiency causes brain damage, despite the brain having the enzymes to synthesize creatine. Such damage occurring despite an endogenous synthesis is not easily explained. This condition is incurable, because creatine may not be delivered to the brain without its transporter. Creatine-derived compounds that crossed the blood-brain barrier in a transporter-independent fashion would be useful in the therapy of hereditary creatine transporter deficiency, and possibly also in neuroprotection against brain anoxia or ischemia. We tested the double hypothesis that: (1) the creatine carrier is needed to make creatine cross the plasma membrane of brain cells and (2) creatine-derived molecules may cross this plasma membrane independently of the creatine carrier. In in vitro mouse hippocampal slices, incubation with creatine increased creatine and phosphocreatine content of the tissue. Inhibition of the creatine transporter with 3-guanidinopropionic acid (GPA) dose-dependently prevented this increase. Incubation with creatine benzyl ester (CrOBzl) or phosphocreatine-Mg-complex acetate (PCr-Mg-CPLX) increased tissue creatine content, not phosphocreatine. This increase was not prevented by GPA. Thus, the creatine transporter is required for creatine uptake through the plasma membrane. Since there is a strong indication that creatine in the brain is mainly synthesized by glial cells and transferred to neurons, this might explain why hereditary transporter deficiency is attended by severe brain damage despite the possibility of an endogenous synthesis. CrOBzl and PCr-Mg-CPLX cross the plasma membrane in a transporter-independent way, and might be useful in the therapy of hereditary creatine transporter deficiency. They may also prove useful in the therapy of brain anoxia or ischemia.

Animals↗

Successful treatment of epilepsy with serotonin reuptake inhibitors: proposed mechanism.

The widely used antidepressants Specific Serotonin Reuptake Inhibitors (SSRI) have been tried with success as anticonvulsants in cases of nonsymptomatic epilepsy. This attempt was performed on the basis of experimental data suggesting the involvement of impairments of the serotonin system in the genesis of epilepsy. This overview summarizes the clinical data and presents biochemical and neurochemical evidences suggesting the mechanism of the therapeutic effects of SSRI in nonsymptomatic epilepsy. In particular, studies on blood-borne neutral amino acids and platelet serotonin transporter (SERT) in epileptics suggest: (a) That a decreased brain availability of tryptophan may be related to some types of epilepsy. (b) That reduction of the density of SERT may be a homeostatic reaction in the brain following epileptic seizures.

Adolescent↗

Calcium accumulation in neurites and cell bodies of rat cerebellar granule cells in culture: effects on GABA(A) receptor function.

Accumulation of calcium in rat cerebellar granule cells in culture was studied by two photon laser scanning microscopy. Depolarizations by high extracellular potassium induced short-lived increases in calcium in both cell bodies and neurites. However, although the increase in neurites subsided completely after the initial peak, in cell bodies there was a persistent plateau until the high potassium stimulus was removed. On the contrary, the calcium signal due to NMDA receptors activation was persistent in both cell bodies and neurites and remained until the agonist was present. The nature of these calcium signals provides an interpretation key for the effects of NMDA receptors activation on GABA(A) receptors. In particular, the persistent calcium increase in neurites may explain the decrease in GABA activated chloride currents which are related to activation of dendritic/synaptic GABA(A) receptors.

Animals↗

Immunocytochemical study by two photon fluorescence microscopy of the distribution of GABA(A) receptor subunits in rat cerebellar granule cells in culture.

An immunocytochemical investigation of the expression of alpha(1), alpha(6), beta(2/3), gamma(2) and delta subunits was performed on rat cerebellum granule cells in culture by the two photon microscopy technique. The first four subunits appear to be expressed abundantly in these cells, whereas the delta one seems to be expressed at a lower level. Another major difference in the distribution of these subunits is that whereas alpha(6), beta(2/3) and gamma(2) appear only on plasma membranes alpha(1) and delta are present mainly in the cell bodies cytoplasm. Still another difference was found in that the presence of gamma(2) on neurites is "polarized", preferentially labelling neurites with the appearance of dendrites. The subunits alpha(6) and beta(2/3) appear to label all types of neurites, with beta(2/3) being by far the most heavily expressed subunit type. A final distinct characteristic is that alpha(6) and, even more, gamma(2) appear to accumulate in the cytoplasmic domains immediately below the cone of emergence of neurites. This suggests a conspicuous transport of such subunits from the site of synthesis in the cell body to the site of final expression in the neurites (dendrites and axon terminals).

Animals↗

Neuronal transmembrane chloride electrochemical gradient: a key player in GABA A receptor activation physiological effect.

It has long been accepted that GABA is the main inhibitory neurotransmitter in the mammalian brain, acting via GABA(A) or GABA(B) receptors. However, new evidences have shown that it may work as an excitatory transmitter, especially in the brain of newly-born animals and acting via GABA(A) receptors. The difference in the end results of GABA(A) receptors activation in the two cases is not due to the receptor associated channels, which in both cases are chloride channels. The different physiological effect in the two cases is due to different electrochemical gradients for chloride. When GABA acting via GABA(A) receptors is inhibitory, either there is no transmembrane electrochemical gradient for chloride or there is one forcing such negative ions into the nerve cell, once chloride channels are open. Viceversa, GABA is excitatory when the electrochemical gradient is such to make chloride ions flow outside the cell, upon opening of the GABA activated chloride channels.In this review this concept is discussed in details and evidence in the scientific literature for the existence of different types of chloride pumps (either internalizing or extruding chloride) is compiled.

Animals↗

Ethanol inhibits the binding of substance P to rat brain cortex NK1 receptors.

The binding of 125I-labeled substance P (SP) to rat brain cortex membranes has been studied under control conditions and in the presence of ethanol. The binding of SP at low concentrations (20-1000 pM) gave two components, one with a KD value of 80 pM and another one with a KD of 500 pM. The higher-affinity component is due to NK1 receptors, as confirmed by the inhibition of the SP binding by the rodent NK1 specific agonist [Sar9 Met(O2)11]SP. Ethanol (1.7 mM) added to the binding assays inhibited by more than 50% the specific binding at a very low SP concentration (20 pM); however, it had no effect at SP concentrations ranging from 50 to 120 pM. This suggests a decrease by ethanol of the affinity of SP to the NK1 receptors involved in this binding component. The ethanol effect disappeared at [EtOH] < or = 0.17 mM.

Animals↗

Antisecretory factor peptide derivatives specifically inhibit [3H]-gamma-amino-butyric acid/36Cl- out-->in permeation across the isolated rabbit Deiters' neuronal membrane.

AIMS: Antisecretory factor (AF) is a 41-kDa protein, its main function being the regulation of intestinal ion/water transport, but it also inhibits chloride and gamma-amino-butyric acid transport across nerve cell membranes. The present experiments were designed to evaluate whether the same AF peptide sequence mediates the permeability effects seen at the nerve cell membrane and in the rat small intestine. METHODS: Four peptides were prepared by the solid phase technique with sequences derived from positions 1-51 of the full-length antisecretory factor AF and tested on nerve cell membranes isolated from rabbit Dieter cells. RESULTS: AF peptides containing the active 36-51 peptide exerted a blocking effect of the out-->in permeation of 36Cl- as well as of [3H]-gamma-amino-butyric acid. The minimal dose causing inhibition, however, varied between 10(-11) m (AF10) and 10(-7) m (AF13). The most potent peptides have been shown previously to be active in inhibiting experimental diarrhoea in vivo in small intestinal ligated loops in rats. The non-active sequence AF23-32 did not inhibit any of the two permeation markers in vitro, a result which supports the lack of activity found also in vivo. CONCLUSION: The results suggest that AF, or AF derivatives, counteract intestinal hypersecretion by blocking anion permeation across large anionic pores. Such a blocking effect could also influence the generation of action potentials in enteric nerve cells controlling the intestinal water and ion transport system.

Amino Acid Sequence↗

Modulation by Zn(2+) and Cd(2+) of GABA(A) receptors of rat cerebellum granule cells in culture.

This study aims to characterize more closely the different populations of GABA(A) receptors present on the cerebellar granule cells of the rat. The effects of two divalent cations, Zn(2+) and Cd(2+), on GABA-activated chloride currents were studied using the whole-cell patch-clamp technique. Zinc cations inhibit differently the peak and the steady-state current elicited by 10 micro M GABA. In fact, Zn(2+) appears to be more potent in inhibiting the steady-state component, with a lower IC(50). The inhibition of the peak component is of the competitive type, whereas the inhibition of the steady-state one is mixed, being partly competitive and partly allosteric. In addition, Cd(2+) has an inhibitory effect on GABA-activated chloride currents. In terms of the peak component, its effect is limited in extent with a maximal inhibition of only 26%, but with a high affinity (IC(50) as low as 0.03 micro M). The steady-state component is inhibited by 20% independently from the Cd(2+) concentration, in the 10(-2)-10(2) micro M range. In this case, the inhibitory mechanism appears to be of the competitive type for the peak component and of the allosteric type for the steady-state one. We suggest these data are a further confirmation that the rapidly and slowly desensitizing components of the GABA-activated chloride currents, corresponding respectively to the peak and the steady-state components, are made up of two different receptor populations.

Animals↗

Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage.

Phosphocreatine can to some extent compensate for the lack of ATP synthesis that is caused in the brain by deprivation of oxygen or glucose. Treatment of in vitro rat hippocampal slices with creatine increases the neuronal store of phosphocreatine. In this way it increases the resistance of the tissue to anoxic or ischemic damage. In in vitro brain slices pretreatment with creatine delays anoxic depolarization (AD) and prevents the irreversible loss of evoked potentials that is caused by transient anoxia, although it seems so far not to be active against milder, not AD-mediated, damage. Although creatine crosses poorly the blood-brain barrier, its administration in vivo at high doses through the intracerebroventricular or the intraperitoneal way causes an increase of cerebral phosphocreatine that has been shown to be of therapeutic value in vitro. Accordingly, preliminary data show that creatine pretreatment decreases ischemic damage in vivo.

Adenosine Triphosphate↗

A simple, inexpensive, and precise spectrophotometric method for evaluating the concentration of ascorbic acid in CSF samples: data from different neurological pathologies.

A method is presented for evaluating the concentration of ascorbic acid in 10 microliters samples of cerebrospinal fluid (CSF) with just ultraviolet (UV) scanning in the 220-300 nm range. The method assumes that the two main UV absorbing CSF components are proteins (with a peak at 280 nm) and ascorbic acid (with a peak at 264 nm). On this basis, the absorbances at those wavelengths are the starting points for a calculation that evaluates the sheer contribution of ascorbic acid to the absorbance at 264 nm. The rapidity of the UV analysis (just a few min), together with the precaution of keeping the samples under argon, along the short preparatory procedure, is especially suitable in the analysis of an air labile substance, such as ascorbic acid. The results of the UV procedure have been checked with parallel HPLC determinations for 19 test CSF samples and the results have shown an excellent correspondence. Finally, data are presented about the evaluation of ascorbic acid concentrations in CSF samples from various neurological pathologies in comparison with normal cases. These data show the interesting result of a significant reduction in the average ascorbic acid CSF level in patients suffering from Alzheimer's disease. However, a decrease in that parameter is found also for a few other pathologies.

Ascorbic Acid↗

Messenger RNAs in synaptosomal fractions from rat brain.

Synaptosomal fractions from rat brain have been analyzed with semi-quantitative RT-PCR methods to determine their content of mRNAs coding for presynaptic, postsynaptic, glial, and neuronal proteins. Each mRNA was determined with reference to the standard HPRT mRNA. In our analyses, mRNAs were considered to be associated with synaptosomes only if their relative amounts were higher than in microsomes prepared in a polysome stabilizing medium, rich in Mg(++) and K(+) ions, or in the homogenate. According to this stringent criterion, the following synaptosomal mRNAs could not be attributed to microsomal contamination and were assumed to derive from the subcellular structures known to harbor their translation products, i.e. GAT-1 mRNAs from presynaptic terminals and glial processes, MAP2 mRNA from dendrites, GFAP mRNA from glial processes, and TAU mRNA from neuronal fragments. This interpretation is in agreement with the involvement of extrasomatic mRNAs in local translation processes.

Animals↗

Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity.

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.

Alkaline Phosphatase↗

Modulation by acute stress of chloride permeation across microdissected vestibular neurons membranes: different results in two rabbit strains and CRF involvement.

Free hand isolation of adult rabbit vestibular Deiters' neurons and dissection of their single membranes allows the study of their ionic permeability characteristics in a microchambers device. In the case of hare-like rabbits, the dissection of such membranes presents evidence of a high basal permeation of labelled chloride, possibly related to mechanical disturbance of the plasma membrane-related cytoskeleton and activation of chloride channels. This did not apply to the laboratory strain of white New Zealand rabbits. However, membranes from hare-like rabbits which were stressed by being rotated on a platform before the experiment, behaved like those from the New Zealand strain. Vice versa, habituation to handling day after day of New Zealand rabbits resulted in a chloride permeation equal to that of unstressed hare-like rabbits. We propose that the stressful conditions result in the release of neurochemical messages to the vestibular Deiters' cells which influence their electrophysiological behavior. The corticotropin releasing factor (CRF), a stress-related peptide present in the climbing fibers, actually blocks the basal chloride permeation across the Deiters' membranes and this effect is partially reversed by its receptor antagonist, alpha-helical CRF [9-41].

Acute Disease↗

Modulation by lanthanum ions of gamma-aminobutyric acid(A) receptors of rat cerebellum granule cells in culture: clues on their subunit composition.

Gamma-aminobutyric acid (GABA) activated chloride currents were studied in rat cerebellum granule cells in culture by the whole cell patch-clamp technique. Both the peak and steady state currents were inhibited by 100 microM lanthanum. In the first case, inhibition is due to an increase of the EC50 for GABA. The inhibitory effect of lanthanum on the peak current at 3 microM GABA increased with the cation concentration. A tendency towards the same behavior was found also for the inhibition of the steady state current, at 3 microM GABA, as a function of lanthanum concentration, although inhibition in this case was lower. The comparison of the results with published data about the effects of lanthanum on recombinant GABA(A) receptors likely to occur in granule cells allows suggestions about the receptor types giving, respectively, the peak and the steady state component.

Animals↗

Regulation of the expression of low affinity GABAA receptors in rat cerebellar granule cells.

GABAA receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding. The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation. A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself. Low affinity GABAA receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABAA receptors mediating the tonic form of inhibition in cerebellar granules.

Animals↗

Holger Hydén's technique of preparation of single Deiters' neurons and study of permeability characteristics of their plasma membranes.

The protocols described here refer to Hydén's technique of isolation and microdissection of vestibular Deiters' neurons from adult mammals. The isolation of Deiters' cells from bovine is described and an example is given of the immunocytochemical visualization of their GABA(A) receptors by monoclonal antibodies against the beta(2/3) subunit. In addition, the protocol of the method for isolation of Deiters' cells from adult rabbit brain stem, the preparation of their plasma membranes and the study of their permeability characteristics is presented. Also in this case, examples of its application to the determination of chloride permeability and its modulation by GABA are given.

Animals↗

Pretreatment of rat brain synaptosomes with GABA increases subsequent GABA uptake via GABA(B) receptor activation.

Pretreatment with 100 microM GABA of synaptosomes purified from rat brain results in an increased uptake of the labelled neurotransmitter in subsequent incubations. The effect is blocked by a GABA(B) receptor antagonist, 2-hydroxy-saclofen. The effect is mimicked by baclofen and the baclofen effect is blocked by saclofen too. Lower GABA concentrations (up to 50 microM) do not result in an increase of subsequent GABA uptake. Treatment of synaptosomes with 8-Br-cAMP results in a decreased GABA uptake. Since the uptake incubations were run with saturating concentrations of labelled GABA, the data indicates that GABA(B) receptor activation in brain synaptosomes up-regulates their GABA uptake capacity by an increase in Vmax. This mechanism appears of physiological relevance under conditions of sustained GABA release and substantial increase of its extracellular concentration.

8-Bromo Cyclic Adenosine Monophosphate↗

GABA and chloride permeate via the same channels across single plasma membranes microdissected from rabbit Deiters' vestibular neurones.

The permeation of labelled gamma-aminobutyric acid (GABA) across single microdissected Deiters' membranes has been studied in a microchamber system. The GABA permeation is via pores which are blocked by 4,4'-diisothiocyanato stilbene-2-2'disulphonic acid (DIDS). As this substance blocks as well chloride permeation across these membranes we tested whether GABA and chloride permeate across the same pores. Membrane pre-treatment with different doses of corticotropin releasing factor (CRF), a membrane permeant cyclic AMP analogue and phalloidin parallelly block the permeation of the two substances. Thus, it is most probable that GABA and chloride pass across the same pores. These pores may be swelling activated ones, opened by the mechanical stress on the membranes in the microchamber system. The passage of GABA across these pores may be of physiological importance in the termination of GABA inhibitory action on the vestibular Deiters' neurones.

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