Search PubMed⌕ Search

Biomedical subjects

M Bertolino

Publications and source records attributed to M Bertolino.

16 recordsLinked to original sources
PubMed1990

Amiloride blocks glutamate-operated cationic channels and protects neurons in culture from glutamate-induced death.

The diuretic amiloride has been suggested as a specific inhibitor of T-type neuronal Ca2+ channels. The effects of amiloride on glutamate receptor-gated cationic channels and glutamate-induced. Ca2(+)-dependent neuronal death were investigated in primary neuronal cultures from neonatal rats. In primary cultures of cerebellar granule neurons of the rat, receiving 50 microM glutamate for 15 min, at 22 degrees C, in the absence of Mg2+, about 80% of neurons were killed in about 24 hr. Exposure of neurons to such a pulse of glutamate, in the presence of various concentrations of amiloride, resulted in a dose-dependent protection from neurotoxicity (EC50 300 microM, complete protection 1 mM). In voltage-clamped cortical and cerebellar neurons of neonatal rats in primary culture, 100 microM amiloride diminished (by about 25%) glutamate- and/or NMDA-evoked cationic currents, recorded in the whole-cell mode. About 80% of the NMDA-(20 microM) stimulated current was inhibited by 700 microM amiloride. The inhibitory effect of amiloride was not voltage-dependent. In outside-out membrane patches, excised from granule cells and held at -50 mV, 100 microM amiloride changed the NMDA-elicited single channel activity into a fast flickering between the open and closed states. The noise analysis of the data revealed that, although resembling the Mg2(+)-induced flickering, the amiloride-induced channel block was more similar to the effects described for the action of local anaesthetics on the nicotinic cholinergic channel. The pharmacological relevance of this action of amiloride requires further characterization; the data point out the necessity of a cautious use of amiloride in studying neuronal function.

Amiloride↗
PubMed1990

Baclofen inhibits with high affinity an L-type-like voltage-dependent calcium channel in cerebellar granule cell cultures.

In primary cultures of cerebellar granule cells, D,L baclofen (p-chlorophenyl-GABA) inhibited approximately 50% of the calcium-45 influx induced with cell depolarization. The half maximal effective concentration for baclofen was 4 nM. Basal calcium influx was not influenced by baclofen thus suggesting that its inhibitory action could be exerted via a voltage dependent calcium channel (VDCC). Whole-cell recordings by patch-clamp technique showed a calcium current that appeared to be similar to the reported L-type VDCC. Nanomolar concentrations of baclofen also inhibited this calcium current by about 60%. However, in order for baclofen to be active, it needed to be placed into the incubation buffer at least five minutes before patching a cell raising the possibility that baclofen may be acting to inhibit the VDCC via a second messenger system.

Animals↗
PubMed1990

Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death.

Exposing primary cultures of cerebellar granule neurons to 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr decreases the Ca2+/phosphatidylserine/diolein-dependent protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37) by approximately 90% in the 100,000 x g supernatant and pellet fractions of neuronal culture homogenates. Immunoblot analysis of the homogenates with polyclonal antibodies raised against either the beta-type PKC peptide or total rat brain PKC reveals a virtual loss of 78-kDa PKC immunoreactivity in the supernatant and a marked decrease of PKC immunoreactivity in the pellet. Exposure of the cultures to 50 microM glutamate for 15 min (no Mg2+) induces the translocation of supernatant PKC immunoreactivity to the pellet. Such translocation persists after glutamate withdrawal and is followed by a progressive increase in neuronal death, which begins 2 hr later. Neuronal death approaches completion in about 24 hr. PMA-induced down-regulation of PKC decreases glutamate-elicited neurotoxicity. Yet, the culture exposure to 100 nM PMA fails to decrease the high-affinity binding of [3H]glutamate to neuronal membranes and does not reduce glutamate-induced activation of ionotropic or metabolotropic receptors (assayed as total membrane current measured in whole-cell voltage-clamped neurons, 45Ca2+ uptake in intact monolayers, inositolphospholipid hydrolysis, and transcriptional activation and translation of c-fos mRNA). Moreover, the immediate cell-body swelling and activation of spectrin proteolysis elicited by glutamate remain unchanged. On the other hand, PMA-induced PKC down-regulation reduces any increase in 45Ca2+ uptake or Ca2(+)-dependent proteolysis (measured as spectrin degradation) after glutamate withdrawal. These results support the view that PKC translocation is operative in glutamate-induced destabilization of cytosolic ionized Ca2+ homeostasis and neuronal death.

Animals↗
PubMed1989

Kynurenic acid inhibits the activation of kainic and N-methyl-D-aspartic acid-sensitive ionotropic receptors by a different mechanism.

The action of kynurenic acid on currents elicited by the activation of amino acid receptors was investigated in primary cultures of cortical neurons prepared from neonatal rats. Kynurenic acid was tested on currents elicited by both N-methyl-D-aspartic acid (NMDA) and kainate, using patch-clamp recording techniques in "outside-out" and "whole-cell" configurations. The inhibition by kynurenic acid was compared with that elicited by amino-phosphono-valeric acid (APV). Whole-cell currents, elicited by increasing doses of NMDA, were antagonized competitively by APV and non-competitively by kynurenic acid (ID50 70 microM); in contrast, kynurenic acid inhibited competitively the whole-cell currents elicited by kainic acid (ID50 500 microM). The non-competitive inhibition by kynurenic acid of the whole cell currents elicited by NMDA was antagonized competitively by glycine, a specific positive allosteric modulator of NMDA receptors; on the other hand glycine failed to change the inhibition by APV of the NMDA-elicited responses. Thus, kynurenic acid inhibits NMDA receptors allosterically (non-competitively) and kainic acid receptors isosterically (competitively).

Animals↗
PubMed1988

Phencyclidine and glycine modulate NMDA-activated high conductance cationic channels by acting at different sites.

Glutamate activates high (40-50 pS) and low (5-15 pS) conductance cationic channels in outside-out patches excised from cultured cortical and cerebellar granule neurons of neonatal rats. In these neurons, the excitatory amino acid N-methyl-D-aspartic acid (NMDA) activates mainly high conductance channels. Phencyclidine (PCP) at 2 microM selectively reduces the number of NMDA-activated channel openings, at 20 microM it reduces the channel open-time. Glycine increases the opening frequency of high conductance NMDA-activated channels. This action is counteracted by PCP. This inhibition by PCP can be eliminated by reversing the polarity of the membrane patch. However, the effect of glycine is voltage independent. These results imply different sites of action for these two modulators.

Animals↗
PubMed1988

Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex.

Using a sensitive histofluorescence staining method that allows for a quantitation of neuronal death, we compared the protective effects of gangliosides (a group of naturally occurring glycosphingolipids), phencyclidine (PCP), and MK-801 (dibenzocyclohepteneimine) on glutamate- and kainate-induced neuronal death in primary cultures of cortical and cerebellar neurons prepared from neonatal rats. PCP and MK-801 block neurotoxicity induced by glutamate doses 50 times higher than the LD50 (LD50 in Mg2+-free medium, 10 microM) but only partially block the kainate neurotoxicity (LD50 in presence of Mg2+, 100 microM). In contrast, pretreatment with gangliosides (GT1b greater than GD1b greater than GM1) results in complete and insurmountable protection against the neurotoxicity elicited by glutamate or kainate. In primary cultures of cerebellar granule cells gangliosides, unlike PCP and MK-801, fail to block glutamate-gated cationic currents and the glutamate-evoked increase of (i) inositol phospholipid hydrolysis (ii) c-fos mRNA content, and (iii) nuclear accumulation of c-fos protein. Protection of glutamate neurotoxicity by gangliosides does not require their presence in the incubation medium; however, it is proportional to the amount of glycosphingolipid accumulated in the neuronal membranes. The ganglioside concentration (30-60 microM) that blocks glutamate-elicited neuronal death also prevents glutamate- and kainate-induced protein kinase C translocation from cytosol to neuronal membranes.

Animals↗
PubMed1988

Voltage-dependent block by strychnine of N-methyl-D-aspartic acid-activated cationic channels in rat cortical neurons in culture.

Single-channel currents were recorded by means of the patch clamp method in outside-out patches excised from rat cortical neurons in primary culture. The excitatory amino acid N-methyl-D-aspartic acid activated mainly 40-50 pS conductance channels. Channel opening durations were characterized by a series of rapid openings and closures induced by the presence of Mg2+ ions. This inhibitory effect was voltage dependent. Strychnine, the antagonist of the glycine-gated Cl- channels, blocks the N-methyl-D-aspartic acid-activated cationic channel in cultured rat cortical neurons. Strychnine action is voltage dependent and it is not counteracted by ethylenediaminetetraacetic acid, ruling out Mg2+ contamination of strychnine. It has been reported recently that glycine increases the rate of openings of N-methyl-D-aspartic acid-activated channels. This action is not affected by the presence of strychnine. Our results show that 1) Mg2+ and strychnine have an apparently similar intermediate blocking action on the NMDA-activated channels, 2) strychnine presumably acts as a sequential open channel blocker producing a different type of block compared with the one reported for Mg2+ ions, and 3) the lack of effect of strychnine on the glycine potentiation of the N-methyl-D-aspartic acid response indicates that this alkaloid does not competitively antagonize glycine but acts as an open channel blocker.

Allosteric Regulation↗
PubMed1986

The action of AD6 on experimental arrhythmias and on action potentials of cardiac fibers.

AD6 is a coumarinic derivative which increases both coronary blood flow and prostacyclin production, while it decreases platelet responsiveness. We tested its action on experimental cardiac arrhythmias. AD6 (2.5-10 mg/kg) was able to antagonize the arrhythmogenic action of aconitine in rats and of adrenaline in cats. AD6 action was also tested in vitro. The drug (20-50 microM) prolonged the functional refractory period of guinea-pig atrial and ventricular muscle and lengthened the refractory period shortened by hypoxia. Intracellular electrophysiological experiments showed that AD6 prolongs action potential duration (APD) of guinea-pig atrial myocardium, sino-atrial node and cat Purkinje fibers. The results obtained in vitro may explain the effect on experimental arrhythmias, therefore suggesting a protective action on cardiac rhythm disturbances.

Aconitine↗
PubMedDate not supplied

[Xerostomia].

Explore the source record for details and available documents.

Humans↗