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A Volterra

Publications and source records attributed to A Volterra.

At least 37 records · Page 2Linked to original sources
PubMed1995

[Ca2+] modulates the ratio between cycloxygenase and lipoxygenase metabolism of arachidonic acid in homogenates of hippocampal astroglial cultures.

While studying the enzymatic processing of arachidonic acid (AA) to eicosanoids in homogenates of hippocampal astrocytes, we observed that all the HPLC peaks corresponding to AA metabolites displayed significantly different levels depending on the presence or not of free Ca2+ in the incubation medium. A specific pattern was noticed, i.e. lipoxygenase (LOX) derivatives, in particular 12-hydroxyeicosatetraenoic acid (12-HETE), showed higher levels in medium containing 1 mM Ca2+, while cycloxygenase (COX) products including prostaglandins (PG) F2 alpha, E2 and D2 and 12-hydroxyhepatadecatrienoic acid (12-HHT), were higher in Ca(2+)-free medium. COX metabolism exceeded LOX metabolism by threefold in Ca(2+)-free medium, while it was only 60% of it in 1 mM Ca2+. The total amount of AA processed under the two conditions was identical. These data suggest that free [Ca2+] influences the pattern of AA metabolites formed in hippocampal astrocytes, with possible important implications in view of the distinct roles played by COX and LOX eicosanoids in synaptic transmission and neurotoxicity in this area.

Animals↗
PubMed1994

Hippocampal hypoglycaemia-activated K+ channels: single-channel analysis of glucose and voltage dependence.

The effect of glucose on kinetics and the voltage-dependent characteristics of glucose-sensitive channels in hippocampal neurons were examined with the cell-attached mode of the patch-clamp technique. Recordings of a 100-pS K+ channel in the presence or absence of glucose demonstrate that the increase in channel open state probability (Po) induced by glucose deprivation (40- to 400-times the control in high-glucose medium) was largely due to a decrease in the global amount of time spent by the channel in a long-lived closed state. The Po value of the same 100-pS channel was also found to increase (by approx. 80-times) following a depolarization of 40 mV from rest, the main factor responsible for this being a dramatic shortening of the long closed-times on depolarization. Another glucose-sensitive channel of smaller conductance (approx. 10 pS) showed a similar dependence of Po on glucose, but different dependence on voltage, with long openings at the same hyperpolarized potentials where the 100-pS channel was almost always closed. Our results indicate that the action of glucose on the kinetics of hippocampal channels closely resembles that of ATP-sensitive channels in pancreatic beta-cells. Furthermore, they indicate that the two types of glucose-sensitive channels found in hippocampal neurons, differing not only in their single-channel conductance but also in the dependence on voltage, could play different roles in the responses of these cells to modified energetic supply.

Animals↗
PubMed1994

Glutamate uptake inhibition by oxygen free radicals in rat cortical astrocytes.

Formation of reactive oxygen species and disfunction of the excitatory amino acid (EAA) system are thought to be key events in the development of neuronal injury in several acute and long-term neurodegenerative diseases. Recent evidence suggests that the two phenomena may be interdependent. The present study is aimed at exploring possible molecular mechanisms underlying oxygen radical-EAA interaction. Exposure of cortical astrocytic cultures to either xanthine + xanthine oxidase (X/XO), a free radical-generating system, or hydrogen peroxide (H2O2) results in a marked decrease of high-affinity glutamate transport. Within 10 min of X/XO application, uptake falls to approximately 60% of its control value. In parallel no detectable release of lactate dehydrogenase occurs. X/XO effect is abolished in the presence of a mixture of scavenger enzymes (superoxide dismutase+catalase) or by the disulfide-reducing agents glutathione and dithiothreitol (DTT), but not by lipophilic antioxidants or ascorbate. The time course of inhibition shows an almost linear decline of glutamate transport during cell exposure to free radicals, while upon their inactivation the decline stops but established inhibition persists for at least 1 hr. In this situation, application of DTT significantly restores transport function. These data suggest that free radicals inhibit glutamate uptake primarily by long-lasting oxidation of protein sulfhydryl (SH) groups. Chemical modifiers of free SH groups, such as p-chloromercuribenzoate and N-ethylmaleimide, also induce uptake inhibition. Na+/K+ ATPase is a known target of oxygen radicals and may be involved in glutamate uptake inhibition. Indeed, ouabain, a blocker of the pump, reduces uptake in astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1994

Glutamate uptake is inhibited by arachidonic acid and oxygen radicals via two distinct and additive mechanisms.

Reuptake of glutamate in astrocytes, a critical mechanism involved in the maintenance of physiological excitatory amino acid neurotransmission, is inhibited by both arachidonic acid (AA) and reactive oxygen species (ROS), via incompletely defined molecular mechanisms. Because ROS are generated during AA metabolism and AA can be released as a result of ROS-mediated phospholipase A2 activation, it seems likely that their effects on uptake are mediated by a common mechanism. However, here we show that rapid (10-min) uptake inhibitions by AA or by ROS generated by the xanthine plus xanthine oxidase (XO) reaction are selectively abolished by distinct agents; bovine serum albumin (BSA) acts only on AA, whereas the scavenger enzymes superoxide dismutase (SOD) and catalase (CAT) and the disulfide-reducing agent dithiothreitol (DTT) act only on ROS. Moreover, when added together, xanthine/XO and AA decrease uptake in a fully additive manner. In particular, the effect of xanthine/XO is seen also in the presence of maximal AA inhibition. No major signs of cell damage or chemical reaction between AA and radicals accompany their cumulative effects on uptake. Finally, uptake inhibition elicited by AA and xanthine/XO together is attenuated but not blocked by either BSA, DTT, or SOD/CAT individually, whereas it is fully blocked and substantially reversed by a combination of SOD/CAT and BSA or SOD/CAT, DTT, and BSA. Together, these data indicate that AA and ROS act on glial glutamate transport via distinct noninteracting mechanisms. Therefore, they could independently and additively contribute to the impairment of reuptake function, a phenomenon observed in pathological conditions such as ischemia/reperfusion injury.

Animals↗
PubMed1992

Hypoglycemia-activated K+ channels in hippocampal neurons.

Channels linking the electrical and metabolic activities of cells (KATP channels) have been described in various tissues, including some brain areas (hypothalamus, cerebral cortex and substantia nigra). Here we report the existence in hippocampal neurons of K+ permeant channels whose activity is regulated by extracellular glucose. They are open at the cell resting potential and respond to transient hypoglycemia with a reversible increase in activity. The one type so far characterized has a conductance of approximately 100 pS in isotonic K+, is inhibited by the sulphonylurea glibenclamide (1 microM), and is activated by the potassium channel opener lemakalim (0.1-1 microM). These data provide a direct demonstration of the presence, in hippocampal neurons, of glucose-sensitive channels that could belong to the KATP family.

Adenosine Triphosphate↗
PubMed1992

A role for the arachidonic acid cascade in fast synaptic modulation: ion channels and transmitter uptake systems as target proteins.

Recent evidence indicates that arachidonic acid (AA) and its metabolites play a fast messenger role in synaptic modulation in the CNS. 12-Lipoxygenase derivatives are released by Aplysia sensory neurons in response to inhibitory transmitters and directly target a class of K+ channels, increasing the probability of their opening. In this way, hyperpolarization is achieved and action potentials are shortened, leading to synaptic depression. Other types of K+ channels in vertebrate excitable cells have been found to be sensitive to arachidonic acid, lipoxygenase products, and polyunsaturated fatty acids (PUFA). In the mammalian CNS, arachidonic acid is released upon stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors. We found that arachidonic acid inhibits the rate of glutamate uptake in both neuronal synaptic terminals and astrocytes. Neither biotransformation nor membrane incorporation are required for arachidonic acid to exert this effect. The phenomenon, which is rapid and evident at low microM concentrations of AA, may involve a direct interaction with the glutamate transporter or its lipidic microenvironment on the outer side of the cell membrane. Polyunsaturated fatty acids mimic arachidonate with a rank of potency parallel to the degree of unsaturation. Since the effect of glutamate on the synapses is terminated by diffusion and uptake, a slowing of the termination process may potentiate glutamate synaptic efficacy. However, excessive extracellular accumulation of glutamate may lead to neurotoxicity.

Animals↗
PubMed1992

A particle beam-liquid chromatography-mass spectrometry method for the determination of lipoxygenase metabolites of arachidonic acid.

An application of the particle beam-liquid chromatography-mass spectrometry technique to the quantification of hydroxyeicosatetraenoic acids (15-, 12-, and 5-HETE) in biological samples is presented. The acids are extracted with Ethyl acetate and then transformed into pentafluorobenzyl esters, thus increasing the sensitivity of their detection by negative chemical ionization mass spectrometry. Reverse-phase HPLC separation of HETEs is performed in about 10 min with a water-methanol gradient. The procedure shows a detection limit of nearly 0.5 pmol, about one order of magnitude lower than that of the widely used HPLC/UV methods. The quantitative determination is linear (r2 greater than 0.998) for all of the HETEs in the range tested (3-1500 pmol) and a CV lower than 8.5% was observed for repeated analysis of samples. As an application of the method, HETEs formed from endogenous arachidonate were evaluated in extracts obtained from coincubates of platelets and neutrophils stimulated with calcium ionophore A23187.

Adult↗
PubMed1992

High sensitivity of glutamate uptake to extracellular free arachidonic acid levels in rat cortical synaptosomes and astrocytes.

By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high-affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1-100 microM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2-1 microgram/ml), a phospholipase A2 activator, or thimerosal (50-200 microM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate, whereas inhibition greater than or equal to 60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1-10 mg/ml), a free fatty acid-binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses.

Albumins↗
PubMed1989

Direct modulation of Aplysia S-K+ channels by a 12-lipoxygenase metabolite of arachidonic acid.

Lipoxygenase metabolites of arachidonic acid have recently been shown to modulate the activity of ion channels in nerve and muscle cells. The mechanism of action of these metabolites is, however, unknown. In sensory neurons of Aplysia, the S-K- channel is under the dual modulatory control of 5-hydroxytryptamine (5-HT), which decreases the number of active S channels through cyclic AMP-dependent phosphorylation, and the neuropeptide FMRFamide, which increases the probability of S-channel opening through the 12-lipoxygenase metabolite 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Here we report that the increase in the probability of S-channel opening with FMRFamide is mimicked by application of 12-HPETE to cell-free membrane patches that lack ATP and GTP. Thus, 12-HPETE can act directly to modulate S-channel activity, independently of protein phosphorylation or dephosphorylation, G-protein activation or cyclic nucleotides.

Adenosine Triphosphate↗
PubMed1989

FMRFamide reverses protein phosphorylation produced by 5-HT and cAMP in Aplysia sensory neurons.

Neurotransmitter can modulate neuronal activity through a variety of second messengers that act on ion channels and other substrate proteins. The most commonly described effector mechanism for second messengers in neurons depends on protein phosphorylation mediated by one of three sets of kinases: the cyclic AMP-dependent protein kinases, the Ca2+-calmodulin-dependent protein kinases, and the Ca2+-phospholipid-dependent protein kinases. In addition, some neurotransmitters and second messengers can also inhibit protein phosphorylation by lowering cAMP levels (either by inhibiting adenylyl cyclase or activating phosphodiesterases). This raises the question: can neurotransmitters also modulate neuronal activity by decreasing protein phosphorylation that is independent of cAMP? Various biochemical experiments show that a decrease in protein phosphorylation can arise through activation of a phosphatase or inhibition of kinases. In none of these cases, however, is the physiological role for the decrease in protein phosphorylation known. Here we report that in Aplysia sensory neurons, the presynaptic inhibitory transmitter FMRFamide decreases the resting levels of protein phosphorylation without altering the level of cAMP. Furthermore, FMRFamide overrides the cAMP-mediated enhancement of transmitter release produced by 5-hydroxytryptamine (5-HT), and concomitantly reverses the cAMP-dependent increase in protein phosphorylation produced by 5-HT. These findings indicate that a receptor-mediated decrease in protein phosphorylation may play an important part in the modulation of neurotransmitter release.

Animals↗
PubMed1989

Arachidonic acid metabolites as mediators of synaptic modulation.

The neurotransmitters histamine, dopamine and the peptide Phe-Met-Arg-Phe-NH2 (FMRFa) cause presynaptic inhibition in the nervous system of the marine mollusk Aplysia Californica by combined down-modulation of a Ca++ conductance and up-modulation of a K+ conductance. The action of FMRFa on the S-type K+ channels of Aplysia sensory neurons is mediated by a metabolite of the 12-lipoxygenase pathway of arachidonic acid, possibly 12-HPETE. A Pertussis toxin-sensitive GTP binding protein couples FMRFa receptor to the activation of the arachidonic cascade. Once produced, 12-HPETE does not require ATP- or GTP-dependent processes to act on the K+ channels, but it may directly modulate the channel via an external membrane receptor. Based on this observation, a role for eicosanoids as possible intercellular messengers in the C.N.S. is discussed.

Animals↗
PubMed1988

Blockade of ion channel expression in Xenopus oocytes with complementary DNA probes to Na+ and K+ channel mRNAs.

Ionic currents were recorded from Xenopus oocytes injected with RNA isolated from chick or mouse brain. Three currents were studied: a rapid tetrodotoxin-sensitive Na+ current (Ina), an early outward K+ current sensitive to 4-aminopyridine (IA), and an inward current activated by the excitatory amino acid receptor agonist kainate. Oligonucleotides (60-80 bases long) complementary to rat brain Na+ channel sequences were prehybridized to chick brain RNA. These DNA sequences, upon injection into oocytes, specifically inhibited expression of INa relative to IA and the kainate-induced current in a dose-dependent manner. By contrast, prehybridization of oligonucleotides complementary to sequences either from the Drosophila Shaker locus (which codes for an early K+ current in Drosophila muscle) or from a homologous clone from mouse brain did not block the expression of the early outward K+ current induced in the oocytes by mRNA from chick or mouse brain. This method provides a convenient means for testing the functional role of cloned DNA species.

Animals↗
PubMed1988

Interaction of 3,8-diazabicyclo (3.2.1) octanes with mu and delta opioid receptors.

A series of 3,8-diazabicyclo (3.2.1) octanes (DBO) (1) substituted at the nitrogen atoms by acyl and aralkenyl groups, were tested in in vitro binding assays towards mu and delta opioid receptors. The most representative terms (1a, 1d, 1g, 1j,) were also evaluated for the analgesic potency in vivo by the hot plate method. Among the compounds tested the most potent was the p.nitrocinnamyl DBO (1d) which displayed a mu/delta selectivity and an analgesic activity respectively 25 and 17 fold those of morphine. On the contrary, the m.hydroxycinnamyl DBO (1g) was markedly less active as agonist than the parent 1a, thus suggesting that structure 1 interacts with opioid receptors in a different fashion than morphine. Compound 1j isomer of 1a which is provided with high mu affinity, but lower analgesic potency, was found to possess a mixed agonist-antagonist activity.

Animals↗
PubMed1988

Role of two different guanine nucleotide-binding proteins in the antagonistic modulation of the S-type K+ channel by cAMP and arachidonic acid metabolites in Aplysia sensory neurons.

The role of guanine nucleotide-binding proteins (G proteins) in the cAMP-dependent action of serotonin (5-HT) and the antagonistic action of the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRF-amide), mediated by the lipoxygenase metabolites of arachidonic acid, was investigated in Aplysia sensory neurons. Intracellular injection of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) mimics the hyperpolarizing action of FMRF-amide due to activation of the S K+ current and alters the transient response to FMRF-amide into an irreversible (or only partially reversible) response. At higher concentrations, GTP[gamma-S] occludes the response to FMRF-amide. Injection of activated pertussis toxin inhibits the response to FMRF-amide but not to 5-HT. Injection of guanosine 5'-[beta-thio]diphosphate inhibits the response to FMRF-amide by approximately equal to 50% and completely blocks the response to 5-HT. Three lines of evidence suggest that the FMRF-amide-activated G protein is involved at an early stage of the arachidonic acid cascade, prior to the release of arachidonate. (i) Pertussis toxin injection blocks the hyperpolarizing response to FMRF-amide but not to exogenously applied arachidonic acid. (ii) Two blockers of the arachidonic acid cascade inhibit the hyperpolarizing responses to both FMRF-amide and GTP[gamma-S] (and unmask a 5-HT-like depolarizing response to the nucleotide). (iii) Concentrations of GTP[gamma-S] that alter the kinetics of the FMRF-amide response have no effect on the hyperpolarizing response to arachidonic acid. We conclude that a pertussis toxin-sensitive G protein most likely acts to couple the FMRF-amide receptor to phospholipase activation and arachidonic acid release, whereas a pertussis toxin-insensitive G protein couples the 5-HT receptor to adenylate cyclase.

Animals↗