Benzothiadiazines derivatives as novel allosteric modulators of kainic acid receptors.

The majority of excitatory neurotransmission in vertebrate CNS is mediated by glutamate binding to different types of receptors. Among them, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainite receptors (KAR) are ionotropic receptors playing important pathophysiological roles. A number of small molecules acting as positive allosteric modulators (PAM) of AMPAR have been proposed as drugs for neurological disorders, however, there is no such abundance of ligands capable of modulating KARs activity. We investigated the ability of IDRA21 and of its derivative, compound 2 (c2), to modulate glutamate-evoked currents at native and recombinantly expressed AMPA and KA receptors. By using the patch clamp technique we analyzed the activity of the two compounds in primary cultures of cerebellar granule neurons and in HEK293 cells transiently transfected with KARs and AMPAR subunits. It resulted that both benzothiadiazine derivatives potentiate AMPAR and KAR mediated currents in native and recombinant receptors, c2 being always more potent and efficacious than IDRA21. The potency of both compounds was higher in native receptors than in recombinant receptors. In HEK293 cells transfected with AMPAR subunits, the efficacy of IDRA21 and c2 was much higher in GluA1 than in GluA2 homomeric receptors while their potency did not change. In recombinant KAR, both compounds had a potency in the high micromolar range, while the efficacy reached a maximum in the GluK2 expressing cells. The benzothiadiazine effect, both in native and recombinant receptors, was detected mainly on plateau current, involving a decrease in AMPAR and KAR desensitization. Our study demonstrates for the first time that two positive allosteric modulators of AMPAR, IDRA21 and its derivative, c2, potentiate KAR activity. Furthermore, we highlighted their subunit selectivity that may enable the design of potent and selective PAMs, which could be relevant for the development of new drugs and for a better understanding of KAR functions in the CNS.

[Milk and butter. From the Neolithic to the current nutritional aspects]. / Il latte e il burro. Dal Neolitico agli attuali aspetti nutrizionali.

The evolution in the history of nutrition knowledge towards dairy products, is strictly related to the socio-cultural development of humans. In fact, milk and butter have accompanied humans since ancient times, which traces of the consumption of such products are dated back about the earliest times after the last (glaciation) ice age, while the application for extra nutritional uses, such as cosmetics and ceremonial rites, are reported in the writings of the Old Testament. Even in Italy, before the Roman Empire, were known rudimentary techniques of production and storage of dairy products. But only with the advent of the Etruscans, and the Romans later, that the use of milk and dairy products reach a wide diffusion in several applications. Since the advent of Christ until today, milk and its derivatives have maintained a privileged place in the human diet, but it is only with the advent of modern medicine and new findings in lipidic chemistry that emerged multiple biological and nutritional properties, very important for human health. After a short summary of the ancient history of the milk and butter, the role of dairy products in cancer, in hypercholesterolemia, and cardiovascular disease are reported. Moreover, the current opinions on saturated fatty acids, the role of polyunsaturated fatty acids and their lipid mediators obtained by the action of cyclooxygenase, lipoxygenase and the cytochrome P450 enzymes, are treated. Even if sometimes mistreated, the milk, but most of all its high fat content derivatives such as butter, is a rich source of biologically active compounds that foster a controversial action against neolplastic and cardiovascular disease. These compounds, mainly contained in the lipid fraction, for the more obvious relationships that exist between nutrition and health status, have been the subject in the last decades of intense scientific investigation in which there were expressed lights and shadows, but recognizing that not all fats are harmful and further thorough studies are necessary, in particular, on the derived lipid mediators. This will allow a significant progress based on new scientific evidences, further orienting researchers and clinicians on evidence-based nutritional science.

Thyroid hormones modulate GABA(A) receptor-mediated currents in hippocampal neurons.

Thyroid hormones (THs) play a crucial role in the maturation and functioning of mammalian central nervous system. Thyroxine (T4) and 3, 3', 5-L-triiodothyronine (T3) are well known for their genomic effects, but recently attention has been focused on their non genomic actions as modulators of neuronal activity. In the present study we report that T4 and T3 reduce, in a non competitive manner, GABA-evoked currents in rat hippocampal cultures with IC50s of 13±4µM and 12±3µM, respectively. The genomically inactive compound rev-T3 was also able to inhibit the currents elicited by GABA. Blocking PKC or PKA activity, chelating intracellular calcium, or antagonizing the integrin receptor αVß3 with TETRAC did not affect THs modulation of GABA-evoked currents. THs affect also synaptic activity in hippocampal and cortical cultured neurons. T3 and T4 reduced to approximately 50% the amplitude and frequency of spontaneous inhibitory synaptic currents (sIPSCs), without altering their decay kinetic. Tonic currents evoked by low GABA concentrations were also reduced by T3 (40±5%, n=14), but not by T4. Similarly, T3 decreased currents elicited by low concentrations of THIP, a low affinity GABAA receptor agonist that preferentially activates extrasynaptic receptors, whereas T4 was ineffective. Thus, our data demonstrate that T3 and T4 selectively affect GABAergic phasic and tonic neurotransmission. Since THs concentrations can be regulated at the level of the synapses these data suggest that the network activity of the whole brain could be differently modulated depending on the relative amount of these two hormones. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Evidence that isopropylthioxanthone (ITX) is devoid of anxiolytic and sedative effect.

Isopropylthioxanthone (ITX) is a well-known photo-initiator in ultraviolet light-cured inks frequently used in milk packaging materials, yoghurt, ready-to-feed infant formula, and other drinks. Traces of ITX have been found in milk and, as a consequence, there was considerable interest in studying the biological activity of this molecule and its potential hazard for the human health. Although the ITX genotoxic effects have been excluded by the European Food Safety Authority (EFSA), the US Environmental Protection Agency (USEPA) is still examining its possible toxic potential depending on a dose-effect ratio. Little is known about the ITX activity on the function of the central nervous system and cerebral neurotransmitters. Using behavioural, biochemical, and electrophysiological tests, the authors have found that: (1) ITX did not exert an in vivo anxiolytic or sedative effect when administered orally to rats; (2) ITX did not affect the binding characteristics of central and peripheral benzodiazepine receptors studied in vitro; and (3) ITX did not influence the ability of gamma-Aminobutyric acid (GABA) to increase the chloride channel permeability studied by patch clamp technique in a single neuron of cultured cerebellar granule cells.

Nongenomic regulation of glutamatergic neurotransmission in hippocampus by thyroid hormones.

Thyroid hormones (THs) are well known for their genomic effects but recently attention has focused also on their nongenomic actions as rapid modulators of membrane receptors. Here we show that thyroxine (T4) and 3,3',5'-l-triiodothyronine (T3) rapidly decrease N-methyl-d-aspartate (NMDA)-evoked currents in rat hippocampal cultures with potency in the micromolar range. The effect is not mediated by glutamate or glycine binding sites as an increase in agonist or glycine concentration does not alter TH potencies. Furthermore THs' effect on NMDA receptors is independent of voltage and of subunit composition. The mechanism of THs' antagonistic effect does not involve PKC phosphorylation of NMDA receptors since neither blocking nor stimulating PKC changed THs' modulation. T3, but not T4, inhibits also kainate-evoked currents in hippocampal neurons in culture. In hippocampal pyramidal neurons in slice, T3, but not T4, significantly reduced the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting their amplitude and decay. In cultured rat cortical neurons THs prevented glutamate-induced neuronal death at concentrations similar to those effective on glutamatergic receptors. Taken together our data show for the first time that THs can rapidly affect ionotropic glutamatergic receptors in hippocampal neurons, an effect that could have an important role in their modulation of brain function in physiological and pathological states.

Evidence that the beta-acids fraction of hops reduces central GABAergic neurotransmission.

Humulus lupulus (hops) is traditionally used as a tranquilizing herbal remedy. Here, we investigated the in vivo and in vitro effect of hop beta-acids on central nervous system function. Oral administration of beta-acids (5-10mg/kg) in rats produced an increased exploratory activity in the open field, a reduction in the pentobarbital hypnotic activity and a worsening of picrotoxin-induced seizures. When dosed at 10mg/kg, beta-acids increased, in the elevated plus maze, open arm entries reducing in parallel those in closed arms. In the forced swimming test, we observed a reduction in the immobility time that could suggest an antidepressant-like activity. Electrophysiological studies performed on cerebellar granule cells in culture showed that the beta-acids fraction decreased GABA-evoked current in a dose-dependent way. The effect was not inhibited by the benzodiazepine antagonist Ro 15-1788. Benzodiazepine receptors involvement was also excluded by [(3)H]-Ro 15-1788 binding assay. In conclusion, the behavioral effects of beta-acids fraction could be explained by a reduction in the GABAergic activity although we cannot rule out the involvement of other neurotransmitter systems.

Synthesis, isolation, and GC analysis of all the 6,8- to 13,15-cis/trans conjugated linoleic acid isomers.

Octadecadienoic acids with conjugated double bonds are often referred to as conjugated linoleic acid, or CLA. CLA is of considerable interest because of potentially beneficial effects reported from animal studies. Analysis of CLA is usually carried out by GC elution of FAME. If the presence of low-level isomers is of interest, a complementary technique such as silverion HPLC is also used. These analyses have been hindered by a lack of well-characterized commercially available reference materials. Described here are the synthesis and isolation of selected 6,8- through 13,15-positional CLA isomers, followed by isomerization of these CLA isomers with iodine to produce all the possible cis,cis, cis,trans, trans,cis, and trans,trans combinations. Also present are the GC retention times of the CLA FAME relative to gamma-linolenic acid (6c,9c,12c-octadecatrienoic acid) FAME using a 100-m CP Sil-88 capillary column (Varian Inc., Lake Forest, CA). These data include all the CLA isomers that have been identified thus far in foods and dietary supplements and should greatly aid in the future analysis of CLA in these products.

NAAG fails to antagonize synaptic and extrasynaptic NMDA receptors in cerebellar granule neurons.

The peptide transmitter N-acetylaspartylglutamate (NAAG) selectively activates the group II metabotropic glutamate receptors. Several reports also suggest that this peptide acts as a partial agonist at N-methyl-D-aspartate (NMDA) receptors but its putative antagonist effects have not been directly tested. To do this, we used whole cell recordings from cerebellar granule cells (CGC) in culture that allow the highest possible resolution of NMDA channel activation. When CGC were activated with equimolar concentrations of NMDA and NAAG, the peptide failed to alter the peak current elicited by NMDA. Very high concentrations of NAAG (100-200 microM) did not significantly reduce the current elicited by 10 microM NMDA or 0.1 microM glutamate, while 400 microM NAAG produced only a very small (less than 15%) reduction in these whole cell currents. Similarly, NAAG (400 microM) failed to significantly alter the average decay time constant or the peak amplitude of NMDA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs). We conclude that high concentrations of the peptide do not exert physiologically relevant antagonist actions on synaptic NMDA receptor activation following vesicular release of glutamate. As an agonist, purified NAAG was found to be at least 10,000-fold less potent than glutamate in increasing "background" current via NMDA receptors on CGC. Inasmuch as it is difficult to confirm that NAAG preparations are completely free from contamination with glutamate at the 0.01% level, the peptide itself appears unlikely to have a direct agonist activity at the NMDA receptor subtypes found in CGC. Recent reports indicate that enhancing the activity of endogenous NAAG may be an important therapeutic approach to excitotoxicity and chronic pain perception. These effects are likely mediated by group II mGluRs, not NMDA receptors.

Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture.

We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro.

Modulation of kainate--activated currents by diazoxide and cyclothiazide analogues (IDRA) in cerebellar granule neurons.

1. Patch-clamp technique was used in primary cultures of cerebellar granule neurons to study the modulation of the cyclothiazide analogue (IDRA21) and of the diazoxide derivative (IDRA 5) on KA-evoked currents. 2. The dose-response of kainic acid (KA) reveals an EC50=90 microM and an Hill coefficient of 1.3. IDRA 21 and cyclothiazide potentiate KA-evoked current in a dose dependent way, being cyclothiazide more potent but less efficacious than IDRA 21. Conversely IDRA 5 acts as a negative modulator of KA evoked -current. 3. Application of IDRA 21 and cyclothiazide results in a current potentiation of 125+/-18% and 80+/-12% respectively, while IDRA 5 decreases KA-current (-21+/-5%). Coapplication of cyclothiazide and IDRA 21 produces a potentiation of 110+/-17%, suggesting a competition of the two drugs for the same site. 4. In the same experimental model we studied the ability of IDRA compounds of promoting toxicity through AMPA-receptor activation. Under basal conditions AMPA treatment (50 microM for 1 hour) results in a negligible excitotoxicity. 5. In contrast similar treatment with AMPA + IDRA 21 (1 mM) or + IDRA 5 (1 mM) or + cyclothiazide (100 microM) induces citotoxicity. The neurotoxic damage induced by IDRA 21 and cyclothiazide is blocked by GYKI 53655 (50 microM) and by NBQX (10 microM). Interestingly GYKI and NBQX are ineffective in reducing IDRA 5 toxicity.

[Immunochemical quantification of human monoclonal components]. / Quantificazione immunochimica di componenti monoclonali umane.

The assay of kappa and lambda light chains by rate nephelometry allows quantitative check of electrophoresis profiles with anomalous bands. Immunofixation has also demonstrated this immunochemical method.

[Observations and significance of extrasystole in very young athletes]. / Osservazioni e significato dell'extrasistolia in atleti giovanissimi.

80 very young football players (from 8 to 12) have been examined for three months by some clinical and instrumental cardiologic tests (starting E.C.G. and after graduated stresses on a football court). The starting E.C.G. showed variable extresystolic arrhythmias in 8 subjects, without any sure signs of a cardiopathy, to point out by deeper tests (such as polygraphic, echocardiographic test and rx heart teleradiography). The above-mentioned arrhythmias felt the effects of training variably, since they regressed in 6 cases, however two subjects needed a pharmacological intervention. They are still talking over the meaning to give to extrasystolic arrhythmias in very young people in evaluation of attitude to agonism and in programming training.