Neuroreceptors and ion channels as targets of alcohol.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Kinya Kuriyama. The presentations were (1) Modulation of neuroreceptors and ion channels by alcohol, by T. Narahashi; (2) Inhibition by ethanol of NMDA and AMPA receptor-channels, by P. Illes, K. Wirkner, W. Fischer, K. Mühlberg, P. Scheibler, and C. Allgaier; (3) Effects of ethanol on metabotropic glutamate receptors, by K. Minami; (4) Acute alcohol actions on the 5-HT3 ligand-gated ion channel, by D. Lovinger; (5) Inhibition of NMDA receptors by MK801 attenuates ethanol-induced taurine release from the hippocampus, by F. Lallemand, R.J. Ward, and P. DeWitte; and (6) Effect of ethanol on voltage-operated Ca2+ channels in hepatic stellate cells, by T. Itatsu, Y. Takei, H. Oide, M. Hirose, X. E. Wang, S. Watanabe, M. Tateyama, R. Ochi, and N. Sato.

Effect of an adenosine A(1) receptor agonist and a novel pyrimidoindole on membrane properties and neurotransmitter release in rat cortical and hippocampal neurons.

Activation of adenosine A(1) receptors by endogenous adenosine plays a neuroprotective role under various pathophysiological conditions including hypoxia. Intracellular recordings were made in rat pyramidal cells of the somatosensory cortex. Hypoxia (5 min) induced a membrane depolarization and a decrease of input resistance. The A(1) receptor agonist N(6)-cyclopentyladenosine (CPA, 100 microM) reversibly inhibited the hypoxic depolarization. The inhibition was also present after blockade of the A(2A), A(2B) and A(3) receptor subtypes by selective antagonists. CPA had no effect on the hypoxic decrease of input resistance. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a selective A(1) receptor antagonist, which did not alter hypoxic depolarization when given alone abolished the inhibitory effect of CPA. Neither CPA nor DPCPX influenced membrane potential or apparent input resistance under normoxic conditions. The novel pyrimidoindole (R)-9-(1-methylbenzyl)-2-(4'-pyridyl)-9H-pyrimido[4,5-b]indole-4-amine (APPPI, 1 and 10 microM) reversibly diminished hypoxic depolarization but had no significant effect on input resistance. The effect of APPPI at a concentration of 1 microM, but not at 10 microM, was blocked by DPCPX (0.1 microM). CPA (100 microM) inhibited [(3)H]-noradrenaline ([(3)H]-NA) release from rat hippocampal brain slices significantly only in the presence of rauwolscine (0.1 microM), an alpha(2)-adrenoceptor antagonist. APPPI (1 and 10 microM) exhibited an inhibitory effect similar to that observed with CPA. The effects of both CPA and APPPI were antagonized by DPCPX (0.1 microM). The present data suggest that mainly presynaptic mechanisms prevent neurons from hypoxic changes by an inhibition of transmitter release. However, in contrast to CPA, APPPI exhibited additional effects, which require further investigation.

Acamprosate inhibits Ca2+ influx mediated by NMDA receptors and voltage-sensitive Ca2+ channels in cultured rat mesencephalic neurones.

Acamprosate has recently been introduced in relapse prophylaxis in weaned alcoholics. Using fura-2 microfluorimetry, the present study investigates whether acamprosate affects N-methyl-D-aspartate (NMDA) or K+-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) in rat cultured mesencephalic neurones. Both application of NMDA (plus glycine) and elevation of extracellular K+ induced rapid increases in [Ca2+]i which respectively were insensitive and sensitive to omega-conotoxin (omega-CTX) MVIIC, a blocker of voltage-dependent Ca2+ channels (VDCCs). Acamprosate (100 microM and 300 microM) significantly attenuated the response induced by NMDA as well as that induced by K+ in a concentration-dependent manner. Concurrent application of omega-CTX MVIIC and acamprosate impaired the K+-induced increase in [Ca2+]i to the same extent as omega-CTX MVIIC alone. The present data suggest that acamprosate inhibits Ca2+ influx through both NMDA receptors and VDCCs.

Ethanol-induced inhibition of NMDA receptor channels.

Ethanol is a potent inhibitor of the N-methyl-D-aspartate (NMDA)-receptor subtype of glutamate receptor in a number of brain areas. The mechanism of ethanol action has been investigated by means of patch-clamp recording of ionic currents and fura-2 measurement of intracellular Ca2+ concentration in cell culture systems; the subunit composition of NMDA receptors and their influence on the effect of ethanol was determined by molecular biology methods. Ethanol does not appear to interact with NMDA either at the glutamate recognition site of the receptor, or at any of the hitherto known multiple modulatory sites, such as the glycine or polyamine site. Moreover, ethanol does not cause an open channel block by itself and fails to interact with Mg2+ at the site where it causes open channel block. The ability of ethanol to inhibit responses to NMDA is dependent on the subunit combination of NMDA receptors. The NR1/NR2A and NR1/NR2B combinations are preferentially sensitive to ethanol inhibition. Chronic treatment with ethanol leads to an increase of the NMDA receptor number at the transcriptional and posttranscriptional level; the receptor function is also facilitated. This causes withdrawal-type seizures after termination of chronic treatment with ethanol. The inhibition of NMDA receptors by ethanol leads to the depression of excitatory synaptic potentials mediated by this type of excitatory amino acid receptor. Ethanol-induced disturbances in certain regions of the brain, i.e. hippocampus, nucleus accumbens or locus coeruleus may lead to cognitive disorders or drug dependence. Brain slices containing the locus coeruleus may be used as an in vitro test system to investigate the addictive properties of ethanol.

NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones.

1. NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT-PCR. 2. NMDA (1-100 microM, plus 10 microM glycine) induced a concentration-dependent increase in [Ca2+]i (EC50 = 5.7 microM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 microM) and nitrendipine (1 microM), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 microM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response. 3. Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 microM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively. 4. Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3' splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable. 5. In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.

Trichloroethanol impairs NMDA receptor function in rat mesencephalic and cortical neurones.

The effects of 2,2,2-trichloroethanol, the active compound of the sedative-hypnotic chloral hydrate, were investigated on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ concentration ([Ca2+]i) in cultured mesencephalic and cortical neurones by means of the fura-2 method. Trichloroethanol inhibited the NMDA response in a concentration-dependent manner in cortical (IC50 = 2.76 mM) and mesencephalic neurones (IC50 = 1.12 mM), with a maximum effect of approximately 85 and 94%, respectively. Ethanol was considerably less potent than trichloroethanol. In conclusion, the trichloroethanol-induced impairment of NMDA receptor function may contribute to the sedative-hypnotic properties of chloral hydrate.

[Analytical comparison of "paraffin" labeled highly toxic lamp oils with paraffinum perliquidum (DAB 10)]. / Analytischer Vergleich "Paraffin"-deklarierter hochtoxischer Lampenöle mit dem Paraffinum perliquidum (DAB 10).

Coloured and odoriferous lamp oils available as household articles repeatedly lead to ingestion intoxications in children in some cases resulting in a bad course. Labels on containers of lamp oils are misleading, e.g. "pure liquid paraffin" or "without noxious substances", causing considerable misconceptions during therapeutic use by treating doctors. Analyses of four arbitrary chosen lamp oils by GC and GC/MS show that the main components (approx. 98%) are the unbranched saturated hydrocarbons n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane. Tests according to the German Pharmacopoeia 10th ed. prove also that "paraffin"-labelled products are not at all identical with medical/pharmaceutical paraffinum perliquidum or liquidum. Lamp oils are low viscous and high toxic petroleum distillates and should be labelled "toxic to humans".

Differences in response of acetylcholinesterase to diisopropylfluorophosphate in the mesencephalic raphe region. A microelectrophoretic, histochemical, and biochemical study in albino rats.

To demonstrate the DFP-effects on the AChE of the mesencephalic raphe region albino rats were investigated using the pharmaco-histochemical method of Butcher et al. (1975). At several times after 1 mg DFP/kg body weight, the histochemical reactions in certain AChE-containing neurons were measured semiquantitatively and compared with biochemical data. Additionally, the multiple forms of water-soluble AChE were separated by microelectrophoresis. Only the electrophoretical results indicate changes of the structural AChE composition 5 d after DFP administration.

The heterogeneity of the nucleus raphes dorsalis in albino rats. A fluorescence histochemical and microelectrophoretic study.

In albino rats, the nucleus raphes dorsalis (NRD) of the midbrain was investigated using a sensitive fluorochroming procedure to detect indolamines in combination with a microelectrophoretic method to demonstrate the isoenzyme pattern of acetylcholinesterase (AChE). Based on the fluorescence histochemical appearance, 3 types of indolaminergic cells were identified in the various areas of the NRD. According to data of the literature and in agreement with our own results, the morphological heterogeneity of the NRD was shown. Significant differences of AChE specific zymograms of NRD regions (subgroups) demonstrate that organization of NRD is heterogeneously proved not only by morphological results but also in respect of enzyme constitution.