Search

The effect of halothane and isoflurane was studied on the function of recombinant neurotransmitter receptors expressed in Xenopus oocytes. Both anaesthetics inhibited nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptor-mediated responses, potentiated 5-hydroxytryptamine type 3 (5-HT3) receptor-mediated responses at low agonist concentrations, and inhibited the function of a chimeric receptor (with the N-terminal domain from the nACh alpha 7 receptor and the transmembrane and C-terminal domains from the 5-HT3 receptor) in a manner similar to that of the nACh alpha 7 receptor. Since the N-terminal domain of the chimeric receptor was from the nACh alpha 7 receptor, the observations suggest that the inhibition involves the N-terminal domain of the receptor.

The effect of pH on ATP-activated current in bullfrog dorsal root ganglion neurons was studied using the whole-cell patch-clamp technique. ATP-activated current amplitude was highly dependent upon extracellular pH. An acid pH increased, whereas alkaline pH decreased, ATP-activated current amplitude. The half-maximal pH (EC50) for potentiation of 2.5 micro;M ATP-activated current was 7.2. Acidification alone did not activate detectable current and, at an acid pH, ATP-activated current was abolished by suramin. Proton-induced enhancement of ATP-activated current was not sensitive to membrane potential between -80 and +40 mV, and did not involve a shift in reversal potential. Lowering pH from 7.2 to 6.5 or elevating pH from 7.2 to 8.0 shifted the ATP concentration/response curve to the left or right, respectively, without changing the maximal response to ATP. Protons increased the time constant of deactivation without affecting the time constant of activation or desensitization of ATP-activated current. Alteration of patch-pipette (intracellular) pH did not affect the enhancement of ATP-activated current by extracellular protons. Diethylpyrocarbonate (DEP), dithiothreitol (DTT), 5, 5'-dithio-bis-(2-nitro-benzoic acid) (DTNB), or N-ethylmaleimide (NEM) did not affect enhancement of ATP-activated current by protons. The results suggest that extracellular protons, at physiological concentrations, can regulate the function of P2X purinoceptors by modulating the affinity of the ATP-binding site.

Dynorphin is an endogenous ligand for kappa-opioid receptors. We investigated the effect of dynorphin 1-13 on different heteromeric subunits of recombinant mouse N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes by using voltage-clamp recording methods. Dynorphin inhibited the NMDA-activated currents of all heteromeric NMDA receptor subunits tested. The different NMDA receptor subunits, however, exhibited a differential sensitivity to dynorphin. For the epsilon-1/zeta-1 subunit combination the EC50 was 19 microM; the other NMDA receptor subunit combinations were less sensitive to dynorphin and had the following order of sensitivity: epsilon-2/zeta-1 > epsilon-4/zeta-1 > epsilon-3/zeta-1. Inhibition of NMDA-activated currents by dynorphin was not competitive with NMDA, and was voltage-independent. NMDA-activated currents were not affected by the synthetic kappa-opioid receptor agonist U50488 ¿trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzene-acetamide¿, the specific kappa-opioid receptor antagonist nor-binaltorphimine1 or the nonspecific opioid receptor antagonist naloxone. In addition, nor-binaltorphimine1 or naloxone did not attenuate dynorphin inhibition of NMDA-activated current. The observations suggest that dynorphin inhibition of NMDA receptor function is mediated by an interaction of dynorphin with NMDA receptors, rather than an action involving kappa-opioid receptors. The data also show that different heteromeric NMDA receptor subunits exhibit a differential sensitivity to dynorphin.

Several lines of evidence suggest a physiological role for Cu2+ in regulating nervous system function. In the present study using whole-cell patch-clamp recording, Cu2+ greatly enhanced current activated by 10 microM ATP in the majority of rat nodose ganglion neurons. The enhancement was concentration-dependent between 1 and 50 microM Cu2+, and had an EC50 of 6.1 microM. Cu2+ shifted the ATP concentration-response curve to the left in a parallel manner. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally-effective concentration of Zn2+. The observations suggest that Cu2+ increases the affinity of the receptor for ATP by acting at the Zn2+ modulatory site. In addition, a subset of neurons in the nodose ganglion express ATP-gated receptor-channels that are insensitive to modulation by physiological concentrations of Cu2+, Zn2+ and protons.

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC50S for inhibition of NMDA-activated currents by ethanol were 129 mM +/- 20 mM in hippocampal neurons and 126 +/- 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC50 of 174 +/- 23 mM, which was not significantly different from the IC50S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC50 of 340 +/- 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC50 of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.

1. The modulation by protons of ATP-gated ion channel responses to ATP and Zn2+ was studied in freshly isolated rat nodose ganglion neurons using the whole cell patch-clamp technique. 2. Reduced external pH enhanced, whereas elevated external pH suppressed, current activated by 10 microM ATP. The pH producing the half-maximal effect (EC50) at this ATP concentration was 7.1. 3. Acidification shifted the ATP concentration-response curve to the left, decreasing the EC50 for ATP, and alkalinization shifted the ATP concentration-response curve to the right, increasing the EC50 for ATP. Fitting the data to a single-site pH model yielded an apparent pKa of the site on the ATP-gated ion channel of 7.6. Between pH 6.8 and 7.8, a change of 0.1 pH unit was calculated to change the ATP EC50 by 4.03 microM. Changing pH did not alter the maximal response to ATP. 4. The potentiating effect of protons appeared to be due to a direct action on the ATP-gated channel, as it could not be explained by an increase in the concentration of one or more species of ATP. 5. Lowering pH also increased the potency of Zn2+ for enhancement of ATP-activated current without altering its maximal response. Changing the pH from 7.3 to 6.8 changed the Zn2+ EC50 from 12 to 1.7 microM. 6. The potentiation of ATP-activated current by protons could not be attributed solely to an increase in the affinity of the receptor for Zn2+, as the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine did not alter the effect of protons. 7. Protons and Zn2+ do not appear to act at the same site on ATP-gated channels, as responses to maximally effective concentrations of Zn2+ were enhanced further by protons and vice versa. 8. These results suggest that protons regulate the function of P2X purinoceptors in rat nodose ganglion neurons by modulating the affinity of the binding sites for ATP and Zn2+ on these receptor channels.

Williams syndrome (WS) is a developmental disorder with variable phenotypic expression associated, in most cases, with a hemizygous deletion of part of chromosomal band 7q11.23 that includes the elastin gene (ELN). We have investigated the frequency and size of the deletions, determined the parental origin, and correlated the molecular results with the clinical findings in 65 WS patients. Hemizygosity at the ELN locus was established by typing of two intragenic polymorphisms, quantitative Southern analysis, and/or FISH. Polymorphic markers covering the deletion and flanking regions were ordered by a combination of genetic and physical mapping. Genotyping of WS patients and available parents for 13 polymorphisms revealed that of 65 clinically defined WS patients, 61 (94%) had a deletion of the ELN locus and were also hemizygous (or noninformative) at loci D7S489B, D7S2476, D7S613, D7S2472, and D7S1870. None of the four patients without ELN deletion was hemizygous at any of the polymorphic loci studied. All patients were heterozygous (or noninformative) for centromeric (D7S1816, D7S1483, and D7S653) and telomeric (D7S489A, D7S675, and D7S669) flanking loci. The genetic distance between the most-centromeric deleted locus, D7S489B, and the most-telomeric one, D7S1870, is 2 cM. The breakpoints cluster at approximately 1 cM to either side of ELN. In 39 families informative for parental origin, all deletions were de novo, and 18 were paternally and 21 maternally derived. Comparison of clinical data, collected in a standardized quantifiable format, revealed significantly more severe growth retardation and microcephaly in the maternal deletion group. An imprinted locus, silent on the paternal chromosome and contributing to statural growth, may be affected by the deletion.

ATP stimulates nociceptive neurons via an action on ligand-gated ion channels. Since tissue injury and inflammation result in both localized acidosis and release of ATP, we studied the effect of acid pH on ATP-gated ion channels in rat nodose ganglion neurons. Lowering pH dramatically increased membrane depolarization and action potential firing elicited by ATP. ATP-activated current was enhanced by acid pH and suppressed by alkaline pH. A pH of 7.2 produced the half-maximal effect. Acidification increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. The observations suggest that the localized acidosis associated with tissue injury may enhance pain perception via an action on ATP-gated ion channels on mammalian sensory neurons.

Evidence indicated that the x gene of human HBV can cause cancer in transgenic mice, moreover, HBxAg was so far the most frequent and strong antigen among those HBV markers expressed in hepatocellular carcinoma (HCC) tissues. Aiming to enhance killing of HCC by effector cells, we established an anti-HBx/anti-CD3 hybrid-hybridomas by fusion of anti-HBx hybridoma cells with FITC-labeled anti-CD3 HAT sensitive cells, and followed by FACStar sterile cell sorting, HAT selection and eventually verified by ELISA and double bridging assay. Using two color cytometric analysis, we found that bispecific monoclonal antibody (BsAb) remarkably enhanced in vitro effector-target cell conjugates (48.3% vs. 8.5%). In in vivo study, BsAb retargeting effector cells were significantly more effective than that of effector cells alone in shrinkage of LTNM4 HCC xenografts (HBxAg positive) in nude mice, not only in fresh inoculated tumors but also in established tumors (p<0.01, p<0.01, respectively). Besides, pronounced apoptotic cell death and infiltration of lymphocytes in the peripheral of tumor nodules can also be witnessed in the tissues treated by BsAb plus effector cells, but not in the controls. The results demonstrated that antiHBx/anti-CD3 BsAb was able to redirect effector cells for lysis of HBxAg positive HCC cells both in vitro and in vivo and it also indicated that shrinkage of tumors in nude mice with therapy of BsAb retargeting effector cells was partially due to initiation of apoptotic cell death.

There has been a long-standing debate concerning whether alcohols produce their effects in the central nervous system (CNS) by acting on lipids or proteins. Lipid theories postulate that alcohols act via some perturbation of the membrane lipids of CNS neurons, whereas protein theories propose that alcohols act by interacting with a neuronal protein site. Although the primary site of action differs in the two theories, both theories postulate that the CNS effects of alcohols ultimately result from alterations in protein function. This review discusses lipid and protein theories of alcohol action and the evidence supporting these theories. In addition, the effects of alcohols on the function of neurotransmitter-gated ion channels are discussed, as several types of these receptor-ion channels have been found to be sensitive to the actions of alcohols, and recent studies on those actions have yielded new insights into the question of whether the primary action of alcohols involves lipids or proteins.

As the number of carbon atoms in an aliphatic n-alcohol is increased from one to five, intoxicating potency, lipid solubility, and membrane lipid disordering potency all increase in a similar exponential manner. However, the potency of aliphatic n-alcohols for producing intoxication reaches a maximum at six to eight carbon atoms and then decreases. The molecular basis of this "cutoff" effect is not understood, as it is not correlated with either the lipid solubility or the membrane disordering potency of the alcohols, which continue to increase exponentially. Since it has been suggested that inhibition of N-methyl-D-aspartate (NMDA) receptors by alcohols may play a role in alcohol intoxication, we investigated whether a series of aliphatic n-alcohols would exhibit a cutoff in potency for inhibition of NMDA receptors. We found that although potency for inhibition of NMDA receptors increased exponentially for alcohols with one to five carbon atoms, potency for inhibition of NMDA receptors reached a maximum at six to eight carbon atoms and then abruptly disappeared. This cutoff for alcohol inhibition of NMDA receptors is consistent with an interaction of the alcohols with a hydrophobic pocket on the receptor protein. In addition, the similarity of the cutoffs for alcohol inhibition of NMDA receptors and alcohol intoxication suggests that the cutoff for NMDA receptor inhibition may contribute to the cutoff for alcohol intoxication, which is consistent with an important role of NMDA receptors in alcohol intoxication.

The ring chromosome 15 syndrome is characterized by mild-to-severe growth failure. We evaluated the status of the insulin-like growth factor I receptor (IGF1R) gene, which had previously been assigned to band 15q26 in several patients with de novo ring 15 chromosomes, to investigate a possible correlation between disruption or loss of the IGF1R gene with the severe growth failure phenotype. The presence or absence of the IGF1R gene on the ring 15 chromosomes of five patients was ascertained by in situ hybridization and gene-dosage (Southern) blotting. The location of the breakpoints was determined by typing polymorphic markers from the distal end of the long arm of chromosome 15 in both the probands and their parents. Deletion mapping determined that all breakpoints were distal to D15S100 and that the IGF1R gene is located between D15S107 and D15S87. Three patients who had suffered severe growth failure in early childhood were hemizygous at the IGF1R locus, while one patient with borderline short stature had two copies of the IGF1R gene. The correlation between IGF1R gene dosage and growth retardation demonstrated here in our ring chromosome 15 patients suggests a possible role for heterozygous IGF1R gene mutations or deletions in other cases of unexplained growth failure.

1. The action of 2,2,2-trichloroethanol on gamma-aminobutyric acid (GABA)-activated Cl- current was studied in mouse hippocampal neurones in tissue culture by use of whole-cell patch-clamp recording. 2. Trichloroethanol increased the amplitude of currents activated by 1 microM GABA or 0.1 microM muscimol. Trichloroethanol, 1-25 mM, potentiated current activated by 1 microM GABA in a concentration-dependent manner with an EC50 of 3.0 +/- 1.4 mM and a maximal response (Emax) of 576 +/- 72% of control. 3. Trichloroethanol potentiated currents activated by GABA concentrations < 10 microM, but did not increase the amplitude of currents activated by concentrations of GABA > or = 10 microM. Despite marked potentiation of currents activated by low concentrations of GABA, trichloroethanol did not significantly alter the EC50, slope, or Emax of the GABA concentration-response curve. 4. Trichloroethanol, 5 mM, potentiated GABA-activated current in neurones in which ethanol, 10-500 mM, did not. The effect of trichloroethanol was not altered by the putative ethanol antagonist, Ro 15-4513. Trichloroethanol did not potentiate currents activated by pentobarbitone. 5. In the absence of exogenous GABA, trichloroethanol at concentrations > or = 2.5 mM activated a current that appeared to be carried by Cl- as its reversal potential changed with changes in the Cl- gradient and as it was inhibited by the GABAA antagonists, bicuculline methiodide and picrotoxin. 6. Since trichloroethanol is thought to be the active metabolite of chloral hydrate and other chloral derivative anaesthetics, potentiation of the GABA-activated current in central nervous system neurones by trichloroethanol may contribute to the sedative/hypnotic effects of these agents.

The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg2+ resulted in a five-fold increase in NMDA-induced peak of [Ca2+]i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg2+. Cyanide enhanced the immediate rise in [Ca2+]i produced by NMDA, followed over a 1-5 min period by a gradual increase of [Ca2+]i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg2+. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [3H]MK-801 to rat forebrain membranes in the presence of Mg2+, whereas in the absence of Mg2+, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca2+ influx and inward current by interacting with the Mg2+ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg2+ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca2+ influx resulting from cellular energy reserve depletion.

For almost a century, alcohols have been thought to produce their effects by actions on the membrane lipids of central nervous system neurons--the well known "lipid theory" of alcohol action. The rationale for this theory is the correlation of potency with oil/water or membrane/buffer partition coefficient. Although a number of recent studies have shown that alcohols can affect the function of certain neuronal neurotransmitter receptors, there is no evidence that the alcohols interact directly with these membrane proteins. In the present study, we report that inhibition of a neuronal neurotransmitter receptor, an ATP-gated ion channel, by a series of alcohols exhibits a distinct cutoff effect. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. The results suggest that alcohols inhibit the function of this neurotransmitter receptor by interacting with a small hydrophobic pocket on the receptor protein.

Eight different mutations were detected in the growth hormone (GH) receptor gene of patients with inherited GH receptor deficiency (GHRD; Laron syndrome) from five continents. All the mutations are located in the extracellular domain of the receptor and are predicted to cause gross structural abnormalities and non-functional receptor molecules. They include three nucleotide changes in the coding region causing translational stop signals, including the newly identified E183X mutation; two nucleotide changes in introns that affect splice junctions; two dinucleotide deletions that result in stop codons downstream; and one single nucleotide change that activates a donor splice site within an exon and results in a transcript missing 24 nucleotides. This latter mutation (E180splice) was first identified in a cohort of patients with GHRD from southern Ecuador. Based on the fact that the E180splice mutation generates a new cleavage site for the restriction enzyme MnlI, a simple diagnostic test has been developed that can be carried out on dried blood spots collected on filter paper. A total of 55 affected individuals from Ecuador has been found to be homozygous for this mutation. Asymptomatic carriers can also be detected, and 104 of 150 individuals screened were found to be carriers. Using this test, the E180splice mutation has recently been detected in one of two oriental Jewish patients from Israel.

The cellular mechanisms by which ethanol affects nervous system function are poorly understood. However, evidence has been accumulating that ethanol can affect the function of neurotransmitter-gated ion channels. Extracellular ATP has recently been reported to produce excitatory actions in the peripheral and central nervous systems by activating ligand-gated ion channels. We studied the effect of ethanol on membrane ion current activated by extracellular ATP in isolated bullfrog dorsal root ganglion neurons, by means of the whole-cell patch-clamp technique. The amplitude of the ATP-activated current was decreased by ethanol in a concentration-dependent manner over the range of 3-500 mM. The average inhibition of 1 microM ATP-activated current by 100 mM ethanol was 64 +/- 3%, and the concentration of ethanol that produced 50% inhibition was 68 mM. Ethanol inhibition of ATP-activated current was not dependent on membrane potential from -80 to +40 mV, and ethanol did not change the reversal potential of ATP-activated current. Ethanol (100 or 400 mM) shifted the ATP concentration-response curve to the right, increasing the EC50 for ATP from 3.0 microM to 6.0 microM or 22.3 microM, respectively, but did not reduce the maximal response to ATP. The results suggest that ethanol inhibits ATP-activated current by increasing the apparent dissociation constant for the ATP receptor.

Despite the increasing recognition that ATP is an important extracellular excitatory mediator in the nervous system, the regulation of ATP receptors is poorly understood. Because the extracellular Zn2+ concentration is regulated in a variety of biological tissues, we studied modulation of the ATP-gated cation channel by Zn2+ in mammalian neurons using the whole-cell patch-clamp technique. In approximately 73% of cells tested, the amplitude of ATP-activated membrane ion current increased up to 5-fold in the presence of micromolar concentrations of Zn2+. The characteristics of this action suggest that Zn2+ increases the apparent affinity of the receptor for ATP. In addition, Zn2+ increased membrane depolarization and action potential firing elicited by ATP. These observations suggest that Zn2+ may play a physiological role in regulating the excitatory action of ATP on mammalian neurons.

The effects of ethanol on excitatory amino acid activated ion channels were investigated using patch-clamp recording methods. Intoxicating concentrations of ethanol (5-50 mM) inhibited ion current activated by the glutamate receptor agonist N-methyl-D-aspartate (NMDA) in a concentration-dependent manner (IC50 = 30 mM). The intoxicating potency of different alcohols was correlated with their potency for inhibiting NMDA-activated current, suggesting that alcohol-induced inhibition of NMDA channel function may contribute to the neural and cognitive impairments associated with intoxication. Analysis of mechanism suggests that ethanol inhibits NMDA-activated current by altering gating of the channel, rather than by affecting channel conductance, ion permeance or regulatory sites on the channel. Anesthetic concentrations of ethanol (> 50 mM) produced a concentration-dependent inhibition of kainate- and quisqualate-activated currents (IC50 = 220 mM), suggesting that this inhibition may contribute to the general anesthetic effects of ethanol. This hypothesis is supported by the observations that the general anesthetic agents, trichloroethanol (the active metabolite of chloral hydrate), pentobarbital and halothane, all inhibit kainate- and quisqualate-activated currents.

The ability of atrial natriuretic peptide (ANP) to modulate K+-stimulated release of [3H]norepinephrine ([3H]NE) from rat hypothalamic slices was investigated. ANP-(1-28) significantly decreased K+-stimulated [3H]NE release in a concentration-dependent manner (maximal inhibition = 22% of control with 100 nM, ED50 = 70 pM). Pretreatment with pertussis toxin did not alter the response to ANP. 8Br-cGMP (10 microM), a cGMP analog, significantly decreased [3H]NE release and when combined with 10 nM ANP-(1-28), an additive effect was observed. Additionally, 3-isobutyl 1-methylxanthine (IBMX) (200 microM), a phosphodiesterase inhibitor, combined with ANP-(1-28) 10 nM, significantly decreased [3H]NE release. These results indicate that ANP-(1-28) modulated release of [3H]NE from rat hypothalamic slices and the effect is most likely mediated by elevation of intraneuronal cGMP.

The interaction of ethanol with glycine at the N-methyl-D-aspartate (NMDA)-activated ion channel was investigated in voltage-clamped rat cultured hippocampal neurons. As shown previously, glycine increased, and ethanol inhibited, the NMDA-activated current in these cells. Concentration-response data for glycine (0.1-100 microM) indicate that the inhibition of NMDA-activated current by ethanol does not involve a competitive interaction with glycine. Thus, ethanol appears to inhibit NMDA-activated current at a locus different from the glycine modulatory site.

The evidence presented above indicates that GABA- and glutamate-activated ion channels are molecular sites of alcohol and anesthetic action. In view of the important role that these channels play in CNS excitability, it seems likely that the actions of alcohol and anesthetics on these channels contribute significantly to the behavioral effects of these agents. Although the behavioral effects of alcohol and anesthetics may well result from a combination of actions on different ion channels and other molecular sites in the CNS, it is of interest to consider whether the actions of these agents on particular types of ion channels may contribute to particular behavioral effects. In this regard, it should be noted that benzodiazepines potentiate GABAA responses, but do not produce intoxication or general anesthesia in their clinical dose range. Benzodiazepines are widely used clinically, primarily for their anxiolytic actions (26), suggesting that the potentiation of GABAA responses by ethanol and barbiturates may contribute to the anxiolytic effects of these agents. Since kainate and quisqualate channels mediate fast excitatory transmission in the CNS, inhibition of kainate and quisqualate receptor-activated responses would be expected to result in general CNS depression. This suggests that inhibition of kainate and quisqualate receptor-mediated responses may contribute to the general anesthetic effects of ethanol, trichloroethanol and barbiturates. NMDA channels are thought to mediate complex excitatory neural phenomena and cognitive function. In view of this, the observation that ethanol inhibits NMDA receptor-mediated responses over the concentration range that produces intoxication and the correlation between the potency of different alcohols for inhibiting NMDA-activated current and their potency for producing intoxication suggest that ethanol-induced inhibition of NMDA receptor-mediated responses may contribute to the intoxicating effects of ethanol. Although these speculations are no doubt oversimplifications, the recognition that GABA- and glutamate-gated ion channels are molecular sites of alcohol and anesthetic action provides a basis for investigating the molecular mechanisms involved in the action of these agents and the behavioral significance of those actions.

Agents that enhance gamma-aminobutyric acid (GABA) neurotransmission can modulate certain effects of opioids, such as analgesia. In this study, the interaction between morphine and GABAergic agents on the release of [3H]norepinephrine ([3H]NE) from rat frontal cortical slices was examined. GABA (10(-4) M), enhanced potassium-stimulated [3H]NE release and reversed the inhibitory effect of 10(-6) M morphine. GABA and muscimol modulated the inhibitory effect of morphine in a noncompetitive manner. Bicuculline methiodide (10(-4) M) reduced the effect of GABA in the absence of morphine, and appeared to reduce the effect of GABA in the presence of morphine, although the latter effect was not statistically significant from the controls. While the GABAA agonist muscimol mimicked the effect of GABA, the GABAB agonist baclofen did not affect the release of [3H]NE in the absence or the presence of 10(-6) M morphine. These results support the involvement of GABAA receptors in modulating the action of opioids on the noradrenergic system in the cerebral cortex of the rat.

Exposure of rat pheochromocytoma (PC12) cells to KCN (1.0-10 mM) over a 30-min period stimulated secretion of dopamine (DA) and decreased intracellular DA content. Addition of KCN (10 mM) to rat frontal cortex slices preloaded with 1-[7-3H]norepinephrine ([3H]NE) increased secretion of NE over a 10- to 30-min incubation period. In PC12 cells release of DA by KCN was nearly abolished in calcium-free media or by prior addition of diltiazem, a calcium channel antagonist. Release of [3H]NE from rat cortical slices by cyanide was only partly inhibited by diltiazem suggesting that intracellular calcium may be involved in this response. In PC12 cells KCN also produced a dose-related release of the DA precursor dihydroxyphenylalanine, without altering intracellular stores. Levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were enhanced at lower concentrations of KCN. These observations indicate cyanide elicits exocytotic release of neurotransmitters in a calcium-dependent manner and also show that cyanide alters catecholamine metabolism. These actions of cyanide may be important in CNS symptoms of intoxication.

Gamma-Aminobutyric acid (GABA) and GABAA agonists enhance stimulated release of [3H]norepinephrine [( 3H]NA) in several regions of the rat brain. In this study, the mechanisms by which GABA and GABAergic agonists augment potassium-stimulated release of [3H]NA from rat frontal cortical slices were examined. GABA enhanced potassium-stimulated [3H]NA release, but did not alter release of [3H]NA evoked by the calcium ionophore A23187, 10(-5) M, either in the presence or the absence of extracellular calcium. The effect of GABA on potassium-stimulated [3H]NA release was apparently reduced by the GABAA antagonist bicuculline methiodide, 10(-4) M, and by the selective inhibitor of GABA uptake SKF 89976A, 10(-5) M, but was abolished only when bicuculline methiodide and SKF 89976A were present in combination. The GABAA agonist muscimol enhanced potassium-stimulated release of [3H]NA in a manner similar to GABA. In addition, nipecotic acid, a substrate for GABA uptake, enhanced potassium-stimulated [3H]NA release. Thus, GABA appears to enhance potassium-stimulated [3H]NA release by acting upon both GABA uptake and GABAA receptors. The GABAA receptors involved in this effect may be a subtype of GABAA receptors since they are not modulated by benzodiazepines. These results support the involvement of the GABA uptake carrier and the GABAA receptor in mediating the enhancement by GABA of potassium-stimulated [3H]NA release in the cortex of the rat.

The actions of alcohol and anesthetics have been studied on excitatory amino acid activated ion channels in mammalian neurons. Ethanol inhibits NMDA-activated current over a concentration range that produces intoxication, and the potency of several alcohols for inhibiting the NMDA-activated current is correlated with their intoxicating potency, suggesting that alcohol-induced inhibition of responses to NMDA receptor activation may contribute to the neural and cognitive impairments associated with intoxication. Studies on the mechanism of ethanol inhibition of NMDA-activated current indicate that ethanol does not appear to block the ion channel, alter the ion selectivity of the channel, or interact with previously described binding sites on the NMDA receptor/ionophore complex. The linear relation between the potency of several alcohols for inhibiting the NMDA-activated current and the hydrophobicity of the alcohols suggests that ethanol may inhibit the NMDA-activated ion current by a novel type of interaction with a hydrophobic site associated with the NMDA channel. In addition, different types of general anesthetic agents exhibit different inhibitory actions on NMDA-, kainate-, and quisqualate-activated currents, suggesting that differences in the profile of inhibition of excitatory amino acid neurotransmission in the CNS among different classes of general anesthetics may contribute to the differences in their behavioral and physiological effects.

The tricyclic antidepressant desmethylimipramine (DMI) interacts with the NMDA receptor/ionophore complex; however, the site of the interaction has not been clearly established. Although evidence from receptor binding assays suggests that DMI interacts with the Zn2+ binding site, other binding studies and electrophysiological studies suggest otherwise. Using the whole-cell patch clamp technique to record from cultured hippocampal neurons, we report that recovery of NMDA-activated current from block by DMI is time-dependent and this time-dependent component was not observed following preexposure of neurons to Zn2+. These observations favor the hypothesis that DMI interacts at a binding site within the NMDA receptor/complex channel pore and not at the Zn2+ binding site.

We conducted a six-month, randomized, double-blind trial of lecithin therapy in early-onset Alzheimer's disease. We hypothesized that such therapy would retard the progression of the clinical and neuropsychological manifestations of this illness. Of the 73 referred patients, 37 met strict requirements for diagnosis and compliance. The 21 placebo and 16 lecithin-treated patients (mean age 63 years) had a comparable degree of severity of dementia (mean Clinical Dementia Rating 1.6). Lecithin therapy produced an increase in mean plasma choline levels from a baseline of 15.9 to 28.8 nmol/ml. Patients were evaluated by the physician using clinical assessments (CDR, Lawton ADL and other rating scales) and by the neuropsychologist who determined the outcome of therapy on a battery of tests (Mini Mental State Examination, Wepman Aphasia Screen, Verbal Fluency Test, Verbal Selective Reminding Test and Spatial Memory Test). Only 6 (37.5%) of the 16 lecithin-treated patients were considered by the neurologist to be clinically stable or improved as compared to 12 (57.1%) of the 21 patients given placebo (difference -19.6%, 95% confidence limits of -51% to 12%). The neuropsychologic scores showed no differences in the stability of the dementing process over time between the lecithin-treated (50.0%) and placebo (47.6%) groups. On the basis of these clinical and neuropsychological findings, it appears that lecithin alone has no important therapeutic effect in early-onset Alzheimer's disease.

Distribution of polychlorinated biphenyls (PCB) in sewage wastes at a municipal sewage treatment plant was studied, showing that the great bulk of PCBs entering such a treatment plant become adsorbed onto the grit chamber solids and the sludge that is passed from the anaerobic digesters. When appreciable quantities of PCBs are present in sewage, as was the case in this study, significant quantities can nevertheless pass with the effluents discharged from the treatment plant. The PCB concentrations in the treatment plant waters undergoing secondary and tertiary treatment tend to be consistent with the limited solubility properties of the PCBs. However, appreciably higher concentrations can be found in the sediments of waters receiving treatment plant discharges and bioaccumulation in fish is demonstrated readily. Also described are quantitative data on PCBs in soils fertilized with PCB-contaminated sludge.

The effects of acute doses of soman (40, 60, or 80 micrograms/kg sc) in rats were evaluated for toxic symptoms as well as for changes in plasma levels of glucose, insulin, glucagon, corticosterone, norepinephrine, and epinephrine. The relationship between changes in these levels and depressed acetylcholinesterase activity in the hypothalamus was determined. Soman 40 micrograms/kg did not manifest significant changes in any of the parameters evaluated. However, both the 60 and 80 micrograms/kg doses of soman caused dose- and time-related increases in plasma levels of glucose, corticosterone, norepinephrine, epinephrine, and a depression of insulin. Many of these increases, as well as the severity of toxicity, appear to be inversely related to the hypothalamic acetylcholinesterase levels. The hyperglycemia following the higher doses of soman is likely due to the combined effects of elevated levels of corticosterone, catecholamines, possibly glucagon, and depressed insulin levels. Stress from the toxic effects of soman is likely partially responsible for the endocrine effects since most of the changes observed are consistent with changes in these levels that would be manifested in an animal stress model.

AC glioma cells, a clonal cell line derived from a rat glioma, responded to 1 mM dibutyryl-cyclic AMP and isobutylmethylxanthine with a change to stellate morphology. A concentration-related morphological change was induced by beta 1- and beta 2-adrenergic agonists with the order of potency being isoproterenol greater than soterenol greater than norepinephrine. Propranolol (nonselective, beta-antagonist), butoxamine (beta 2-antagonist) and metoprolol (beta 1-antagonist) significantly decreased the cell response to isoproternol. Schild analysis of the response, using the competitive antagonist metoprolol, gave pA2 values of 7.5 and 8.5 for the agonists norepinephrine and soterenol, respectively, with slopes of the curves being less than unity. These observations indicate that both beta 1- and beta 2-adrenergic receptors mediate the change in cellular morphology.

The effects of repeated administration of soman on plasma glucose levels, acetylcholinesterase (AChE) activity in erythrocytes and hypothalamus, and plasma levels of corticosterone, glucagon, insulin, epinephrine, and norepinephrine were studied in male rats. Rats were given soman subcutaneously (sc), either 30 micrograms/kg every 24 hr for 5 or 12 days or 40 micrograms/kg every 24 hr for 5 days. All doses of soman markedly depressed AChE activity in the hypothalamus and completely inhibited AChE activity in erythrocytes. Soman 30 micrograms/kg given for 5 days did not alter plasma levels of any hormone assayed and produced few signs of intoxication. Soman 40 micrograms/kg given for 5 days elevated plasma levels of glucose and corticosterone and produced signs of severe cumulative intoxication. Daily administration of 30 micrograms/kg of soman for 12 days inhibited hypothalamic AChE activity 75%, lowered plasma insulin, and produced signs of moderate intoxication. Repeated administration of soman produced endocrine alterations only when significant signs of intoxication were evident. The absence of increases in plasma levels of catecholamines and corticosterone in rats exhibiting signs of moderate intoxication, and of catecholamines in rats exhibiting signs of severe intoxication, may indicate an impairment by soman of the normal endocrine response to stress.