Lapses of the Heart: Frequency and Subjective Salience of Impressions Reported by Patients after Cardiac Arrest.

After cardiac arrest (CA), some patients report impressions with highly realistic features, often referred to as near-death experience (NDE). The frequency of such episodes seems to be variable, with various types of content. In a prospective study, we subjected 126 CA cases treated at the Department of Emergency Medicine of the Medical University of Vienna under carefully controlled conditions to a structured interview. We included all patients admitted due to CA, whose communicative abilities were restored and who agreed to participate in the study. The questionnaire inquired as to living conditions, attitudes towards issues of life and death, and last recollections before and first impressions after the CA. The majority of the subjects (91 = 76%) replied to inquiries concerning impressions during CA with "nothing" or "blackout", but 20 (16%) gave a detailed account. A German version of the Greyson questionnaire specifically addressing NDE phenomena (included towards the end of the interview) resulted in ≥7 points in five patients (4%). Three patients reported a meeting with deceased relatives (one with 6 Greyson points), one an out-of-body episode, and one having been sucked into a colorful tunnel. Eleven of these twenty cases had their cardiopulmonary resuscitation (CPR) started within the first min of CA, a higher fraction than cases without experience. Reported experience after CA was of high significance for the patients; many of them changed their point of view on issues of life and death.

Disturbed neurotransmitter homeostasis in ether lipid deficiency.

Plasmalogens, the most prominent ether (phospho)lipids in mammals, are structural components of most cellular membranes. Due to their physicochemical properties and abundance in the central nervous system, a role of plasmalogens in neurotransmission has been proposed, but conclusive data are lacking. Here, we targeted this issue in the glyceronephosphate O-acyltransferase (Gnpat) KO mouse, a model of complete deficiency in ether lipid biosynthesis. Throughout the study, focusing on adult male animals, we found reduced brain levels of various neurotransmitters. In the dopaminergic nigrostriatal tract, synaptic endings but not neuronal cell bodies were affected. Neurotransmitter turnover was altered in ether lipid-deficient murine as well as human post-mortem brain tissue. A generalized loss of synapses did not account for the neurotransmitter deficits, since the levels of several presynaptic proteins appeared unchanged. However, reduced amounts of vesicular monoamine transporter indicate a compromised vesicular uptake of neurotransmitters. As exemplified by norepinephrine, the release of neurotransmitters from Gnpat KO brain slices was diminished in response to strong electrical and chemical stimuli. Finally, addressing potential phenotypic correlates of the disturbed neurotransmitter homeostasis, we show that ether lipid deficiency manifests as hyperactivity and impaired social interaction. We propose that the lack of ether lipids alters the properties of synaptic vesicles leading to reduced amounts and release of neurotransmitters. These features likely contribute to the behavioral phenotype of Gnpat KO mice, potentially modeling some human neurodevelopmental disorders like autism or attention deficit hyperactivity disorder.

Soaping the NMDA receptor: Various types of detergents influence differently [(3)H]MK-801 binding to rat brain membranes.

Membranes prepared from rat brain were treated with increasing concentrations of cationic, neutral, anionic and zwitterionic surfactants. Potent inactivation of [(3)H]MK-801 binding to NMDA receptors (NRs) was provided by the cation cetyl pyridinium (IC50 25 µM) and the neutral digitonin (IC50 37 µM). A 2 h incubation of rat brain membranes at 24°C with 100 µM of the neutral Triton X-100 resulted in about 50% reversible inhibition (without inactivation). Reversible inhibition was also effected by the anion deoxycholate (IC50 700 µM), and by the zwitterions N-lauryl sulfobetaine (12-SB(±), 400 µM) and CHAPS (1.5 mM), with inactivation at higher concentrations. Keeping the NR cation channel in the closed state significantly protected against inactivation by cations and by 12-SB(±), but not by the other detergents. Inactivation depended differentially on the amount of the membranes, on the duration of the treatment, and on the temperature. Varying the amount of membranes by a factor 8 yielded for cetyl trimethylammonium (16-NMe3(+)) IC50s of inactivation from 10 to 80 µM, while for deoxycholate the IC50 of inactivation was 1.2 mM for all tissue quantities. Some compounds inactivated within a few min (16-NMe3(+), digitonin, CHAPS), while inactivation by others took at least half an hour (Triton X-100, deoxycholate, 12-SB(±)). These last 3 ones also exhibited the steepest temperature dependence. Knowledge about the influence of various parameters is helpful in selecting appropriate conditions allowing the treatment of brain membranes with amphiphiles without risking irreversible inactivation.

Differential influence of 7 cations on 16 non-competitive NMDA receptor blockers.

The specific binding of the NMDA receptor (NR) channel ligand [(3)H]MK-801 to rat brain membranes is sensitive to positively charged buffer ingredients as to tris(hydroxymethyl)aminomethane (Tris), to Na(+), or to protons. Here we demonstrate that 16 non-competitive NR antagonists, including 5 long-chain diamines, classical NR channel blockers and several less known compounds, differ widely in their sensitivities to cationic buffer constituents. Although chemically distinguished either as extended di-cationic or as compact mono-cationic, their sensitivities to cationic buffer ingredients did not suggest this grouping. While the di-cationic compounds are known for their sensitivity to spermine (polyamine inverse agonists), also some of the mono-cationic blockers exhibited this feature. They might share as common target a recently described negatively charged extracellular GluN1/GluN2B interface.

Pentamidine analogs as inhibitors of [(3)H]MK-801 and [(3)H]ifenprodil binding to rat brain NMDA receptors.

The anti-protozoal drug pentamidine is active against opportunistic Pneumocystis pneumonia, but in addition has several other biological targets, including the NMDA receptor (NR). Here we describe the inhibitory potencies of 76 pentamidine analogs at 2 binding sites of the NR, the channel binding site labeled with [(3)H]MK-801 and the [(3)H]ifenprodil binding site. Most analogs acted weaker at the ifenprodil than at the channel site. The spermine-sensitivity of NR inhibition by the majority of the compounds was reminiscent of other long-chain dicationic NR blockers. The potency of the parent compound as NR blocker was increased by modifying the heteroatoms in the bridge connecting the 2 benzamidine moieties and also by integrating the bridge into a seven-membered ring. Docking of the 45 most spermine-sensitive bisbenzamidines to a recently described acidic interface between the N-terminal domains of GluN1 and GluN2B mediating polyamine stimulation of the NR revealed the domain contributed by GluN1 as the most relevant target.

GABA(A) receptor subtype-selectivity of novel bicuculline derivatives.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are targets of clinically important drugs modulating GABA induced ion flux by interacting with distinct allosteric binding sites. ROD 185 is a previously investigated structural analogue of the GABA site antagonist bicuculline, and a positive allosteric modulator acting via the benzodiazepine binding site. Here, we investigated 13 newly synthesized structural analogues of ROD 185 for their interaction with rat GABA(A) receptors. Using [(3)H]flunitrazepam binding assays, we identified four compounds exhibiting a higher affinity for the benzodiazepine binding site than ROD 185. Two electrode voltage clamp electrophysiology at recombinant GABA(A) receptors indicated that most of these compounds positively modulated GABA-induced currents at these receptors. Additionally, these experiments revealed that this compound class not only interacts with the benzodiazepine binding site at αßγ receptors but also with a novel, so far unidentified binding site present in αß receptors. Compounds with a high affinity for the benzodiazepine binding site stimulated GABA-induced currents stronger at αßγ than at αß receptors and preferred α3ß3γ2 receptors. Compounds showing equal or smaller effects at αßγ compared to αß receptors differentially interacted with various αß or αßγ receptor subtypes. Surprisingly, five of these compounds interacting with αß receptors showed a strong stimulation at α6ß3γ2 receptors. The absence of any direct effects at GABA(A) receptors, as well as their potential selectivity for receptor subtypes not being addressed by benzodiazepines, make this compound class to a starting point for the development of drugs with a possible clinical importance.

Holographic quantitative structure-activity relationships of tryptamine derivatives at NMDA, 5HT(1A) and 5HT(2A) receptors.

Tryptamine derivatives (Ts) were found to inhibit the binding of [³H]MK-801, [³H]ketanserin and [³H]8-OH-DPAT to rat brain membranes. [³H]MK-801 labels the NMDA (N-methyl-D-aspartate) receptor, a ionotropic glutamate receptor which controls synaptic plasticity and memory function in the brain, whereas [³H]ketanserin and [³H]8-OH-DPAT label 5HT(2A) and 5HT(1A) receptors, respectively. The inhibitory potencies of 64 Ts (as given by IC50 values) were correlated with their structural properties by using the Holographic QSAR procedure (HQSAR). This method uses structural fragments and connectivities as descriptors which were encoded in a hologram thus avoiding the usual problems with conformation and alignment of the structures. Four correlation equations with high predictive ability and appropriate statistical test values could be established. The results are visualized by generation of maps reflecting the contribution of individual structural parts to the biological activities.

[(3)H]metyrapol and 4-[(131)i]iodometomidate label overlapping, but not identical, binding sites on rat adrenal membranes.

Metyrapone, metyrapol, and etomidate are competitive inhibitors of 11-deoxycorticosterone hydroxylation by 11ß-hydroxylase. [(3)H]Metyrapol and 4-[(131)I]iodometomidate bind with high affinity to membranes prepared from bovine and rat adrenals. Here we report inhibitory potencies of several compounds structurally related to one or both of these adrenostatic drugs, against the binding of both radioligands to rat adrenal membranes. While derivatives of etomidate inhibited the binding of both radioligands with similar potencies, derivatives of metyrapone inhibited the binding of 4-[(131)I]iodometomidate about 10 times weaker than the binding of [(3)H]metyrapol. By X-ray structure analysis the absolute configuration of (+)-1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanol [(+)-11, a derivative of metyrapol] was established as (R). We introduce 1-(2-fluorophenyl)-2-methyl-2-(pyridin-3-yl)-1-propanone (9; Ki = 6 nM), 2-(1-imidazolyl)-2-methyl-1-phenyl-1-propanone (13; 2 nM), and (R)-(+)-[1-(4-iodophenyl)ethyl]-1H-imidazole (34; 4 nM) as new high affinity ligands for the metyrapol binding site on 11ß-hydroxylase and discuss our results in relation to a proposed active site model of 11ß-hydroxylase.

Development and automation of a novel NET-PET tracer: [11C]Me@APPI.

INTRODUCTION: The norepinephrine transporter (NET) is an important target for research in neurology and psychology and is involved in the pathophysiology of many neurodegenerative diseases such as Alzheimer's disease and attention deficient hyperactivity disorder. For visualization of NET abundance and deregulation, a novel PET tracer--[(11)C]Me@APPI--has been developed. METHODS: For precursor synthesis, a 4-step synthesis starting from N-phenyl-o-phenylenediamine was set up. Radiosynthesis was established and optimized using standard methods and subsequently automated in a GE TRACERlabFx C Pro synthesizer. Preclinical testing was performed comprising affinity and selectivity testing on human membranes as well as stability and blood-brain-barrier-penetration using in-vitro models. RESULTS: Precursor molecule (APPI:0) and reference compound (Me@APPI) were synthesized with 26.5% and 21.4% overall yield, respectively. So far, 1.25±0.72 GBq [(11)C]Me@APPI with 54.35±7.80 GBq/µmol specific activity were produced (n=11). Affinity of reference compounds was determined as 8.08±1.75 nM for Me@APPI and 19.31±2.91 nM for APPI:0, respectively (n≥9). IAM-chromatography experiments (n=3) revealed a P(m) value of 1.51±0.34 for Me@APPI. Stability testing using human liver microsomes revealed that 99.5% of the tracer was found to be still intact after 60 minutes (n=4). CONCLUSION: Present data indicate that [(11)C]Me@APPI has promising properties to become a clinically useful NET-PET-tracer. Further in-vitro and in-vivo evaluations are currently under way.

Exploring the polyamine regulatory site of the NMDA receptor: a parallel synthesis approach.

The elongated structures of polyamine inverse agonists such as 1,12-diaminododecane (N12N) and 5-(4-aminobutyl)-2-thiopheneoctanamine (N4T8N) lend themselves to a combinatorial chemistry approach to explore a potential polyamine pharmacophore at the NMDA receptor. Herein we describe more than 100 new analogues of N4T8N obtained by breaking up the long octanamine arm into a dipeptide chain of equivalent length. Solid-phase parallel synthesis based on cross-linked polystyrene and a Wang anchor allowed the low-scale preparation of four small libraries based on the combination of two amino acid residues (out of Gly, Leu, Phe, Lys, phenylglycine, Tyr, Trp, His, and Arg). The obtained compounds were tested as modulators of [(3) H]MK-801 binding to rat brain membranes and of NMDA-induced currents in cultured rat hippocampal neurons. Compounds with two aromatic residues acted as binding inhibitors (inverse agonists). Compounds with two Lys residues acted as binding stimulators (agonists) and had stimulatory and inhibitory effects on NMDA-induced currents, depending on the holding potential. High sensitivity of binding inhibition to spermine was conferred by a Tyr residue, whereas a His residue favored high potency at acidic pH.

Structure-activity relationship of etomidate derivatives at the GABA(A) receptor: Comparison with binding to 11beta-hydroxylase.

At the GABA(A) receptor, low concentrations of etomidate potentiate the inhibitory effect of GABA on specific binding of the closed channel ligand [(3)H]ethynylpropylbicycloorthobenzoate ([(3)H]EBOB). Here, we present SARs for etomidate and structurally related compounds inducing this effect. In the absence of GABA, similar SARs, but 14-20 times weaker potencies were observed. We discuss these SARs in comparison to the much higher potencies of these compounds as inhibitors of 11beta-hydroxylase.

NMDA receptor affinities of 1,2-diphenylethylamine and 1-(1,2-diphenylethyl)piperidine enantiomers and of related compounds.

We resolved 1,2-diphenylethylamine (DPEA) into its (S)- and (R)-enantiomer and used them as precursors for synthesis of (S)- and (R)-1-(1,2-diphenylethyl)piperidine, flexible homeomorphs of the NMDA channel blocker MK-801. We also describe the synthesis of the dicyclohexyl analogues of DPEA. These and related compounds were tested as inhibitors of [(3)H]MK-801 binding to rat brain membranes. Stereospecificity ranged between factors of 0.5 and 50. Some blockers exhibited stereospecific sensitivity to the modulator spermine. Our results may help to elucidate in more detail the NMDA channel pharmacophore.

Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner.

The dopamine transporter (DAT), a membrane protein specifically expressed by dopaminergic neurons and mediating the action of psychostimulants and dopaminergic neurotoxins, is regulated by Zn(2+) which directly interacts with the protein. Herein, we report a host-cell-specific direction of the Zn(2+) effect on wild type DAT. Whereas low mumolar Zn(2+) decreased dopamine uptake by DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC cells. Inhibition or stimulation was lost in a DAT construct without the binding site for Zn(2+). Also reverse transport was differentially affected by Zn(2+), dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells. Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an activator of protein kinase C, attenuated the inhibitory effect of Zn(2+) on uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells. Patch-clamp experiments under non-voltage-clamped conditions revealed a significantly higher membrane potential of HEK293 than SK-N-MC cells and a reduced membrane potential after phorbol ester treatment. Lowering chloride in the uptake buffer switched the stimulatory effect of Zn(2+) in SK-N-MC cells to an inhibitory, whereas high potassium depolarization of HEK293 cells switched the inhibitory effect of Zn(2+) to a stimulatory one. This study represents the first evidence that DAT regulation by Zn(2+) is profoundly modulated by the membrane potential and chloride.

New selective inhibitors of steroid 11beta-hydroxylation in the adrenal cortex. Synthesis and structure-activity relationship of potent etomidate analogues.

Derivatives of etomidate were evaluated as inhibitors of adrenal steroid 11beta-hydroxylations. Stereoselective coupling by Mitsunobu produced chirally pure analogues to study the effect of configuration, modification of the ester, and substitution in the phenyl ring, with the aim to probe specific sites for introducing a radionuclide. Iodophenyl metomidate (IMTO) labeled with iodine-131 served as radioligand for structure-affinity relationship studies. We have characterized the kinetic parameters of specific (131)I-IMTO binding on rat adrenal membranes and used the displacement of (131)I-IMTO binding to evaluate functionalized MTO analogues. Our results indicated that (1) ( R)-configuration is essential for high affinity, (2) highest potency resides in the ethyl, 2-propyl, and 2-fluoroethyl esters, and (3) substitution of the phenyl ring is well tolerated. The clinically used inhibitors metyrapone and ketoconazole inhibited (131)I-IMTO binding with low affinity. Incubation of selected analogues with human adrenocortical NCI-h295 cells demonstrated a high correlation with the inhibitory effect on cortisol secretion.

New polyamine-sensitive inhibitors of the NMDA receptor: syntheses and pharmacological evaluation.

Derivatives of 5-(4-aminobutyl)-2-thiophene-octylamine, a potent polyamine-sensitive inhibitor of the NMDA receptor, were synthesized and evaluated as inhibitors of [(3)H]MK-801 binding to rat brain membranes. Alkylations of the terminal amino groups reduced inhibitory potency; only incorporation of the amino group of the short 4-aminobutyl arm into a piperidine ring was tolerated. Substitution of the thiophene nucleus with methyl or ethyl, and its replacement by a benzene nucleus, was of minor influence. The corresponding diguanidines exhibited high potency independent of chain length, whereas their sensitivity to spermine was sharply dependent on chain length. Insertion of an amide bond into the long octylamine arm increased sensitivity to spermine and to Tris buffer. Our results indicate that spermine sensitivity of [(3)H]MK-801 binding inhibition is responsive to subtle changes in inhibitor structure and represents a promising target for pharmaceutical research.

Polyamines and the NMDA receptor: modifying intrinsic activities with aromatic substituents.

Thirty-four spermidine (SPD) and spermine (SPM) derivatives with aromatic substituents were synthesized and tested as inhibitors of specific binding of the NMDA channel blocker [3H]MK-801 to membranes prepared from rat hippocampus and cerebral cortex. SPD and SPM derivatives with aromatic substituents at the primary amino groups were the most potent inhibitors (IC50 3.9-4.7 microM). These compounds most likely act directly at the NMDA ion channel, since 30 microM SPM had no pronounced influence on their inhibiting activities. SPD derivatives with aromatic substituents at the secondary amino group were either inactive or highly SPM-sensitive inhibitors (IC50 10-82 microM), depending on the size of the substituent. Our results support the hypothesis that an aromatic interaction site near the center of polyamine derivatives contributes to polyamine inverse agonism.

Synthesis of thieno[2,3-b]pyridinones acting as cytoprotectants and as inhibitors of [3H]glycine binding to the N-methyl-D-aspartate (NMDA) receptor.

The standard glycine site antagonist of the N-methyl-D-aspartate (NMDA) receptor, 3-phenyl-4-hydroxyquinolin-2(1H)-one (21), was used as a template for bioisostere benzene/thiophene exchange. Phenylacetylation of aminothiophene carboxylic acid methyl esters and subsequent cyclization delivered the three possible thienopyridinone isomers. 4-Hydroxy-5-phenylthieno[2,3-b]pyridin-6(7H)-one (3a), with the shortest distance between the sulfur and the nitrogen atom, was the most potent isomer (K(i) against the binding of [(3)H]glycine to rat membranes 16 microM), comparable in potency to the model quinolinone (21, 12 microM). Replacement of the phenyl substituent of 21 by a 2-thienyl residue resulted in a 2-5-fold loss in potency and was abandoned. In the thieno part of the thienopyridinone nucleus, the most successful substituents were halogen (Cl or Br) close to the sulfur atom and short alkyl chains at the other position, resulting in 7h, 8h, 8i, and 8m, with K(i) values between 5.8 and 10.5 nM. Introduction of a 3'-phenoxy moiety yielded several compounds with still higher potencies (18h, 18i, 18l, and 18m; K(i) between 1.1 and 2.0 nM). Quantitative structure-activity relationship (QSAR) calculations resulted in a consistent interpretation of the potencies of most compounds. Several of these 3'-phenoxy derivatives protected mouse fibroblast cell lines with transfected NMDA receptors from glutamate-induced toxicity. In addition, we report in vivo results for four of these compounds.

Characteristic functional networks in high- versus low-proficiency second language speakers detected also during native language processing: an explorative EEG coherence study in 6 frequency bands.

An EEG coherence study was performed with a twofold objective: first, to scrutinize the theoretical concept of "cortical efficiency" in connection with second language (L2) acquisition and, second, to detect cooperations between cortical areas in specific frequency bands indicative for highly proficient L2 processing. Two groups differing only in their level of L2 proficiency were contrasted during presentation of natural language videos in English (L2) and German (native language, L1), with explorative coherence analysis in 6 frequency bands (0.5-31.5 Hz). The coherence brain maps revealed more pronounced and widespread increases in coherences in the alpha1-band (8-10 Hz) in low-proficiency than in the high-proficiency L2 speakers. Surprisingly, this difference was obtained also during L1 processing and corroborated for both languages by multivariate permutation tests. These tests revealed additional differences between the low- and the high-proficiency group also for coherences within the beta1- (13-18 Hz) and the beta2-band (18.5-31.5 Hz), again during L2 and L1 processing. Since the same group differences were observed during L1 and L2 processing, our high-proficiency group might have profited from a more generic advantage in language or text processing strategy. This strategic advantage was most evident at alpha1 frequencies, possibly related to a specific way of processing internal mental states (top-down processing).

Decreased EEG coherence between prefrontal electrodes: a correlate of high language proficiency?

To investigate the influence of proficiency level on the cortical organization of foreign language processing, two groups of German speaking students, differing only in their proficiency in English as a second language, were subjected to EEG coherence analysis during foreign and native language processing (news reports, alpha1 frequency band). In the group with minor experience with English, coherence increase was observed with all electrode combinations, with left hemisphere (LH) predominance. In the high proficiency group, coherence increase was limited to temporal electrodes over LH. In the latter group only, coherence between prefrontal electrodes was significantly lower during the language tasks than during the baseline task (silence, noisy screen). Both results were obtained with foreign as well as native language processing. We suggest that reduced EEG coherence in highly proficient foreign language speakers reflects a more efficient operating strategy not only for their second, but also for their native language.