Genetic deletion of TRPA1 receptor attenuates amyloid beta- 1-42 (Aß1-42)-induced neurotoxicity in the mouse basal forebrain in vivo.

Amyloid ß 1-42 peptide (Aß1-42) accumulates in Alzheimer's disease (AD) that is toxic to the basal forebrain cholinergic (BFC) neurons in substantia innominata-nucleus basalis magnocellularis complex (SI-NBM). Transient Receptor Potential Ankyrin1 (TRPA1) receptor is present in murine brain, however its role in neurotoxic processes is unclear. We investigated the Aß1-42-induced neurotoxicity in TRPA1 wild-type (TRPA1+/+) and knockout (TRPA1-/-) mice. Expression and neuroanatomical localization of TRPA1 receptor were examined using RT qPCR. Cholinergic fibre loss was determined on acetylcholinesterase (AChE) stained brain slices, and choline acetyltransferase (ChAT) immunohistochemistry was used to assess the cholinergic cell loss. Novel object recognition (NOR), radial arm maze (RAM) and Y-maze tests were used to investigate memory loss. Aß1-42-injected WT mice showed marked loss of cholinergic fibres and cell bodies, which was significantly attenuated in TRPA1-/- animals. According to the NOR and RAM tests, pronounced memory loss was detected in Aß1-42-injected TRPA1+/+ mice, but not in TRPA1-/- group. Our findings demonstrate that TRPA1 KO animals show substantially reduced morphological damage and memory loss after Aß1-42 injection in the SI-NBM. We conclude that TRPA1 receptors may play an important deteriorating role in the Aß1-42-induced cholinergic neurotoxicity and the consequent memory loss in the murine brain.

Impact of the Conformational Variability of Oligopeptides on the Computational Prediction of Their CD Spectra.

Although successful in the structural determination of ordered biomolecules, the spectroscopic investigation of oligopeptides in solution is hindered by their complex and rapidly changing conformational ensemble. The measured circular dichroism (CD) spectrum of an oligopeptide is an ensemble average over all microstates, severely limiting its interpretation, in contrast to ordered biomolecules. Spectral deconvolution methods to estimate the secondary structure contributions in the ensemble are still mostly based on databases of larger ordered proteins. Here, we establish how the interpretation of CD spectra of oligopeptides can be enhanced by the ability to compute the same observable from a set of atomic coordinates. Focusing on two representative oligopeptides featuring a known propensity toward an α-helical and ß-hairpin motif, respectively, we compare and cross-validate the structural information coming from deconvolution of the experimental CD spectra, sequence-based de novo structure prediction, and molecular dynamics simulations based on enhanced sampling methods. We find that small conformational variations can give rise to significant changes in the CD signals. While for the simpler conformational landscape of the α-helical peptide de novo structure prediction can already give reasonable agreement with the experiment, an extended ensemble of conformers needs to be considered for the ß-hairpin sequence.

Assessing structure, function and druggability of major inhibitory neurotransmitter gamma-aminobutyrate symporter subtypes.

Ambient level of gamma -aminobutyric acid (GABA), the major inhibitory neurotransmitter of the brain is mediated by neuronal and glial GABA transporters (GATs), members of the sodium and chloride ion-dependent solute carrier family. The neuronal GABA transporter subtype (GAT-1) has already been proven to be the target for the antiepileptic drug Tiagabine. However, druggability of glial GAT-2 and GAT-3 is yet to be established. Recent advances in structure elucidation of a bacterial orthologue leucine transporter in complex with different substrates substantiate homology modeling of human GATs (hGATs). These modeling studies can provide mechanistic clues for structure-based prediction of the potential of medicinal chemistry campaigns. A recently identified characteristic structural feature of the occluded conformation of hGATs is that similar extra- and intracellular gates are formed by middle-broken transmembrane helices TM1 and TM6. Binding crevice formed by unwound segments of broken helices facilitates symport of GABA with Na+ ion via fitting of GABA to TM1-bound Na+ closely inside. Favored accommodation of substrate inhibitors with high docking score predicts efficient inhibition of the neuronal hGAT-1 if the TM1-TM8 binding prerequisite for GABA was used. Docking, molecular dynamics and transport data indicate, that amino acids participating in substrate binding of the neuronal hGAT-1 and the glial hGAT-2 and hGAT-3 subtypes are different. By contrast, substrate binding crevices of hGAT-2 and hGAT-3 cannot be distinguished, avoiding sensible prediction of efficient selective substrate inhibitors. Glial subtypes might be specifically distinguished by interfering Zn2+ binding in the second extracellular loop of hGAT-3. Formation of the unique ring-like Na+-GABA complex in the occluded binding crevices anticipates family member symporters exploring chemiosmotic energy via reversible chemical coupling of Na+ ion.

Desynchronisation of spontaneously recurrent experimental seizures proceeds with a single rhythm.

Here we investigate the temporal properties of recurrent seizure-like events (SLEs) in a low-[Mg(2+)] model of experimental epilepsy. Simultaneous intra- and extracellular electric signals were recorded in the CA3 region of rat hippocampal slices whereby cytosolic [Ca(2+)] transients were imaged by fluorescence detection. Recurrence pattern analysis was applied to give a measure of synchrony of simultaneously recorded intra- and extracellular electric signals and the SLE frequencies were extracted by complex wavelet analysis. Slices from the juvenile, but not the young adult rats, displayed several high-amplitude triplets of electric and [Ca(2+)] transients, termed paroxysmal spikes, followed by an SLE. Occurrence of the full-blown SLE was associated with decreased synaptic activity between the paroxysmal spikes that were seen as incomplete SLE starting sequences. The time series of recurrent SLEs provide evidence for a single SLE rhythm as continuously declining from about 200 Hz to below 1 Hz at the onset and termination of SLE, respectively, with an intermediate spectral discontinuity, tentatively identified with the tonic/clonic transition. All other frequency components were the harmonics of the fundamental rhythm, whereby the gamma and the theta band oscillations were not detected as separate activities. Recurrence showed decreasing temporal synchrony of intra- and extracellular signals during the SLE, suggesting that coincidence is destroyed by the SLE. Blockade of gap junctions with 200 microM carbenoxolone ceased recurrent SLEs. Release of gap junction blockade shortened both SLEs and their tonic phase indicating that persistent changes occurred via an altered gap junction coupling. We conclude that the initially precise temporal synchrony is gradually destroyed during ictal events with a single rhythm of continuously decreasing frequency. Blockade of gap junction coupling might prevent epileptic synchronisation.

Universality of receptor channel responses.

Rate parameters estimated for neurotransmitter-gated receptor channel opening and receptor desensitization are classified according to their dependence on the temporal resolution of the techniques applied in the measurements. Because allosteric proteins constituting receptor channels impose restrictions on the types of model suitable to describe the dynamic response of channels to neurotransmitters, Markovian, non-linear or fractal dynamic models and their possible extension to receptor channel response in excitable membranes are discussed.

The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

Assembly and enzymatic properties of the catalytic domain of human complement protease C1r.

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.

A better enzyme to cope with cold. Comparative flexibility studies on psychrotrophic, mesophilic, and thermophilic IPMDHs.

3-isopropylmalate dehydrogenase (IPMDH) from the psychrotrophic bacterium Vibrio sp. I5 has been expressed in Escherichia coli and purified. This cold-adapted enzyme is highly homologous with IPMDHs from other organisms, including mesophilic E. coli and thermophilic Thermus thermophilus bacteria. Its molecular properties are similar to these counterparts. Whereas the E. coli and T. thermophilus enzymes are hardly active at room temperature, the Vibrio IPMDH has reasonable activity below room temperature. The thermal stabilities, conformational flexibilities (hydrogen-deuterium exchange), and kinetic parameters of these enzymes were compared. The temperature dependence of the catalytic parameters of the three enzymes show similar but shifted profiles. The Vibrio IPMDH is a much better enzyme at 25 degrees C than its counterparts. With decreasing temperature i.e. with decreasing conformational flexibility, the specific activity reduces, as well; however, in the case of the Vibrio enzyme, the residual activity is still high enough for normal physiological operation of the organism. The cold-adaptation strategy in this case is achieved by creation of an extremely efficient enzyme, which has reduced but still sufficient activity at low temperature.

Ca2+ signalling and changes of mitochondrial function during low-Mg2+-induced epileptiform activity in organotypic hippocampal slice cultures.

Several lines of evidence indicate that augmented neuronal activity is associated with increased mitochondrial function, however, the mechanisms of coupling are still unclear. In this study we used a low extracellular Mg2+ concentration and short stimulus trains to evoke neuronal hyperactivity in the form of seizure-like events (SLE) in hippocampal slice cultures. Simultaneous microfluorimetric and electrophysiological techniques were applied to gain insight into changes of Ca2+ concentration in different compartments and into mitochondrial function. SLEs were associated with a large decrease of the extracellular Ca2+ concentration ([Ca2+]e), a spiking increase of the cytoplasmic and a smoothed elevation of the mitochondrial Ca2+ concentration (cytoplasmic concentration [Ca2+]i; intramitrochondrial concentration [Ca2+]m). Following an initial apparent decline in the mitochondrial membrane potential (DeltaPsi) and NAD(P)H autofluorescence, mitochondria depolarized and NADH production was augmented. Furthermore, SLEs were associated with increased oxidation of dihydroethidine (HEt). Our data suggest that intramitochondrial Ca2+ accumulation stimulates NADH production and production of radical oxygen species (ROS). Interestingly, mitochondrial depolarization followed [Ca2+]i and [Ca2+]m changes with a delay implying that electrogenic extrusion of Ca2+ from the mitochondrial matrix might be responsible for the depolarization of the mitochondrial membrane.

Quinazolone-alkyl-carboxylic acid derivatives inhibit transmembrane Ca(2+) ion flux to (+)-(S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid.

Comparison of the kinetics of the inward Ca(2+) ion flux to (S)-alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid [(S)-AMPA] in cerebrocortical homogenates and that of the previously reported transmembrane Na(+) ion influx mediated by an AMPA receptor in hippocampal homogenates established that the agonist-induced opening of the AMPA receptor channels occurs in two kinetically distinguishable phases. Here we report that the 2-methyl-4-oxo-3H-quinazoline-3-acetic acid (Q1) inhibits the major slow-phase response specifically, whereas the acetyl piperidine derivative (Q5) is a more potent inhibitor of the fast-phase response. Both the quinazolone-3-propionic acid (Q2) and the quinazolone-3-acetic acid methyl ester (Q3) enhanced the slow-phase response to (S)-AMPA. The information provided by docking different Q1, Q2, and Q5 models at the ligand-binding core of iGluRs were used to define agonistic and antagonistic modes of interactions. Based on the effects of quinazolone-3-alkyl-carboxylic acid derivatives on specific [(3)H]Glu binding and kinetically distinguishable Ca(2+) ion permeability responses to (S)-AMPA and molecular modeling, the fast- and the slow-phase (S)-AMPA-elicited Ca(2+) ion fluxes were corresponded to different subunit compositions and degrees of S1S2 bridging interaction relative to substitution of kainate thereupon. Substitutions of agonists and antagonists into the iGluR2 S1S2 ligand binding core induced different modes of domain-domain bridging.

Coupled intra- and extracellular Ca2+ dynamics in recurrent seizure-like events.

We considered the evolution of Ca2+ oscillation dynamics in recurrent seizure-like events. Dynamic system behaviour was characterized in the state space reconstructed from intra- and extracellular [Ca2+] fluctuations simultaneously measured in cultured rat hippocampal slices under low-[Mg2+] conditions. When associated in the seizure-like event, these fluctuations occurred on a restricted set, the attractor, embedded in the full state space with less than five degrees of freedom. Instantaneous relative phase differences indicated field potential-driven phase jumps locked onto seizure-like events. To account for recurrent dynamics, calculations were performed on different extensions of a model for Ca2+ oscillation. These identified bidirectional, asymmetrical coupling of extracellular with intracellular (cytosolic, Ca2+ store, mitochondrial) Ca2+ dynamics as critical in its development.

Low-[Mg2+]-induced Ca2+ fluctuations in organotypic hippocampal slice cultures.

We show here by whole field monitoring of free intracellular Ca2+ ([Ca2+]i), locally recorded field potential (fp) and external [Ca2+], that low-[Mg2+] induces seizure like events (SLEs) accompanied by simultaneous fluctuations of [Ca2+]i and [Ca2+]e in cultured hippocampal slices. Within a SLE, complex [Ca2+]e fluctuations are seen throughout phases of Ca2+ depletion (tonic) and Ca2+ recovery (clonic) of the extracellular space. Information theory entropy-based analyses revealed strong asymmetric associations of [Ca2+]i and [Ca2+]e kinetics. By contrast, signal-associations between SLEs were found to be weak and of symmetric nature distinguishing seizure-like and interictal events by extensive coupling and decoupling of [Ca2+]i and [Ca2+]e fluctuations, respectively.

Persistent depolarization and Glu uptake inhibition operate distinct osmoregulatory mechanisms in the mammalian brain.

The ways of coupling neuronal with glial compartments in natural physiology was investigated in microdialysis experiments by monitoring extracellular concentration of amino acids in the brain of anaesthetized rats. We hypothesized that extracellular [Glu], [Gln] and [Tau] patterns would be state-dependent. This was tested by stimulation of N-methyl-D-aspartate (NMDA) receptors, by inhibition of Glu uptake or by local depolarization with a high-K(+) dialysate, coupled with the addition of Co(2+) to block Ca(2+) influx. The results showed that (1) extracellular [Gln] was low whereas [Glu] and [Tau] were high during infusion of NMDA (0.5-1.0 mM) or high-K(+) (80 mM) in the hippocampus and ventrobasal thalamus, (2) hippocampal extracellular [Glu], [Gln] and [Tau] were increased in response to the Glu uptake inhibitor, L-trans-pyrrolidine-2, 4-dicarboxilic acid (tPDC, 0.5-3.0 mM), in a concentration-dependent manner, (3) high-K(+)-induced increase of extracellular [Glu] was partially blocked by the addition of 10 mM CoCl(2) with the high-K(+) dialysate in the hippocampus. Searching for main correlations between changes in [Glu], [Gln] and [Tau] by calculating partial correlations and with the use of factor analyses we found, the primary response of the mammalian brain to persistent depolarization is the neuronal uptake of [Gln] and release of [Tau] thereupon, acting independently of Glu changes. When glial and neuronal uptake of Glu is blocked, releases of Tau occur from neuronal as well as glial compartments accompanied by increases of [Gln] in the mammalian brain.

Deramciclane inhibits N-methyl-D-aspartate receptor function.

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca(2+) ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 microM) significantly inhibited [(3)H]D-aspartate release and transmembrane Ca(2+) flux to N-methyl-D-aspartate in the absence of Mg(2+). By contrast, inhibition of [(3)H]D-aspartate release and transmembrane Ca(2+) flux evoked by 0.1 mM (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg(2+) and 10 microM cyclothiazide by 10 microM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 microM) did not inhibit [(3)H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.

Serum albumin-lipid membrane interaction influencing the uptake of porphyrins.

It is frequently observed in pharmaceutical practice that entrapped substances are lost rapidly when liposomes are used as carriers to introduce substances into cells. The reason for the loss is the interaction of serum components with liposomes. To elucidate the mechanism of this phenomenon the partition of mesoporphyrin (MP) was systematically studied in model systems composed of various lipids and human serum albumin (HSA). As surface charge is an important factor in the interaction, neutral (1, 2-dimyristoyl-sn-glycero-3-phosphatidylcoline, DMPC) and negatively charged (1,2-dimyristoyl-sn-glycero-3-phosphatidylcoline/1, 2-dimyristoyl-sn-glycero-3-phosphatidylglycerol, DMPC/DMPG = 19/1 w/w) lipids were compared. The liposome/apomyoglobin system was the negative control. The size distribution of sonicated samples was carefully analyzed by dynamic light scattering. Constants of association of MP to the proteins and to the liposomes were determined: K(p,1) = (2.5 +/- 0.7) x 10(7) M(-1), K(p,2) = (1.0 +/- 0.7) x 10(8) M(-1), K(L,1) = (1.3 +/- 0.3) x 10(5) M(-1), and K(L,2) = (3.2 +/- 0.6) x 10(4) M(-1) for HSA, apomyoglobin, DMPC, and DMPC/DMPG liposomes, respectively. These data were used to evaluate the partition experiments. The transfer of MP from the liposomes to the proteins was followed by fluorescence spectroscopy. In the case of apomyoglobin, the experimental points could be interpreted by ruling out the protein-liposome interaction. In the case of HSA, the efflux of MP from the liposomes was strongly inhibited above a critical HSA concentration range for negatively charged vesicles. This effect was interpreted as the result of HSA coat formation on the liposome surface. This direct interaction is significant for small liposomes. The interpretation is fully supported by differential scanning calorimetry experiments.

Kinetically distinguishable AMPA receptors in rat hippocampus are associated with the loss of glutamate-sensitive conformational transitions.

We describe a stopped-flow method to study alpha-amino-7-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-kainate receptor-mediated Na+ ion flux through native membranes. Resealed plasmalemma vesicles and nerve endings from the rat hippocampus were mixed rapidly with a membrane impermeant form of the fluorescence indicator, sodium binding benzofurane oxazole and the changes in fluorescence intensity in response to various [Glu] on the time scale of 0.04 ms-10 s were monitored at a sampling rate of 6.55 kHz. Inhibitors like ouabain (1 mM) and 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (dizocilpine, 50 microM) enhanced Na+ ion translocation under low-[Na+] and physiological conditions, respectively. Dependence of AMPA-kainate receptor kinetics on [Glu] was described in a model of channel activation by faster and slower desensitizing receptors. The model accounted for almost all of the Na+ ion flux activity in the 30 microM-10 mM range of [Glu]. We found that the values of the initial rate constant for Na+ ion influx, JA, and rate constant for desensitization, alpha, for the faster desensitizing receptor were dependent on data sampling rate, whereas the initial rate constant for Na+ ion flux through the slower desensitizing receptor, JB, varied much less with the sampling rate. These phenomena can be described by (1) a fractal model of short-lived AMPA-kainate receptor channel with many closely spaced states (fractal dimension approximately 1.8) and (2) a model of long-lived AMPA-kainate receptor channel with two discrete states.

Inhibition of [3H]D-aspartate release by deramciclane.

The effects of the 5-HT2C receptor inverse agonist deramciclane on the gamma-aminobutyric acid (GABA) uptake and excitatory amino acid release processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane non-competitively inhibited the uptake of [3H]GABA with a Ki value of 13.7 +/- 0.5 microM and partially displaced specifically bound [3H](R,S)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid ([3H]NNC-328) with high affinity (IC50 = 2.0 +/- 0.7 nM). Depolarization by 4-aminopyridine or by 4-aminopyridine with (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate [(S)-AMPA] induced the release of [3H]D-aspartate. Deramciclane (10 microM) partially (approximately 50%) inhibited the release of [3H]D-aspartate without affecting [3H]D-aspartate uptake. These results suggest a role for presynaptic inhibition of excitatory amino acid release and GABA uptake in the anxiolytic properties of deramciclane.

Disulfide-linked propeptides stabilize the structure of zymogen and mature pancreatic serine proteases.

Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with propeptides that remain attached to the active enzyme via a disulfide bridge. It is likely, although not proven, that these propeptides are functionally important in the active enzymes, as well as in the zymogens. A mutant chymotrypsin was constructed to test this hypothesis, but it was demonstrated that the lack of the propeptide had no effect on the catalytic efficiency, substrate specificity, or folding of the protein [Venekei, I., et al. (1996) FEBS Lett. 379, 139-142]. In this paper, we investigate the role of the disulfide-linked propeptide in the conformational stability of chymotrypsin(ogen). We compare the stabilities of the wild-type and mutant proteins (lacking propeptide-enzyme interactions) in their zymogen (chymotrypsinogen) and active (chymotrypsin) forms. The mutants exhibited a substantially increased sensitivity to heat denaturation and guanidine hydrochloride unfolding, and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. From guanidine hydrochloride denaturation experiments, we determined that covalently linked propeptide provides about 24 kJ/mol of free energy of extra stabilization (DeltaDeltaG). In addition, the mutant chymotrypsinogen lacked the normal resistance to digestion by pepsin. This may also explain (besides keeping the zymogen inactive) the evolutionary conservation of the propeptide-enzyme interactions. Tryptophan fluorescence, circular dichroism, microcalorimetric, and activity measurements suggest that the propeptide of chymotrypsin restricts the relative mobility between the two domains of the molecule. In pancreatic serine proteases, such as trypsin, that lose the propeptide upon activation, this function appears to be accomplished via alternative interdomain contacts.

Role of intracellular Ca(2+) stores shaping normal activity in brain.

The role of intracellular Ca(2+) stores in the control of brain activity was investigated in microdialysis experiments by monitoring changes in the extracellular concentration of amino acids (AA) in the hippocampus of the rat after intracerebroventricular (icv) administration of the intracellular Ca(2+) release blocker, dantrolene in vivo, as well as in D-aspartate release and transmembrane Ca(2+) flux measurements in dantrolene-treated (50 microM) hippocampal homogenates containing resealed plasmalemma fragments and nerve endings in vitro. Microdialysis data demonstrate that icv injection of 0.6 mM dantrolene significantly decreases ( approximately 20%) the background (Glu) in the hippocampus. Both the (Glu; approximately 300%) and the inhibitory effect of dantrolene thereupon ( approximately 50%) was significantly increased when 0.5 mM of the Glu uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid, was dialysed into the hippocampus. NMDA and (S)-AMPA induced [(3)H]-D-aspartate release in hippocampal homogenates. Preincubation of these homogenates with 50 microM dantrolene was found to reduce the response to NMDA, but not to (S)-AMPA, in a NMDA-dependent manner. Increased rates of transmembrane influx and efflux of Ca(2+) in hippocampal homogenates with half-times of 4 ms and 200 ms, respectively, can be observed by the addition of 100 microM NMDA as recorded using a stopped-flow UV/fluorescence spectrometer in combination with the Ca(2+) indicator dye, bisfura-2. Both the Ca(2+) influx and efflux rates of the NMDA response were reduced (25-fold and >5-fold, respectively) in homogenates preloaded with 50 microM dantrolene. These results suggest a role for NMDA-inducible intracellular Ca(2+) stores in the control of normal brain activity in vivo.

Possible involvement of GABA(A) and GABA(B) receptors in the inhibitory action of lindane on transmitter release from cerebellar granule neurons.

Cerebellar granule cells in culture express receptors for GABA belonging to the GABA(A) and GABA(B) classes. In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 microM THIP as this is known to influence the properties of both GABA(A) and GABA(B) receptors. It was found that lindane regardless of the culture condition inhibited evoked (40 mM K+) release of neurotransmitter ([3H]D-aspartate as label for glutamate). In naive cells both GABA(A) and GABA(B) receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA(A) and GABA(B) receptor active drugs had any effect on the inhibitory action of lindane. This lack of effect was not due to inability of baclofen itself to inhibit transmitter release. It is concluded that lindane dependent on the state of the GABA(A) and GABA(B) receptors is able to indirectly interfere with both GABA(A) and GABA(B) receptors. In case of the latter receptors it was shown using [3H]baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.