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Biomedical subjects

J Heid

Publications and source records attributed to J Heid.

At least 19 recordsLinked to original sources
PubMed2007

Challenges and perspectives of computer-assisted instruction in medical education: lessons learned from seven years of experience with the CAMPUS system.

OBJECTIVES: In this paper we discuss solutions to the problem that medical teachers and students do not use modern computer-assisted instruction systems in medical education as much as expected by their developers. METHODS: As an example for a modern problem-based CAI system we introduce the CAMPUS shell system for case-based training in medicine. RESULTS: CAMPUS has received several awards and positive evaluation results. Nevertheless, the usage of such systems in courses and for self-study could be increased. CONCLUSIONS: Curricular integration of CAI as well as further improvements on existing CAI systems to increase the usage in medical education is essential.

Case-Control Studies↗
PubMed2001

Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABA(B) responses in mice lacking GABA(B(1)).

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Animals↗
PubMed2001

C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABA(b) receptors.

Assembly of fully functional GABA(B) receptors requires heteromerization of the GABA(B(1)) and GABA(B(2)) subunits. It is thought that GABA(B(1)) and GABA(B(2)) undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABA(B(1)) retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABA(B(1)) trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABA(B(1)) that when deleted or mutagenized allows for surface delivery in the absence of GABA(B(2)). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in K(ATP) channels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABA(B(2)) is able to mask the RSRR signal and to direct the GABA(B(1)) monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABA(B(2)) participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABA(B(1)) and GABA(B(2)) supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABA(B) receptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

Amino Acid Motifs↗
PubMed2001

Positive allosteric modulation of native and recombinant gamma-aminobutyric acid(B) receptors by 2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog CGP13501.

The compounds CGP7930 [2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol] and its close analog CGP13501 were identified as positive modulators of gamma-aminobutyric acid(B) (GABA(B)) receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[(35)S]thiotriphosphate) (GTP gamma[(35)S]) binding to membranes from a GABA(B(1b/2)) expressing Chinese hamster ovary (CHO) cell line at low micromolar concentrations and are ineffective in the absence of GABA. The structurally related compounds propofol and malonoben are inactive. Similar effects of CGP7930 are seen in a GTP gamma[(35)S] binding assay using a native GABA(B) receptor preparation (rat brain membranes). Receptor selectivity is demonstrated because no modulation of glutamate-induced GTP gamma[(35)S] binding is seen in a CHO cell line expressing the metabotropic glutamate receptor subtype 2. Dose-response curves with GABA in the presence of different fixed concentrations of CGP7930 reveal an increase of both the potency and maximal efficacy of GABA at the GABA(B(1b/2)) heteromer. Radioligand binding studies show that CGP7930 increases the affinity of agonists but acts at a site different from the agonist binding site. Agonist affinity is not modulated by CGP7930 at homomeric GABA(B(1b)) receptors. In addition to GTP gamma[(35)S] binding, we show that CGP7930 also has modulatory effects in cellular assays such as GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in Xenopus laevis oocytes and Ca(2+) signaling in human embryonic kidney 293 cells. Furthermore, we show that CGP7930 enhances the inhibitory effect of L-baclofen on the oscillatory activity of cultured cortical neurons. This first demonstration of positive allosteric modulation at GABA(B) receptors may represent a novel means of therapeutic interference with the GABA-ergic system.

Allosteric Regulation↗
PubMed2000

Ca(2+) requirement for high-affinity gamma-aminobutyric acid (GABA) binding at GABA(B) receptors: involvement of serine 269 of the GABA(B)R1 subunit.

The gamma-aminobutyric acid (GABA) receptor type B (GABA(B)R) is constituted of at least two homologous proteins, GABA(B)R1 and GABA(B)R2. These proteins share sequence and structural similarity with metabotropic glutamate and Ca(2+)-sensing receptors, both of which are sensitive to Ca(2+). Using rat brain membranes, we report here that the affinity of GABA and 3-aminopropylphosphinic acid for the GABA(B)R receptor is decreased by a factor >10 in the absence of Ca(2+). Such a large effect of Ca(2+) is not observed with baclofen or the antagonists CGP64213 and CGP56999A. In contrast to baclofen, the potency of GABA in stimulating GTPgammaS binding in rat brain membranes is also decreased by a factor >10 upon Ca(2+) removal. The potency for Ca(2+) in regulating GABA affinity was 37 microM. In cells expressing GABA(B)R1, the potency of GABA, but not of baclofen, in displacing bound (125)I-CGP64213 was similarly decreased in the absence of Ca(2+). To identify residues that are responsible for the Ca(2+) effect, the pharmacological profile and the Ca(2+) sensitivity of a series of GABA(B)R1 mutants were examined. The mutation of Ser269 into Ala was found to decrease the affinity of GABA, but not of baclofen, and the GABA affinity was found not to be affected upon Ca(2+) removal. Finally, the effect of Ca(2+) on the GABA(B) receptor function is no longer observed in cells coexpressing this GABA(B)R1-S269A mutant and the wild-type GABA(B)R2. Taken together, these results show that Ser269, which is conserved in the GABA(B)R1 protein from Caenorhabditis elegans to mammals, is critical for the Ca(2+)-effect on the heteromeric GABA(B) receptor.

Amino Acid Sequence↗
PubMed1999

Gamma-hydroxybutyrate is a weak agonist at recombinant GABA(B) receptors.

Gamma-hydroxybutyrate (GHB) is a neuromodulator with high affinity binding sites in the mammalian brain. However, the receptor for GHB has not yet been identified. There are indications that GHB and gamma-aminobutyric acid (GABA) mediate their effects via the same receptor. We tested this hypothesis using GABA(B)R1/R2 receptors co-expressed with Kir3 channels in Xenopus oocytes. GHB activated these receptors with an EC50 of approximately 5 mM and a maximal stimulation of 69% when compared to the GABA(B) receptor agonist L-baclofen. GHB and L-baclofen did not amplify each others effect nor did they stimulate the GABA(B) receptor in a linearly additive manner. CGP54626A, 2-OH saclofen and CGP35348, three competitive GABA(B) receptor antagonists, inhibited the GHB induced response completely. A concentration of 30 mM GHB displaced [125I]CGP64213 binding at GABA(B)R1 expressed in COS cells by 21%. These results indicate that GHB is a weak partial agonist at the GABA binding site of GABA(B)R1/R2.

Anesthetics, Intravenous↗
PubMed1999

Ligands for the isolation of GABA(B) receptors [corrected] .

Since the discovery that the most abundant inhibitory neurotransmitter in the mammalian brain, GABA (gamma-aminobutyric acid), interacts not only with ionotropic GABA(A) receptors, but also with metabotropic GABA(B) receptors (Bowery et al., 1980) much work has been devoted to the elucidation of the structure of GABA(B) receptors by either affinity chromatography purification or by expression cloning. In 1997 Kaupmann et al. succeeded in cloning two splice variants designated GABA(B) R1a (960 amino acids) and GABA(B) R1b (844 amino acids). Although the amino acid sequences are now known, precise information on the three-dimensional environment of the GABA(B) R1 binding site is still lacking. Recent experiments demonstrated that the amino acids of the seven transmembrane helices are not essential for ligand binding as a soluble GABA(B) receptor fragment is still able to bind antagonists (Malitschek et al., 1999). For the isolation and purification of the soluble N-terminal extracellular domain (NTED) of GABA(B) receptors potent ligands for affinity chromatography were synthesised with the aim of obtaining a crystalline receptor fragment-ligand complex for X-ray structure determination. The most promising ligand [125I]CGP84963 (K(D) = 2 nM) combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as a photoaffinity moiety separated by a spacer consisting of three GABA molecules from 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion.

Animals↗
PubMed1999

The N-terminal domain of gamma-aminobutyric Acid(B) receptors is sufficient to specify agonist and antagonist binding.

The recently identified gamma-aminobutyric acid type B receptors (GABA(B)Rs) share low sequence similarity with the metabotropic glutamate (mGlu) receptors. Like the mGlu receptors, the N-terminal extracellular domain (NTED) of GABA(B)Rs is proposed to be related to bacterial periplasmic binding proteins (PBPs). However, in contrast to the mGlu receptors, the GABA(B)Rs lack a cysteine-rich region that links the PBP-like domain to the first transmembrane domain. This cysteine-rich region is necessary for the PBP-like domain of mGlu receptors to bind glutamate. To delimit the ligand-binding domain of GABA(B)Rs, we constructed a series of chimeric GABA(B)R1/mGluR1 and truncated GABA(B)R1 receptor mutants. We provide evidence that despite the lack of a cysteine-rich region, the NTED of GABA(B)Rs contains all of the structural information that is necessary and sufficient for ligand binding. Moreover, a soluble protein corresponding to the NTED of GABA(B)Rs reproduces the binding pharmacology of wild-type receptors. This demonstrates that the ligand-binding domain of the GABA(B)Rs can correctly fold when dissociated from the transmembrane domains.

Animals↗
PubMed1998

GABA(B)-receptor subtypes assemble into functional heteromeric complexes.

B-type receptors for the neurotransmitter GABA (gamma-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABA(B) receptors, so it is expected that GABA(B)-receptor (GABA(B)R) subtypes exist. Two GABA(B)-receptor splice variants have been cloned (GABA(B)R1a and GABA(B)R1b), but native GABA(B) receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult. Here we describe a new GABA(B) receptor subtype, GABA(B)R2, which does not bind available GABA(B) antagonists with measurable potency. GABA(B)R1a, GABA(B)R1b and GABA(B)R2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABA(B) receptors in vivo and show that GABA(B)R2 and GABA(B)R1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABA(B) receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.

Amino Acid Sequence↗
PubMed1998

Human gamma-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels.

gamma-Aminobutyric acid type B receptors (GABABRs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABABRs inhibit neurotransmitter release by down-regulating high-voltage activated Ca2+ channels, whereas postsynaptic GABABRs decrease neuronal excitability by activating a prominent inwardly rectifying K+ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABABRs, hGABABR1a (hR1a) and hGABABR1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABABRs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABABRs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABABR1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.

Amino Acid Sequence↗
PubMed1998

Developmental changes of agonist affinity at GABABR1 receptor variants in rat brain.

Recently, two N-terminal splice variants of the metabotropic receptor for GABA (gamma-amino-butyric acid) were cloned. Here, we describe an antiserum that recognizes the two receptor variants. We demonstrate that these proteins are identical with GABAB receptors that are photoaffinity labeled with [125I]CGP71872 in rat brain. The C-terminal epitopes recognized by the antiserum are conserved in several vertebrate species but not in chicken. No hints for the existence of additional closely related receptor subtypes or variants are found in double-labeling experiments with antibody and photoaffinity ligand. Western blot analysis reveals widespread expression of the GABABR1 receptor proteins in rat brain with the highest level of expression at early postnatal stages. The binding affinity of the GABAB receptor agonist L-baclofen at native R1a and R1b variants is similar. In early postnatal development the affinity at R1a and R1b is 10-fold lower than in adult brain and gradually increases with aging.

Animals↗
PubMed1997

Expression cloning of GABA(B) receptors uncovers similarity to metabotropic glutamate receptors.

GABA (gamma-amino-butyric acid), the principal inhibitory neurotransmitter in the brain, signals through ionotropic (GABA(A)/ GABA(c)) and metabotropic (GABA(B)) receptor systems. Here we report the cloning of GABA(B) receptors. Photoaffinity labelling experiments suggest that the cloned receptors correspond to two highly conserved GABA(B) receptor forms present in the vertebrate nervous system. The cloned receptors negatively couple to adenylyl cyclase and show sequence similarity to the metabotropic receptors for the excitatory neurotransmitter L-glutamate.

Adenylyl Cyclases↗
PubMed1987

Substance P depresses neuronal activity in the rat olfactory bulb in vitro and in vivo: possible mediation via gamma-aminobutyric acid release.

The rat olfactory bulb is an area displaying a particularly high density of substance P receptors in the glomerular cell layer whose functions are unknown. In pilot in vivo experiments we discovered that iontophoretically administered substance P potently depressed the spontaneous firing rate of most unidentified neurons of the rat olfactory bulb. To further elucidate the mechanism of this unexpected depressant effect, we studied the peptide's action in vitro on coronal sections of this brain region. Bath applied and microiontophoretically administered substance P depressed the spontaneous discharge of unidentified glomerular neurons in a dose-dependent fashion. This inhibiting effect is mediated indirectly via the release of another transmitter because it was abolished completely if the standard perfusion medium was replaced by a medium containing zero calcium and high magnesium. It appears that substance P acts by means of releasing GABA which in turn evokes the observed cell depression because the depressant effects were completely abolished by bath-applied bicuculline (10 microM) and picrotoxin (100 microM). In conclusion we propose that substance P indirectly depresses neuronal activity in the glomerular cell layer of the rat olfactory bulb by releasing gamma-aminobutyric acid.

Action Potentials↗
PubMed1986

Dose pipecolic acid interact with the central GABA-ergic system?

Several previous studies have suggested a strong GABA-mimetic action of the endogenous brain imino acid, L-pipecolic acid (L-PA). In the present study, these observations were evaluated using electrophysiological and neurochemical methods. In contrast to published data our electrophysiological studies on rat cortical neurones in situ showed only a weak, but bicuculline-sensitive depressant action of L-PA on cortical neurones. Furthermore, L-PA proved to have no affinity for any of the three components of the GABA-benzodiazepine-chloride channel receptor complex. However, using a modification of published methods a weak affinity for the GABA-B receptor site was demonstrated (IC50 = 1.8 X 10(-3) M). L-PA showed no anticonvulsive activity in several tests; in particular, it did not protect mice from seizures induced by inhibition of L-glutamate-1-decarboxylase (EC 4.1.1.15: GAD). L-PA had a very weak action on brain GABA levels of mice, and did not modify the rate of GABA synthesis. In conclusion, these results are not compatible with a strong in vivo interaction between L-PA and GABA-mediated inhibitory transmission.

Animals↗
PubMed1983

Decreased activity of locus coeruleus neurons in the rat after DSP-4 treatment.

The activity of noradrenergic neurons of the rat locus coeruleus was investigated at 10 and 50 days after the administration of DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzyl-amine), a selective noradrenergic neurotoxin. The mean neuronal firing rate in control animals was 2.4 Hz. In contrast, DSP-4 animals had lower rates of 1.2 Hz at 10 days and 1.7 Hz at 50 days. Histological examinations revealed no morphological changes of locus coeruleus cell bodies at either the 10- or 50-day time points. These results suggest that DSP-4 can impair neuronal activity of the locus coeruleus without altering the structural appearance of locus coeruleus perikarya.

Action Potentials↗
PubMed1980

Are only alpha 2-adrenergic receptors present in bovine retina?

The retina represents a part of the central nervous system (CNS) with a well studied, geometrically defined structure and a specialized function--the processing of light signals. Neurotransmitters such as glutamate, aspartate, glycine, gamma-aminobutyric acid (GABA), dopamine and acetylcholine are considered to be involved in the neuronal activity of the retina. Receptors for acetylcholine, GABA, dopamine and benzodiazepines have also been demonstrated. Thus, the retina can be considered as a model for the study of neuronal processing in general, in which the input, light signals, can be selected and regulated in a defined way. We report here that alpha-adrenergic receptors in the bovine retina have been characterized using radioreceptor assays. 3H-phentolamine (an alpha 1- and alpha 2-antagonist), 3H-clonidine (a preferential alpha 2-agonist) and 3H-WB 4101 [(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane, an alpha 1-antagonist] were used as radioligands, and WB 4101 and prazosin (alpha 1-antagonists), tolazine (an alpha 2-antagonist), yohimbine and its stereoisomers rauwolscine (preference for alpha 2-receptors) and corynanthine (preference for alpha 1-receptors), were used as inhibitors of radioaligand binding. Only alpha 2-adrenergic receptors were found.

Adrenergic alpha-Agonists↗