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Biomedical subjects

S Heinemann

Publications and source records attributed to S Heinemann.

At least 37 records · Page 2Linked to original sources

Ionotropic glutamate-receptor gene expression in hypothalamus: localization of AMPA, kainate, and NMDA receptor RNA with in situ hybridization.

In situ hybridization and Northern blots were used to study the ionotropic subtypes of the glutamate receptor in the rat hypothalamus. Widespread expression of AMPA, kainate, and NMDA receptor RNA was found in the hypothalamus with the transcripts the same size and number as found in other regions of the brain. Most of the glutamate-receptor subunits studied were expressed in greater amounts in hippocampus than in hypothalamus; GluR5, on the other hand, showed a greater expression in hypothalamus than in hippocampus. On the basis of Northern blot analysis, all regions of the brain examined, including hypothalamus, cerebral cortex, cerebellum, olfactory bulb, and hippocampus, expressed all eight of the subunits studied. Each subunit showed different relative expressions in the different regions. In the hypothalamus, GluR1 and GluR2 were among the most widely expressed of the non-NMDA ionotropic receptors. Other AMPA-preferring receptors, GluR3 and -R4, were also found, but to a lesser extent. Scattered cells expressed the kainate-preferring receptors GluR5, -R6, and -R7. The NMDA receptor NMDAR1 was detected throughout the hypothalamus. In many regions of the hypothalamus, only scattered cells showed detectable expression of the glutamate-receptor mRNA as detected by autoradiographic silver grains over neurons; unlabeled cells were mixed among labeled cells. Every region of the hypothalamus had several different glutamate receptors. The expression of many different types of ionotropic glutamate receptors throughout the hypothalamus suggests that multiple modes of ion channel regulation by glutamate probably operate here and provides further support for the importance of the excitatory transmitter glutamate in hypothalamic regulation.

Animals↗

Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells.

Adenosine potentiates the stimulated release of mast cell mediators. Pharmacologic studies suggest the presence of two adenosine receptors, one positively coupled to adenylate cyclase and the other coupled to phospholipase C activation. To identify mast cell adenosine receptor subtypes, cDNAs for the A1 and A2a adenosine receptors were obtained by screening a mouse brain cDNA library with the use of PCR-derived probes. Mouse bone marrow-derived mast cell cDNA libraries were constructed and screened with the use of A1 and A2a cDNA probes, which revealed the presence of A2a, but not A1, receptor clones. A putative A2b receptor was identified by using low stringency mast cell library screening. Northern blotting of mast cell poly(A)+ RNA with the use of receptor subtype probes labeled single mRNA bands of 2.4 kb and 1.8 kb for the A2a and A2b receptors, respectively. In situ cells. An A2a receptor-specific agonist failed to enhance mast cell mediator release, which suggests that the secretory process is modulated through the A2b and/or another receptor subtype. By using RNase protection assays, we found that mast cells that had been cultured in the presence of N-ethylcarboxamidoadenosine for 24 h exhibited a decrease in both A2a and A2b receptor RNA levels. Cells that had been cultured for 1 to 2 days in the presence of dexamethasone demonstrated increased amounts of A2a receptor mRNA, but no identifiable change in A2b receptor mRNA. Mast cells possess at least two adenosine receptor subtypes that may be differentially regulated.

Adenosine↗

Molecular cloning and chromosomal localization of the human alpha 7-nicotinic receptor subunit gene (CHRNA7).

We have isolated cDNA and genomic clones coding for the human alpha 7 neuronal nicotinic receptor subunit, the major component of brain nicotinic receptors that are blocked by alpha-bungarotoxin. The human alpha 7 neuronal nicotinic cDNA encodes a mature protein of 479 amino acids that is highly homologous to the rat alpha 7 neuronal nicotinic subunit (90%). We have mapped the human alpha 7-nicotinic receptor subunit gene to chromosome 15, band q14, a region frequently rearranged in patients carrying a bisatellite 15 chromosome, large inv dup (15), whose clinical features include mental retardation and seizures.

Amino Acid Sequence↗

Cellular distribution of nicotinic acetylcholine receptor subunit mRNAs in the human cerebral cortex as revealed by non-isotopic in situ hybridization.

The pharmacology of telencephalic nicotinic acetylcholine receptors (nAChRs) has become an important issue in recent years. While in the human brain a direct pharmacological assessment is difficult to achieve the visualization of nAChRs has been enabled by histochemical techniques providing an ever increasing and improving resolution. Receptor autoradiography was used to visualize binding sites on the level of cortical layers whereas immunohistochemistry has allowed for the cell type-specific and ultrastructural localization of receptor protein. Further investigations have to elucidate the cellular sites of NAChR biosynthesis by visualizing subunit-specific transcripts. Using autopsy samples of the human precentral cortex (Area 4) as a paradigm we have applied digoxigenin-labeled cRNA probes to localize transcripts for the alpha 3- and alpha 4-1-subunits of the nAChR. In accordance with findings in the monkey cortex, the alpha 3-subunit seems to be expressed mainly in pyramidal neurons of layers III-VI of the human cerebral cortex. Transcripts for the alpha 4-1-subunit, by contrast, appear to be present in a large number of neurons throughout all layers of the cerebral cortex, consonant with its ubiquitous distribution in the rodent brain. The present findings show that also in human autopsy brains the cell type-specific detection of nAChR transcripts is possible. For the future, this technique will enable to investigate the expression of receptor transcripts in diseased human brains as compared to controls.

Adult↗

N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1.

We investigated the transmembrane topology of the glutamate receptor GluR1 by introducing N-glycosylation sites as reporter sites for an extracellular location of the respective site. Our data show that the N-terminus is extracellular, whereas the C-terminus is intracellular. Most importantly, we found only three transmembrane domains (designated TMD A, TMD B, and TMD C), which correspond to the previously proposed TMDs I, III, and IV, respectively. Contrary to earlier models, the putative channel-lining hydrophobic domain TMD II does not span the membrane, but either lies in close proximity to the intracellular face of the plasma membrane or loops into the membrane without transversing it. Furthermore, the region between TMDs III and IV, in previous models believed to be intracellular, is an entirely extracellular domain.

Amino Acid Sequence↗

The inactivation behaviour of voltage-gated K-channels may be determined by association of alpha- and beta-subunits.

Voltage-gated K-channels of the Shaker related subfamily have two subunits, membrane integrated alpha- and peripheral beta-subunits. alpha-Subunits may assemble as tetramers and form in in vitro expression systems functional K-channels. beta-Subunits cannot from channels by themselves. Like for alpha-subunits, the rat nervous system apparently expresses a family of beta-subunit proteins. We have demonstrated that one rat K-channel beta-subunit, Kv beta 1, contains an inactivating domain. Upon association of alpha- and Kv beta 1-subunits, delayed-rectifier type K-channels are converted to rapidly inactivating A-type K-channels. The beta-subunit inactivation domain acts via a ball and chain type mechanism previously proposed for N-type inactivation of alpha-subunits. The association of alpha- and beta-subunits endows the nervous system with an unprecedented flexibility and diversity of K-channels which may play an important role in the regulation of nervous excitability.

Amino Acid Sequence↗

[Doppler ultrasound evaluation of blood flow direction in distal occlusion of arteries of the lower leg].

It is necessary to observe the direction of the blood flow for measurement of the ankle pressure by the Dopplermethod. Obliteration of the ankle pressure by the Dopplermethod. Obliteration of the anterior tibial artery can be compensated by collaterals from the posterior tibial artery. Through this the blood flow is reversed in the dorsalis pedis artery. If this possibility is disregarded, the pressure of the anterior tibial artery will yield false data. The situation of the blood flow can be clarified by a compression test of the posterior tibial artery. This also applies to obliterations of the other arteries of the ankle.

Arterial Occlusive Diseases↗

A single amino acid determines the subunit-specific spider toxin block of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels.

Joro spider toxin (JSTX) is one of the most potent antagonists of glutamatergic AMPA/KA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate) receptor channels in invertebrates and vertebrates. A differential blocking effect on certain types of glutamatergic synapses--e.g., parallel and climbing fiber synaptic inputs to rat cerebellar Purkinje neurons--has been shown by using a synthetic analog of the spider toxin. By investigating the molecular basis of the JSTX action on the recombinant AMPA/KA receptors GluR1-GluR4 and GluR6 expressed in Xenopus oocytes, we found that submicromolar concentrations of JSTX exert a subunit-specific block. Thus, receptor subunits forming a receptor channel with a linear current-voltage (I-V) relationship (GluR1/2, GluR2/3, and GluR6) were not affected, while receptor subunits with rectifying I-V relationships (GluR1, GluR3, GluR4, and GluR1/3) were reversibly blocked by JSTX. By using receptor-subunit mutants obtained by site-directed mutagenesis, we have identified a single amino acid position (glutamine in the proposed second transmembrane domain) that is critical for the JSTX block. Since this site has previously been shown to control the I-V relationship of the AMPA/KA receptor channel and to participate in the regulation of the channel's permeability for calcium ions, our findings suggest that JSTX binds close to the central pore region of the channel.

Amino Acid Sequence↗

Fat emulsions for parenteral nutrition II: Characterisation and physical long-term stability of Lipofundin MCT LCT.

Lipofundin MCT LCT emulsions for parenteral nutrition were characterized in terms of particle size (bulk population), presence of large particles ( 1 mum) and droplet charge (zeta potential as measure of electrostatic stabilization). Photon Correlation Spectroscopy (PCS), microscopy and Laser Diffractometry (LD) were employed as sizing techniques and compared to each other. Zeta potentials were determined by Laser Doppler Anemometry (LDA). The characterization data showed a good batch-to-batch reproducibility. The physical stability of Lipofundin MCT LCT was monitored over a period of 2 years. The mean diameter of the bulk droplet population did not increase. No formation of larger droplets was found by microscopy and LD. A negligible coalescence could only be detected by the very sensitive PCS measurements (two time window analysis). The zeta potential stayed above -45 mV providing sufficient electrostatic stabilization during the whole storage period.

Journal Article↗

Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor.

We have determined the gene structure for the NMDA receptor subunit gene NMDAR1. We found eight splice variants that arise from different combinations of a single 5' terminal exon insertion and three different 3' terminal exon deletions, relative to NMDAR1. We analyzed the modulation by Zn2+ of currents through homomeric receptors assembled from these splice variants and found that, in addition to its well-known inhibitory effect at high concentrations, Zn2+ potentiates agonist-induced currents at submicromolar concentrations (EC50 = 0.50 microM). This potentiation is observed only with a subset of NMDAR1 splice variants that show additional differences in pharmacological properties. Zn2+ potentiation is rapidly reversible, noncompetitive with either glutamate or glycine, and voltage independent. Zn2+ potentiation is mimicked by Cd2+, Cu2+, and Ni2+, but not by Mn2+, Co2+, Fe3+, Sn2+, or Hg2+. Our results suggest a possible role for Zn2+ as a positive modulator of NMDA receptors in certain regions of the brain.

Animals↗

[Long-term treatment of sleep apnea with evening administration of oral retard theophylline].

In 776 patients suffering from sleep apnoea long-term treatment with sustained-release theophylline was investigated. Responders show a significant reduction of apnoea phases. Using an individual theophylline dosage with morning theophylline concentrations between 5 and 8 micrograms/ml, only few side effects occurred. Positive effects on cardiopulmonary capacity were seen.

Administration, Oral↗

Expression of glutamate receptor genes in the mammalian retina: the localization of GluR1 through GluR7 mRNAs.

Seven distinct cDNAs encoding functional subunits of the AMPA/kainate-type glutamate receptors have been recently cloned. This in situ hybridization study was done to determine which subunits are expressed in the retina and, where possible, which neurons express them. Hybridization of 35S-UTP-labeled cRNA probes with transverse sections revealed that mRNAs for all seven receptor subunits (GluR1-GluR7) are expressed in both cat and rat retinas. GluR1 and GluR2 produced labeling over the entire inner nuclear layer (INL) and ganglion cell layer (GCL). GluR3-GluR7 have more limited distributions, indicative of expression by only a subset of neurons. All of the subunits are expressed by the cells at the inner edge of the INL, where amacrine cells reside, yet the layers with the horizontal, bipolar, and ganglion cells contain different subsets of subunits. These findings suggest that these glutamate receptor subunits are employed at many of the retinal synapses, including the photoreceptor input to the outer plexiform layer and the bipolar cell's contacts with the processes at the INL. It is also possible that some glial cells in the INL express some of the subunits. Since different combinations of GluR1-GluR3 have been shown to play an important role in the calcium permeability in response to glutamate, we investigated whether single cells coexpressed those subunits. By hybridizing adjacent semithin (1 micron) sections of the cat retina with probes for GluR1-GluR3, it was possible to observe coexpression of all three subunits, or of pairs of these subunits, in cells within the INL and GCL.

Animals↗

Calcium influx through subunits GluR1/GluR3 of kainate/AMPA receptor channels is regulated by cAMP dependent protein kinase.

Excitatory synaptic transmission in the central nervous system (CNS) is mediated by three major classes of glutamate receptors, namely the ionotropic NMDA (N-Methyl-D-Aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid) receptors and the metabotropic receptor type. Among the ionotropic receptors, NMDA receptors are thought to mediate their physiological response mainly through the influx of extracellular calcium, while KA/AMPA receptor channels are mainly thought to carry the influx of monovalent cations. Recently, we have challenged this view by showing that cloned KA/AMPA receptor subunits GluR1 and GluR3 form ion channels which are permeable to calcium. We now directly demonstrate large increases in intracellular calcium concentrations induced by calcium fluxes through KA/AMPA receptor channels in solutions with physiological calcium concentrations. Calcium fluxes were observed through glutamate receptor channels composed of the subunits GluR1 and GluR3, which are both abundantly present in various types of central neurones. The calcium influx was fluorometrically monitored in Xenopus oocytes injected with the calcium indicator dye fura-2. Bath application of the membrane permeable analogue of adenosine cyclic monophosphate (cAMP) potentiated the current and also the flux of calcium through open KA/AMPA receptor channels. Further pharmacological experiments suggested that this effect was mediated by the activation of protein kinase A. Our results provide a molecular interpretation for the function of calcium permeable KA/AMPA receptor channels in neurones and identify two of the subunits of the KA/AMPA receptor channel which are regulated by the cAMP dependent second messenger system.

Animals↗

Fat emulsions for parenteral nutrition. I: Evaluation of microscopic and laser light scattering methods for the determination of the physical stability.

Droplet size distribution was studied as a measure of the physical stability of fat emulsions. Test emulsions with increasing fractions of larger particles were prepared and investigated by conventional light microscopy and laser light scattering techniques (Photon Correlation Spectroscopy (PCS), Laser Diffractometer). Light microscopy proved to be most sensitive for detecting a few single large particles. All the techniques placed the test emulsions in the same order of increasing content of larger droplets. The application of a two time window analysis for PCS markedly improved the ability to differentiate between emulsions which were very similar in size distribution. In contrast to the applied semi-quantitative microscopic method, the laser techniques characterise the emulsion in absolute figures. Additional zeta potential measurements by Laser Doppler Anemometry (LDA) can quantify reduced electrostatic repulsion as a possible cause for increased formation of larger droplets.

Journal Article↗

Cloning of a putative glutamate receptor: a low affinity kainate-binding subunit.

Kainate, a glutamate receptor agonist, is a potent neuroexcitatory agent that produces epileptiform activity and selective neuronal degeneration. Binding studies using neuronal membrane homogenates or brain sections have identified sites having either high or low affinity for [3H]kainate. Here we report the cloning of a gene, GluR7, with approximately 75% sequence identity with the previously cloned GluR5 and GluR6 subunit genes. Transcripts of the GluR7 gene are evident in brain areas that bind [3H]kainate and are susceptible to kainate-induced neurotoxicity. We have performed ligand binding studies with membranes of transfected HeLa cells expressing GluR6 or GluR7 subunits. Our data show that the GluR6 and GluR7 subunits have a rank order of agonist affinity (domoate greater than kainate much greater than L-glutamate, quisqualate much greater than AMPA, NMDA) and a dissociation constant for kainate (95 and 77 nM, respectively) characteristic of the low affinity kainate-binding sites described in the brain.

Amino Acid Sequence↗