Biomedical subjects

L A Raymond

Publications and source records attributed to L A Raymond.

At least 37 records · Page 2Linked to original sources

PubMed1999

D1 dopamine receptor-induced cyclic AMP-dependent protein kinase phosphorylation and potentiation of striatal glutamate receptors.

Dopamine receptor activation regulates cyclic AMP levels and is critically involved in modulating neurotransmission in the striatum. Others have shown that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor-mediated current is potentiated by cyclic AMP-dependent protein kinase (PKA) activation. We made whole-cell patch clamp recordings from cultured striatal neurons and tested whether D1-type dopamine receptor activation affected AMPA receptor-mediated currents. After a 5-min exposure to the D1 agonist SKF 81297 (1 microM), kainate-evoked current amplitude was enhanced in approximately 75% of cells to 121+/-2.5% of that recorded prior to addition of drug. This response was inhibited by the D1 antagonist SCH 23390 and mimicked by activators of PKA. Moreover, by western blot analysis using an antibody specific for the phosphorylated PKA site Ser845 of GluR1, we observed a marked increase in phosphorylated GluR1 following a 10-min exposure of striatal neurons to 1 microM SKF 81297. Our data demonstrate that activation of D1-type dopamine receptors on striatal neurons promotes phosphorylation of AMPA receptors by PKA as well as potentiation of current amplitude. These results elucidate one mechanism by which dopamine can modulate neurotransmission in the striatum.

Action Potentials↗

PubMed1999

Motor skill impairment in SIV-infected rhesus macaques with rapidly and slowly progressing disease.

A number of studies have shown that simian immunodeficiency virus (SIV) infection in rhesus macaques parallels many aspects of HIV disease in humans. The purpose of this study was to further characterize the rhesus macaque infected with neurovirulent SIV as a model of neuroAIDS. Using a motor skill task, our objective was to detect SIV-related movement impairments in behaviorally trained macaques. The motor skill task required retrieval of a food pellet from a cup in a rotating turntable across a range of speeds. Nine monkeys were infected with neurovirulent strains of SIVmac (R71/17E): four monkeys served initially as controls pre-inoculation. Seven monkeys developed simian AIDS within 4 months of inoculation (rapid progressors), and two survived more than 18 months post-inoculation (slow progressors). Of the rapid progressors, five exhibited significant deficits in this task, most showing a gradual decline in performance terminating in a sharp drop to severely impaired levels of performance. One slow progressor (AQ15) showed no performance declines. The other slow progressor (AQ94) showed a significant decrease in maximum speed that was concurrent with the onset of clinical signs. For AQ94, the role of sickness behavior related to late stage simian AIDS could not be ruled out. These results demonstrate that motor system impairment can be detected early in the course of SIV infection in rhesus macaques, further establishing the SIVmac-infected macaque monkey as a viable model of neuroAIDS.

Animals↗

PubMed1999

Motor evoked potentials in a rhesus macaque model of neuro-AIDS.

Previous work using bone marrow passaged SIVmac239 (simian immunodeficiency virus) has shown that macrophage tropic strains of this virus enter the rhesus macaque brain early following inoculation (Sharma et al, 1992; Desrosiers et al, 1991; Zhu et al, 1995; and Narayan et al, 1997). As part of an effort to more fully characterize the extent of neurologic impairment associated with SIV infection of the brain, we used transcranial electrical stimulation of motor cortex and the spinal cord to evoke EMG potentials in two forelimb (EDC and APB) and two hindlimb (LG and AH) muscles. The latencies, magnitudes and thresholds of motor evoked potentials (MEPs) recorded from nine monkeys infected with neurovirulent SIVmac R71/17E were compared to pre-inoculation records from the same monkeys. Seven of nine monkeys developed simian AIDS within 4 months of inoculation and were euthanized. Two monkeys remained free of AIDS-related clinical illness for over 18 months following inoculation. Six of the seven monkeys with rapidly progressing disease showed post-inoculation latency increases ( > or = 2 s.d. of control) in at least one cortical MEP. Increases in cortical MEP latency ranged from 21-97% in different monkeys. All seven rapidly progressing animals showed post-inoculation increases in at least one spinal cord MEP latency. Maximum spinal cord MEP latency increases ranged from 22-147%. Increases in central conduction time (CCT) ranged up to 204% and exceeded two standard deviations of control in four monkeys. Neither of the two monkeys with slowly progressing disease showed significant increases in either cortical or spinal cord MEP latency or CCT. Only the monkeys with rapidly progressing disease exhibited classic AIDS-related neuropathology, although there was no consistent relationship between the severity of neuropathology and the extent of MEP abnormalities. In conclusion, our results demonstrate clear deficits in the functional integrity of both central and peripheral motor system structures associated with SIV infection and further support the use of SIV-infected rhesus macaques as a model of neuro-AIDS.

Animals↗

PubMed1998

Neurovirulent simian immunodeficiency virus induces calbindin-D-28K in astrocytes.

Astrocyte activation has been postulated to be a major contributor to functional changes in the brain of AIDS patients. We assessed astrocyte activation in the simian immunodeficiency virus (SIV) model. Four groups of macaque brains were examined: uninoculated controls, animals inoculated with virus that did not cause disease, animals inoculated with virus that caused AIDS but did not cause encephalitis, and animals with SIV encephalitis. We examined expression of calbindin-D-28K, a calcium binding protein that is upregulated in astrocytes during excitotoxic events, as well as glial fibrillary acidic protein (GFAP). The presence of calbindin in astrocytes was confirmed by double-labeling using confocal microscopy. Increases in calbindin staining were most apparent in the white matter, but increases in GFAP staining were most apparent in middle layers of the cerebral cortex. Six of the seven animals with SIV encephalitis had calbindin immunoreactive astrocytes in the subcortical white matter, corpus callosum, internal capsule, cerebral peduncle, pontine white matter, and cerebellar white matter. Very rarely, a few, very lightly calbindin-immunoreactive astrocytes were present in the uninoculated control brains. The increase in calbindin expression by astrocytes in SIV encephalitis suggests that these cells are subject to calcium toxicity. In uninoculated control macaques, and in macaques inoculated with virus that did not cause disease, GFAP-immunoreactive astrocytes were present throughout the subcortical white matter and in layer I, but very few were found in layers III-V of the cerebral cortex. Two animals that died of AIDS without encephalitis had somewhat higher numbers of GFAP immunoreactive astrocytes in middle cortical layers. In seven animals that received passaged neurovirulent virus and developed both AIDS and encephalitis, the number of GFAP-immunoreactive astrocytes in middle cortical layers was high, indicating widespread astrocyte activation.

Animals↗

PubMed1998

Fractionator analysis shows loss of neurons in the lateral geniculate nucleus of macaques infected with neurovirulent simian immunodeficiency virus.

Infection of macaques with neurovirulent strains of simian immunodeficiency virus (SIVmac) is an experimental model for the neurological manifestations of AIDS. Loss of neurons has been reported in the cerebral cortex following immunodeficiency viral infection, but thalamic structures which may contribute to electrophysiological changes and neurological deficits have not been examined. In this study, the lateral geniculate nucleus (LGN) of macaques inoculated with macrophage-tropic, neurovirulent virus SIVmac239 (R71 and 17E) was examined for neuron loss using the optical fractionator method. Estimates of the number of neurons in the P layers of the lateral geniculate nucleus of age-matched control macaques ranged from 1.0 to 1.3 x 10(6), while the number of neurons in SIV infected macaques ranged from 0.8 to 1.1 x 10(6), reflecting neuron loss of up to 28%. Neuron loss was not observed in the magnocellular layer. The total number of glia and glial density were unchanged. Loss of neurons in the lateral geniculate nucleus was correlated with the pattern of neuropathological changes. Neuron loss was most severe in animals with encephalitis concentrated in the brain stem and subcortical white matter and was less apparent in animals with diffuse encephalitis. Neuron loss in the lateral geniculate nucleus did not explain changes observed in the visual evoked potential, which was severely affected in two animals which showed a loss of 24 and 26%, while it was normal in a third animal which showed neuron loss of 28%.

Animals↗

PubMed1998

Auditory brainstem responses in a Rhesus Macaque model of neuro-AIDS.

Nine rhesus macaques (Macaca mulatta) were inoculated with a combination of two passaged strains of SIVmac (R71 and 17E), both of which are known to be neurovirulent. Auditory brainstem responses (ABRs) were recorded at regular intervals from these animals both before and after inoculation. Increases in ABR peak and interpeak latency were observed corresponding to progression of SIV disease. Post-inoculation increases in latency were observed for all five peaks of the ABR and for interpeak intervals I-V and III-V. The largest increases in latency were associated with end-stage disease. Within 14 weeks of inoculation, all but two animals developed end-stage simian AIDS and were euthanized. Histopathological examination revealed multifocal lesions in the cerebral gray and white matter as well as in the auditory structures of the brainstem. In most animals, ABR changes were accompanied by evidence of underlying neuropathology. However, cases of severe neuropathology with no ABR abnormalities and vice versa were also noted. Though in a much shorter time frame, SIVmac R71/17E produced both physiological and histopathological abnormalities similar to those associated with HIV disease in humans. These results further support the SIVmac R71/17E infected rhesus macaque as an animal model of HIV related neurological disease in humans.

AIDS Dementia Complex↗

PubMed1997

Genomic organization, chromosomal mapping, and analysis of the 5' promoter region of the human MAdCAM-1 gene.

MAdCAM-1, the endothelial addressin cell adhesion molecule-1, interacts preferentially with the leukocyte beta7 integrin LPAM-1 (alpha4beta7), but also with L-selectin, and with VLA-4 (alpha4beta1) on myeloid cells, and serves to direct leukocytes into mucosal and inflamed tissues. Overlapping cosmid and phage lambda genomic clones were isolated, revealing that the human MAdCAM-1 gene contains five exons where the signal peptide, two Ig domains, and mucin domain are each encoded by separate exons. The transmembrane domain, cytoplasmic domain, and 3' untranslated region are encoded together on exon 5. The mucin domain contains eight repeats in total that are subject to alternative splicing. Despite the absence of a human counterpart of the third IgA-homologous domain and lack of sequence conservation of the mucin domain, the genomic organizations of the human and mouse MAdCAM-1 genes are similar. An alternatively spliced MAdCAM-1 variant was identified that lacks exon 4 encoding the mucin domain, and may mediate leukocyte adhesion to LPAM-1 without adhesion to the alternate receptor, L-selectin. The MAdCAM-1 gene was located at p13.3 on chromosome 19, in close proximity to the ICAM-1 and ICAM-3 genes (p13.2-p13.3). PMA-inducible promotor activity was contained in a 700 base pair 5' flanking fragment conserved with the mouse MAdCAM-1 gene including tandem NF-kB sites, and an Sp1 site; and in addition multiple potential AP2, Adh1 (ETF), PEA3, and Sp1 sites. In summary, the data establish that the previously reported human MAdCAM-1 cDNA does indeed encode the human homologue of mouse MAdCAM-1, despite gross dissimilarities in the MAdCAM-1 C-terminal structures.

Amino Acid Sequence↗

PubMed1997

Differential sensitivity of recombinant N-methyl-D-aspartate receptor subtypes to zinc inhibition.

Zinc has been shown to be present in synaptic vesicles of a subset of glutamatergic boutons and is believed to be core-leased with glutamate at these synapses. A variety of studies have suggested that zinc might play a role in modulation of excitatory transmission, as well as excitotoxicity, by inhibiting N-methyl-D-aspartate (NMDA)-type glutamate receptors. To further investigate the modulatory effects of zinc on NMDA receptors of different subunit compositions, we coexpressed the recombinant subunit NR1 with NR2A and/or NR2B in HEK 293 cells. In whole-cell patch-clamp recordings from these transfected cells, zinc inhibited peak glutamate-evoked current responses in a noncompetitive manner, but there were significant differences between the receptor subtypes in sensitivity to zinc inhibition. For NR1/NR2A, approximately 40% of the peak current was inhibited by zinc in a voltage-independent manner with an IC50 value of 5.0 +/- 1.6 nM and at a V(H) value of -60 mV; the remainder was blocked at a second, voltage-dependent site with an IC50 value of 79 +/- 18 microM. In contrast, NR1/NR2B currents showed nearly complete inhibition at a voltage-independent site with an IC50 value of 9.5 +/- 3.3 microM. Cells cotransfected with NR1, NR2A, and NR2B showed zinc sensitivity intermediate between that characteristic of NR1/NR2A and that of NR1/NR2B. Furthermore, zinc accelerated the macroscopic desensitization of both NR1/NR2A and NR1/NR2B in a dose-dependent manner, apparently independently of glycine-sensitive desensitization and Ca2(+)-dependent inactivation; maximal effects were to decrease desensitization time constants for NR1/NR2A by approximately 75% and for NR1/NR2B by approximately 90%. Differential modulation of NR1/NR2A and NR1/NR2B currents by zinc may play a role in regulating NMDA receptor-induced synaptic plasticity and neurotoxicity.

Animals↗

PubMed1997

Dopaminergic modulation of excitatory postsynaptic currents in rat neostriatal neurons.

Gamma-aminobutyric acid (GABA)-containing medium spiny neurons constitute approximately 90% of the neuronal population in the neostriatum (caudate and putamen) and play an important role in motor programming. Cortical glutamatergic afferents provide the main excitatory drive for these neurons, whereas nigral dopaminergic neurons play a crucial role in regulating their activity. To further investigate the mechanisms underlying the dopaminergic modulation of medium spiny neuronal activity, we tested the effect of dopamine receptor agonists on excitatory synaptic transmission recorded from these neurons. Excitatory postsynaptic currents (EPSCs) were evoked by local stimulation and recorded from medium spiny neurons in postnatal rat striatal thin brain slices. Recordings were made using the whole cell patch-clamp technique under voltage clamp and conditions that selected for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- and kainate-type glutamate receptor-mediated components of the EPSC. Incubation of slices in 10 microM dopamine resulted in a 33 +/- 11% (mean +/- SE) decrease in the amplitude of evoked EPSCs, an effect that developed during seconds. The relative variability in amplitude of dopamine's effects on medium spiny neuron EPSCs may reflect activation of different receptor subtypes with opposing effects. In contrast to the results with dopamine, incubation of slices in SKF 38393, a D1-type dopamine receptor selective agonist, resulted in dose-dependent potentiation of the medium spiny neuron EPSC that developed during several minutes. At a concentration of 5 microM, SKF 38393 resulted in a 29 +/- 4.5% increase in EPSC amplitude, an effect that was blocked by preincubation with the D1-selective antagonist, SCH 23390 (10 microM). On the other hand, 5 microM SKF 38393 had no apparent effect on medium spiny neuron currents activated by exogenous application of glutamate or kainate. However, because of the inherent limitations of rapid agonist perfusion in the brain slice preparation (caused by slow agonist diffusion and rapid glutamate receptor desensitization) and because of anatomic evidence that colocalizes D1 and glutamate receptors to medium spiny neuron dendrites, our results leave open the possibility that the effect of D1 receptor activation on the EPSC is mediated via modulation of postsynaptic glutamate receptor responsiveness. The significant potentiation by D1 receptor agonists of EPSC amplitude at the cortico-striatal medium spiny synapse that we observed, in part, may underlie the role of D1 receptors in facilitating medium spiny neuronal firing, with implications for understanding regulation of movement.

Animals↗

PubMed1996

Molecular cloning and characterization of human tissue inhibitor of metalloproteinase 4.

The tissue inhibitors of metalloproteinases (TIMPs) constitute a family of proteins, of which three members have so far been described. Using the expressed sequence tag sequencing approach, we have identified a novel TIMP-related cDNA fragment and subsequently cloned a fourth human TIMP (TIMP-4) from a human heart cDNA library. The open reading frame encodes a 224-amino acid precursor including a 29-residue secretion signal. The predicted structure of the new protein shares 37% sequence identity with TIMP-1 and 51% identity with TIMP-2 and -3. The protein has a predicted isoelectric point of 7.34. The open reading frame-directed expression of TIMP-4 protein in MDA-MB-435 human breast cancer cells showed metalloproteinase inhibitory activity on reverse zymography. By Northern analysis, only the adult heart showed abundant TIMP-4 transcripts with a 1. 4-kilobase predominant transcript band; very low levels of the transcripts were detected in the kidney, placenta, colon, and testes, and no transcripts were detected in the liver, brain, lung, thymus, and spleen. This unique expression pattern suggests that TIMP-4 may function in a tissue-specific fashion in extracellular matrix homeostasis.

Amino Acid Sequence↗

PubMed1996

Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line.

Excessive activation of glutamate receptors is thought to play a critical role in neuronal excitotoxicity. To compare the cytotoxic potential of different glutamate receptor subtypes and correlate receptor biophysical properties with cytotoxicity, we have expressed recombinant receptors in human embryonic kidney 293 (HEK-293) cells. Survival of transfected cells was analyzed under conditions of defined agonist concentration and exposure time. For HEK-293 cells transfected with N-methyl-D-aspartate (NMDA) receptors, the EC50 for NMDA-induced cytotoxicity was 300 microM. Experiments using ion substitution, or cells expressing mutant NMDA receptors with low calcium permeability, suggested that both calcium and sodium influx through NMDA receptors contributed to cytotoxicity. In contrast, cytotoxicity was not observed in cells transfected with calcium permeable alpha-amino 3-hydroxy-5-methyl-4-isoxazole propionate- or kainate-type glutamate receptors even at saturating agonist concentrations, unless inhibitors of agonist-dependent desensitization were included. These results directly demonstrate that calcium permeability and desensitization kinetics play important roles in determining the excitotoxic potential of different glutamate receptor subtypes.

2-Amino-5-phosphonovalerate↗

PubMed1996

Evans blue antagonizes both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors and modulates receptor desensitization.

The biphenyl derivative of 1,3-naphthalene disulfonic acid, known as Evans blue (EB), has been shown previously to specifically antagonize currents mediated by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors (1). In contrast, we demonstrate herein that EB potently inhibits glutamate-evoked currents mediated by the kainate-type receptor GluR6 (IC50 150 nM) as well as the AMPA-type receptor GluR1 (IC50 = 220 nM) in whole-cell patch clamp recordings from transfected human embryonic kidney 293 cells. In addition to diminishing GluR6-mediated peak current amplitude, EB significantly altered receptor desensitization by slowing the rate of onset by approximately 2-fold (1 microM EB), slowing the rate of recovery by approximately 2-fold (0.1 microM EB), and increasing the steady state to peak current amplitude ratio by approximately 50-fold (1 microM EB). Interestingly, relatively little EB inhibition of GluR6 currents was observed in recordings from cells pretreated with the lectin concanavalin A, which eliminates kainate receptor desensitization. Similarly, currents recorded from GluR1-transfected cells were also relatively insensitive to EB inhibition if desensitization was first blocked by cyclothiazide. Moreover, for both GluR6 and GluR1, EB inhibition of agonist-evoked current was largely reversed if transfected cells were subsequently exposed to concanavalin A or cyclothiazide, respectively. Although EB may not be as selective an antagonist as previously believed, the relationship between EB-induced peak current inhibition and effects on receptor desensitization may be useful in further elucidating structures or mechanisms involved in the rapid desensitization of AMPA- and kainate-type glutamate receptors.

Cell Line↗

PubMed1995

Neodymium:YAG laser treatment for hemorrhages under the internal limiting membrane and posterior hyaloid face in the macula.

BACKGROUND: A dense premacular hemorrhage may occur from proliferative diabetic retinopathy, a ruptured retinal artery macroaneurysm, or Valsalva retinopathy. Spontaneous clearing of the hemorrhage in Valsalva retinopathy usually occurs, taking several months. In diabetic retinopathy, a traction macular detachment may result as early as 5 weeks after the hemorrhage. In diabetic retinopathy or a macroaneurysm, only a fibrotic epiretinal membrane overlying the macula may develop. Observation or vitrectomy is the current way of managing a dense premacular hemorrhage. This study was undertaken to investigate an alternative, neodymium:YAG (Nd:YAG) laser membranotomy, in treating this type of hemorrhage. METHODS: Six eyes in six patients had Q-switched Nd:YAG laser treatment to open trapped hemorrhage overlying the macula. One to three laser membranotomies were performed. Colored fundus photographs were obtained before and after the hemorrhage was treated. In some cases, fluorescein angiography was done before the hemorrhage was treated. The size of the pretreated hemorrhage was estimated from the photographs and expressed in disc diameters. Patients with diabetes had panretinal laser before Nd:YAG laser membranotomy. RESULTS: The premacular bleeding originated from proliferative diabetic retinopathy in four eyes and from a retinal artery macroaneurysm in two. Average estimated area of the pretreated hemorrhage was 14 disc diameters. Five eyes had marked clearing of hemorrhage and rapid improvement of vision after Nd:YAG laser membranotomy. Average follow-up after laser treatment was 20 months. One diabetic eye required vitrectomy for rebleeding. One eye had little visual improvement due to pre-existing subretinal bleeding. CONCLUSION: Neodymium:YAG laser membranotomy seems helpful in rapid clearing of premacular hemorrhage in certain eyes. A randomized prospective study is needed to evaluate observation, vitrectomy, and Nd:YAG laser treatment of dense premacular hemorrhage.

Adult↗

PubMed1994

Transmembrane topology of the glutamate receptor subunit GluR6.

Ionotropic glutamate receptors mediate most rapid excitatory synaptic transmission in the mammalian central nervous system. These receptors are divided into alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), kainate, and N-methyl-D-aspartate receptors based on pharmacological and electrophysiological characteristics. Ionotropic receptor subunits are integral membrane proteins that have been proposed to have a large extracellular ligand-binding N-terminal domain, four hydrophobic transmembrane domains, and an extracellular C-terminal domain. In this study we have shown that both AMPA receptor subunits (GluR1-4) and kainate receptor subunits (GluR6/7) are glycosylated in adult rat brain; however, the kainate receptor subunits are glycosylated to a greater extent. Examination of the sequences of AMPA and kainate receptors revealed that kainate receptors have several additional consensus sites for N-linked glycosylation; interestingly, one of these is located in the proposed major intracellular loop of the receptor subunits. To test the proposed transmembrane topology model for these receptors, we have used site-specific mutagenesis of the GluR6 subunit to remove the consensus glycosylation site located within the proposed intracellular loop. Mutagenesis of this site demonstrates that it is glycosylated in transiently transfected human embryonic kidney cells, which express functional kainate receptors. Since N-linked glycosylation has only been found to occur on extracellular domains of plasma membrane proteins, these results suggest that the proposed transmembrane topology model for the glutamate receptor subunits is incorrect. Combining these results with other recent data, we have proposed an alternative transmembrane topology model.

Amino Acid Sequence↗

PubMed1994

Glutamate receptor modulation by protein phosphorylation.

Glutamate-gated ion channels mediate most excitatory synaptic transmission in the mammalian central nervous system and play major roles in synaptic plasticity, neuronal development, and in some neuropathological conditions. Recent studies have suggested that protein phosphorylation of neuronal glutamate receptors by cyclic AMP-dependent protein kinase (PKA) and protein kinase C (PKC) may regulate their function and play a role in some forms of synaptic plasticity. To test whether these protein kinase effects are due to direct phosphorylation of the receptors and to further examine the sites and mechanisms by which the receptors are modulated, we transiently expressed recombinant glutamate receptors in HEK-293 cells and studied their biochemical and biophysical properties. Our results indicate that the kainate-preferring receptor GluR6 is phosphorylated by PKA, primarily on a single serine in the proposed major intracellular loop. Moreover, using the whole cell patch clamp recording technique, we have shown that phosphorylation at this site increases the amplitude of the GluR6-mediated glutamate current without significantly altering its dose-response, current-voltage relation or desensitization kinetics. In other experiments, we have demonstrated that the NMDA receptor subunit NR1 is phosphorylated by PKC on several distinct sites, and most of these sites are located within a single alternatively spliced exon in the C-terminal domain. These findings suggest that RNA splicing can regulate NMDA receptor phosphorylation and that, contrary to the previously proposed membrane topology model, the NR1 C-terminus is intracellular. Furthermore, in HEK-293 cells co-transfected with NR2A and NR1 subunits containing the C-terminal exon with the PKC phosphorylation sites, our preliminary studies indicate that the NMDA-evoked current is potentiated by intracellular PKC. We are currently examining PKC effects on the NMDA-evoked current responses of mutant NR1 receptors that lack the C-terminal phosphorylation sites. These studies provide evidence that glutamate receptors are directly phosphorylated and functionally modulated by protein kinases. Moreover, by identifying phosphorylation sites within the receptor proteins, our results provide information about the structure and membrane topology of these receptors.

Animals↗

PubMed1993

Phosphorylation and modulation of recombinant GluR6 glutamate receptors by cAMP-dependent protein kinase.

Glutamate-gated ion channels mediate most excitatory synaptic transmission in the central nervous system and play crucial roles in synaptic plasticity, neuronal development and some neuropathological conditions. These ionotropic glutamate receptors have been classified according to their preferred agonists as NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and KA (kainate) receptors. On the basis of sequence similarity and pharmacological properties, the recently cloned glutamate receptor subunits have been assigned as components of NMDA (NMDAR1, 2A-D), AMPA (GluR1-4) and KA (GluR5-7, KA1, KA2) receptors. Protein phosphorylation of glutamate receptors by protein kinase C and cyclic AMP-dependent protein kinase (PKA) has been suggested to regulate their function, possibly playing a prominent role in certain forms of synaptic plasticity such as long-term potentiation and long-term depression. Here we report that the GluR6 glutamate receptor, transiently expressed in mammalian cells, is directly phosphorylated by PKA, and that intracellularly applied PKA increases the amplitude of the glutamate response. Site-specific mutagenesis of the serine residue (Ser 684) representing a PKA consensus site completely eliminates PKA-mediated phosphorylation of this site as well as the potentiation of the glutamate response. These results provide evidence that direct phosphorylation of glutamate receptors modulates their function.

Amino Acid Sequence↗

PubMed1993

Phosphorylation of amino acid neurotransmitter receptors in synaptic plasticity.

The precise regulation of synaptic efficacy in the mammalian central nervous system is fundamental for learning, memory, motor control and sensory processing, as well as synaptogenesis. Currently, the molecular mechanisms underlying synaptic plasticity involved in these crucial processes are topics of intense investigation. The modulation of neurotransmitter receptors has received considerable attention, since these receptors mediate signal transduction at the postsynaptic membranes of chemical synapses. Over the past several years, evidence has suggested that protein phosphorylation of neurotransmitter receptors is a common mechanism for the regulation of receptor function. In this reaction, protein kinases catalyse the transfer of a highly charged phosphate moiety from ATP to serine, threonine or tyrosine residues of a neurotransmitter receptor, thereby altering the charge and/or conformation of the receptor and regulating its function. Phosphorylation of neurotransmitter receptors is reversible, can occur rapidly, and might result in prolonged changes in receptor function. Thus, this modification might play an important role in both short- and long-term changes in synaptic transmission.

Animals↗

PubMed1990

Fluorescein angiography using ultra-high speed film.

We used a new ultra-high speed black-and-white film, Kodak T-MAX P3200 (ASA 3200), in routine fluorescein angiography on 51 patients and one normal volunteer. The increased film speed permitted a lower flash intensity than with other available films. Photophobia was improved subjectively in 25% of patients who had undergone prior angiography at a higher flash setting. In addition, patient cooperation was improved as evidenced by a decrease in the frequency of photographic artifacts and uninterpretable photographs compared with prior angiograms done using Kodak TRI-X Pan film (ASA 400). T-MAX yielded excellent resolution, but its grain size was slightly greater than that of TRI-X Pan. The use of ultra-high speed film and reduced light intensity may benefit the patient and improve photographic quality in some individuals with photophobia.

Evaluation Studies as Topic↗