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Biomedical subjects

R E Paulsen

Publications and source records attributed to R E Paulsen.

At least 19 recordsLinked to original sources
PubMed2001

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF.

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Detection of high molecular weight DNA fragments characteristic of early stage apoptosis in cerebellar granule cells exposed to glutamate.

In cerebellar granule cells a rapid necrotic cell death has been observed during and immediately after glutamate exposure, followed by a delayed apoptotic type of neuronal death in a subpopulation of the surviving neurons. In some experimental models the DNA fragmentation characteristic of apoptosis is readily detected. In other systems apoptosis may occur only in a limited number of cells, rendering DNA fragmentation undetectable using conventional DNA-staining techniques (e.g., ethidium bromide). We have used a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. This method is based on the introduction of thymine dimers into DNA after separation by pulse field gel electrophoresis, followed by detection with thymine dimer specific antibodies. Applying this method to cerebellar granule cells in culture, we detected an increase in the amount of HMW DNA fragments characteristic of apoptosis as early as 4 h after glutamate exposure. The N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK801 protected against the fragmentation, whereas no protection was observed using the non-NMDA-receptor antagonist CNQX.

Animals↗
PubMed1999

3-Nitropropionic acid: an astrocyte-sparing neurotoxin in vitro.

3-Nitropropionic acid (NPA), an inhibitor of succinate dehydrogenase, is dietary neurotoxin. It is not known if neurons and astrocytes differ in their vulnerability to NPA, therefore, we investigated its toxicity in primary cultures of cerebellar granule cells and astrocytes. NPA inhibited succinate dehydrogenase and tricarboxylic acid cycle activity to the same degree in neurons and astrocytes. Even so NPA acid was 16 times more toxic to neurons than to astrocytes (LC50: 0.7 and 11 mM, respectively). The neurotoxicity of NPA was mediated by NMDA-receptor activation, calcium influx, and formation of reactive oxygen species, as revealed by the protective effect of NMDA-receptor blockade, the accumulation of 45Ca, and the protective effect of N-t-butyl-alpha-phenylnitron (PBN), a scavenger of reactive oxygen species. Cytotoxic concentrations of NPA caused a reduction in the intracellular level of glutathione, which probably contributed to the oxidative damage in both neurons and astrocytes. The relative resistance of astrocytes to NPA appeared to be related to their low tricarboxylic acid cycle activity (5%-10% of that in neurons) and to the inability of NPA to cause astrocytic calcium overload. We conclude that NPA acid predominantly is an astrocyte-sparing neurotoxin.

Amino Acids↗
PubMed1997

Infusion of D-cycloserine into temporal-hippocampal areas and restoration of mnemonic function in rats with disrupted glutamatergic temporal systems.

Partial transections of the fiber connections between the temporal cortex and the lateral entorhinal cortex at a site of the white matter corresponding to the perirhinal cortex result in impaired visual memory accompanied by reduced concentrations of glutamate in both the temporal cortex and lateral entorhinal cortex. Intraperitoneal administration of the glycinergic receptor agonist D-cycloserine produces complete restoration of memory function, as measured by a brightness discrimination task in rats with temporal cortex/lateral entorhinal cortex transections. The purpose of the present study was to identify in which brain structures the compensatory activity might take place. The results show that infusion of cycloserine into either the temporal cortex or lateral entorhinal cortex fully ameliorated the impairment of temporal cortex/lateral entorhinal cortex lesions, whereas infusion into the hippocampal region caused only a mild improvement of the retention performance. Infusion of cycloserine into the frontal cortex or saline into the temporal cortex or lateral entorhinal cortex had no ameliorating effects on the memory dysfunction of rats bearing temporal cortex/lateral entorhinal cortex transections. It is concluded that the temporal cortex, lateral entorhinal cortex and perirhinal cortex are highly critical in forming visual memory.

Analysis of Variance↗
PubMed1997

Chronic pre-explant blockade of the NMDA receptor affects survival of cerebellar granule cells explanted in vitro.

Rat pups were treated with the competitive NMDA antagonist CGP 39551 with daily injections on postnatal days 1 to 8. Cultures of cerebellar granule cells were prepared from these pups as well as from control pups of the same age and body weight. Granule neurons explanted from CGP 39551-treated pups showed a decreased survival, both at short (2 days) or longer (8 days) time in vitro, irrespective of trophic (high K+) or non-trophic (low K+) culture conditions. Granule cells from control or treated animals underwent apoptotic death when shifted from high to low K+ after maturation in vitro and were rescued by lithium (5 mM). Under the same experimental conditions, the block of protein synthesis through cycloheximide only partially protected from apoptotic death granule neurons from control rats, whereas it was totally effective on cultures derived from CGP 39551-treated animals. This suggests that a different balance between apoptotic/necrotic cell death may be the result of the same experimental conditions in the two types of cultures. Finally, the acquisition of excitotoxic sensitivity to glutamate and the protection given by MK-801 were the same in both types of cultures. The present results demonstrate that the previous block of the NMDA receptor negatively affects the subsequent survival of granule cells once they are explanted in vitro, whereas some features related to the maturation of these neurons in vitro are not impaired.

2-Amino-5-phosphonovalerate↗
PubMed1996

Inhibition of free radical production or free radical scavenging protects from the excitotoxic cell death mediated by glutamate in cultures of cerebellar granule neurons.

Glutamate kills sensitive neurons through several steps downstream to receptor activation: increased free Ca2+ levels, activation of various enzymes and accumulation of reactive oxygen species (ROS). We have evaluated in a well established model of neuronal cultures the neuroprotective effects of blocking these mechanisms, either singularly or by combining multiple enzyme inhibition and/or ROS scavenging. In vitro cultures of cerebellar granule cells exposed to a toxic concentration of glutamate (100 microM for 15 min in the absence of Mg2+) combined with several pharmacological treatments. Inhibition of nitric oxide synthase (NOS) and phospholipase A2 (PLA2) were effective in decreasing cell death and the combined treatments showed some degree of additivity. By contrast, inhibition of xanthine oxidase (XO) with allopurinol was uneffective. Antioxidants (in particular vitamin e or vitamin E analogs). protected neurons up to more than 50%. A synergistic effect was demonstrated by the combination of vitamin E and C. On the other hand, antioxidants did not increase the protection granted by enzyme inhibitors, suggesting that they act downstream to NOS and PLA2. In conclusion, NOS and PLA2 activated by Ca2+ influx give rise to reactive oxygen species whose deleterious action can be counteracted either by inhibiting these enzymes or by scavenging the excess of free radicals produced by them. Finally, a moderate protection was obtained by blocking protein synthesis with cycloheximide, suggesting a partial contribution of apoptotic mechanisms to the excitotoxic cell death.

Animals↗
PubMed1996

A microdialysis study in rat brain of dihydrokainate, a glutamate uptake inhibitor.

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K+-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K+-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca(2+)-dependency, whereas 50% of the increase in glutamate efflux during K+-depolarization was Ca(2+)-dependent. The Ca(2+)-dependent efflux is related to transmitter release, whereas the Ca(2+)-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K+-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.

Animals↗
PubMed1996

Contributing mechanisms for cysteine excitotoxicity in cultured cerebellar granule cells.

Several possible mechanisms for cysteine toxicity on rat cerebellar granule cells were studied and compared with the excitotoxic effect of glutamate. It was shown that the excitotoxic potency of both cysteine and glutamate increased in the presence of elevated concentrations of bicarbonate or increased pH. Pharmacological studies showed that the cysteine toxicity was specifically coupled to the NMDA receptor, whereas the glutamate toxicity was mediated to a smaller extent also by non-NMDA receptors. Treatment of cerebellar granule cells with cysteine led to an increased extracellular level of glutamate. In addition, cysteine sensitized NMDA receptors by reducing disulfide bonds in the receptor to sulfhydryl groups. A mechanism for cysteine excitotoxicity may therefore be formation of cysteine-sensitized NMDA receptors that are stimulated either by cysteine and/or by endogenous glutamate. This mechanism may also be important for the effects observed during regulated physiological release of cysteine.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1995

Activation of a reporter gene responsive to NGFI-B in cultured neurons and astrocytes.

NGFI-B is an immediate early gene and orphan member of the nuclear receptor superfamily. It is induced in several tissues, including brain, and in cultured cerebellar granule cells in response to different stimuli. Since both the induction of its mRNA as well as the level and function of its gene product are under the control of the inducing stimulus, we wanted to study the final outcome of the stimulus, i.e., transcriptional activity, by means of a specific, artificial reporter gene in cultured CNS cells. Cultured cerebellar granule cells and astrocytes were transfected with an NGFI-B responsive reporter gene to study the role of NGFI-B as a transcriptional activator after stimulation of the protein kinase A and C pathways. In both cell types, stimulation of either protein kinase A or C with forskolin (10 microM) or phorbol 12-myristate 13-acetate (0.1 microM), respectively, gave up to fivefold induction of the reporter gene. In the granule cells a combined treatment gave a strong synergistic induction of the reporter gene. The astrocytes showed only weak synergy, indicating cell-specific regulation of the target gene by the two kinases.

Animals↗
PubMed1995

Distribution of morphine 6-glucuronide and morphine across the blood-brain barrier in awake, freely moving rats investigated by in vivo microdialysis sampling.

Microdialysis was used to sample morphine 6-glucuronide (M6G) and morphine in striatal extracellular fluid after systemic administration in awake, freely moving rats. Morphine or M6G (25-67 mumol/kg) was given subcutaneously, and blood and striatal dialysate were sampled repeatedly during 120 min. Blood samples were obtained by indwelling catheters in the inferior vena cava. Opiates in serum or brain dialysate were analyzed with high-performance liquid chromatography. The functional intactness of the blood-brain barrier was verified by the use of sodium technetate (Na99mTcO4). The fractional penetration into the brain of morphine and M6G was approximately 350- and 90-fold higher than that of Na99mTcO4, respectively, with a relative difference in the transfer of morphine and M6G of about 4. No hydrolysis of M6G to morphine was detected. Striatal dialysate-to-serum ratios of M6G did not differ after 25 or 67 mumol/kg. Serum AUC0-120 min was 10 times higher for M6G than for morphine. This reflects both a smaller volume of distribution (Vd) for M6G and a decreased rate of elimination compared with morphine. The median t1/2 from serum was 36 and 32 min for morphine and M6G, respectively. The striatal dialysate AUC0-120 min of M6G was 2.9 times greater than that of morphine after an equimolar subcutaneous dose. Dialysate tmax was delayed approximately 40 min relatie to serum tmax for both drugs, and the median t1/2 from the dialysate was 82 and 48 min for M6G and morphine, respectively. These results represent direct evidence for the penetration of M6G into the brain after systemic administration to living rats.

Animals↗
PubMed1994

Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA.

Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol-and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar granule cells acutely treated with TPA. TPA had no effect on basal, GTP gamma S-, NaF-, and calcium-stimulated phospholipase C when compared with membranes prepared from vehicle-treated cells. The effects of pulsing with TPA (30-min pulse, 10 nM) on agonist-stimulated PI hydrolysis were studied 1, 3, and 5 or 6 d after TPA treatment. TPA treatment results in a statistically significant decrease in glutamate-stimulated PI hydrolysis, and a slight reduction of carbachol-stimulated PI hydrolysis when compared to temporally matched controls. Measurements in membranes prepared from TPA-treated vs control cells 1, 3, and 5 d after treatment showed that calcium- and NaF-stimulated phospholipase C activity was significantly decreased at all days tested, whereas GTP gamma S-stimulated phospholipase C activity was significantly decreased only at d 3. These data demonstrate differences in the acute vs long-term effects of TPA treatment on agonist-stimulated PH hydrolysis, and suggest that the acute effects may be mediated at the level of the receptor, whereas long-term effects of TPA on PI hydrolysis may be mediated by deficits in effector function.

Animals↗
PubMed1993

Changes in evoked potentials and amino acid content during fluorocitrate action studied in rat hippocampal cortex.

Fluorocitrate inhibits the glial tricarboxylic acid cycle and thereby the synthesis of glutamine, which is the main precursor for transmitter glutamate. We investigated the possibility that there is a functional correlate to fluorocitrate action by recording evoked field potentials in rat hippocampal slices. The excitatory postsynaptic potential (field-EPSP) was markedly depressed after 7-8 h of fluorocitrate action. The population spike was also reduced, but a major part of the reduction may be the result of weaker synaptic activation rather than reduced excitability of the postsynaptic cells. The activity of thin unmyelinated fibres was only slightly affected. Preceding the changes in the field-EPSP there was a decrease in the glutamine content in the fluorocitrate treated slices relative to controls. Only a small decrease in tissue glutamate was seen concomitantly with the synaptic failure, probably because the transmitter pool of glutamate in those fibres stimulated makes little contribution to the total tissue glutamate.

Adenosine Triphosphate↗
PubMed1993

Evidence using in vivo microdialysis that aminotransferase activities are important in the regulation of the pools of transmitter amino acids.

The effect of aminooxyacetic acid (AOAA), an inhibitor of pyridoxal phosphate-dependent enzymes (including the aminotransferases), on the K(+)-evoked release of amino acids was studied during microdialysis of neostriatum in anesthetized rats. K(+)-evoked (100 mM) release of aspartate, glutamate, and GABA was inhibited by 74%, 70%, and 63%, respectively, by 20 mM Mg2+ and are therefore reflecting release from the transmitter pools of these amino acids. Treatment with AOAA decreased the K(+)-evoked release of aspartate, glutamate, and GABA instantly, with a delayed decrease in the efflux of glutamine and alanine, arguing that the synthesis of transmitter amino acids in particular is sensitive to the activity of pyridoxal phosphate-dependent enzymes. Interestingly, GABA release increased severalfold following the initial decrease, probably reflecting inhibition by AOAA on GABA aminotransferase, the enzyme most sensitive to inhibition by AOAA, and responsible for enzymatic inactivation of transmitter GABA.

Alanine↗
PubMed1992

Memory dysfunction following disruption of glutamergic systems in the temporal region of the rat: effects of agonistic amino acids.

It has previously been shown that disruptions of fiber connections between the temporal cortex (TC) and the lateral entorhinal cortex (LEC) in rats result in severely impaired retention of a simultaneous brightness discrimination task. This memory impairment is accompanied by reduced high affinity D-aspartate uptake in both TC and LEC. The purpose of this study was to investigate whether systemic administration of glutamergic agonists might ameliorate the mnemonic dysfunction seen to follow TC/LEC transections. The results from Experiment 1 show that agonists acting selectively at the NMDA receptors (NMDA and glycine) or the quisqualate receptors (AMPA) produced complete amelioration of the memory deficit. Injection of kainic acid only produced a slight improvement of memory. The results from Experiment 2 show that the positive effects of agonists are probably not attributable to peripheral adrenergic mechanisms, because blockade of sympathetic terminal release did not prevent mitigating effect of glycine. The results are discussed in terms of possible central nervous mechanisms interfered with by the various agonists.

Amino Acids↗
PubMed1992

Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B.

NGFI-B is an early response gene which encodes a protein that has strong homology with nuclear receptors in the DNA binding domain and in carboxyl-terminal domains responsible for ligand binding and regulation of transcriptional activity. Previously, we have demonstrated that NGFI-B is transcriptionally active in cells grown in vitro in the absence of exogenously added ligand. However, the ligand for NGFI-B does not appear to be a component of cell culture medium, as NGFI-B remained active when expressed in cells grown in medium lacking phenol red, serum, essential vitamins, or essential amino acids. To define the transactivation domains, a mutational analysis was conducted which revealed that a serine/threonine-rich area of 18 amino acids within the amino terminus, termed TAB-1, is an important transcriptional activation domain. The mutation of two adjacent serine and threonine residues within TAB-1 significantly decreased transactivation by NGFI-B. An examination of the role of the carboxyl terminus in regulating NGFI-B transcriptional activity revealed that, in accordance with other nuclear receptors, mutants lacking portions of the carboxyl terminus had greatly decreased activity. The similarity with other receptors was further supported by studies with the mutant B delta 414-597 which encodes a fully active, truncated receptor analogous to a hormone-independent, constitutively active glucocorticoid receptor truncation mutant. This NGFI-B truncation mutant had activity similar to wild type NGFI-B in a number of mammalian cell lines; however, in contrast, it was 8-fold more active than the wild type receptor in the Drosophila S2 cell line, suggesting that insect cells either lack the NGFI-B ligand or obligatory accessory factors.

3T3 Cells↗
PubMed1992

Participation of non-zinc finger residues in DNA binding by two nuclear orphan receptors.

Steroid-thyroid hormone receptors typically bind as dimers to DNA sequences that contain repeated elements termed half-sites. NGFI-B, an early response protein and orphan member of this receptor superfamily, binds to a DNA sequence that contains only one half-site (5'-AAAGGTCA-3'). A domain separate from the NGFI-B zinc fingers, termed the A box, was identified and is required for recognition of the two adenine-thymidine (A-T) base pairs at the 5' end of the NGFI-B DNA binding element. In addition, a domain downstream of the zinc fingers of the orphan receptor H-2 region II binding protein, termed the T box, determined binding to tandem repeats of the estrogen receptor half-site (5'-AGGTCA-3').

Amino Acid Sequence↗