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J H Neale

Publications and source records attributed to J H Neale.

At least 55 records · Page 3Linked to original sources
PubMed1986

High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture.

We examined the membrane action of the endogenous dipeptide and putative neurotransmitter N-acetylaspartylglutamate (NAAG) on the excitatory amino acid receptors of cultured mouse spinal cord neurons using electrophysiological methods. Responses to NAAG (1 microM-5 mM) were compared to those elicited by N-methyl-D-aspartate (1 microM-1 mM) and L-glutamate (0.5-500 microM). Under voltage clamp, concentration-response curves of agonist-evoked currents demonstrated that NAAG was much less potent than either L-glutamate or N-methyl-D-aspartate (NMDA), so that inward currents could be evoked only at NAAG concentrations above 300 microM. Analysis of the dipeptide by high-pressure liquid chromatography showed no evidence of contamination by excitatory amino acids, suggesting that NAAG has an intrinsic, although weak, neuroexcitatory action on spinal neurons. Previous studies have shown that activation of NMDA receptors produces a voltage-dependent response. The current-voltage relationship of responses evoked by NAAG was also voltage-dependent. The peptide-activated conductance decreased with hyperpolarization in the presence of extracellular Mg2+, such that little inward current could be evoked at a membrane potential of -80 mV. In addition, responses to NAAG were completely antagonized by 250 microM DL-2-amino-5-phosphonovaleric acid, a specific NMDA-receptor antagonist. Application of NAAG in Mg2+-free medium resulted in an inward current with a large increase in membrane current noise. The spectral density function of this current noise could be fitted with a single Lorentzian with a decay time constant near 5 msec and a calculated single-channel conductance of 50-60 pS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Immunohistochemical localization of the pro-opiomelanocortin-gene product, glycylglutamine, in the intermediate pituitary.

Glycylglutamine, the carboxyterminal sequence of beta-endorphin1-31, is produced as a free dipeptide during the posttranslational synthesis of beta-endorphin1-27. Antisera which recognize glycylglutamine were raised in rabbits and used for immunohistochemistry. With these antisera, glycylglutamine immunoreactivity was demonstrated in cells of the rat intermediate pituitary. In contrast, anterior pituitary cells, which exhibited beta-endorphin immunoreactivity, did not react with the anti-glycylglutamine sera. The conclusion that the antisera distinguished glycylglutamine immunoreactivity from beta-endorphin1-31 immunoreactivity is based upon cellular specificity, fixation requirements and blocking studies. The antisera demonstrated the differential expression of this dipeptide product of the proopiomelanocortin prohormone. The efficacy of carbodiimide as an immunohistochemical fixative for small molecules is also shown.

Animals↗
PubMed1985

Localization of elevated glutaminase immunoreactivity in small DRG neurons.

Glutamate has long been considered to be a neurotransmitter candidate in vertebrate spinal sensory nerve cells. We report here the first immunohistochemical evidence in support of this hypothesis. We find that up to 30% of the moderately small dorsal root ganglion neurons in the rat contain elevated levels of glutaminase immunoreactivity. This enzyme, which mediates the synthesis of glutamate from glutamine, is not found at these high levels in large diameter neurons of the same ganglia. In contrast, another enzyme associated with glutamate metabolism, aspartate aminotransferase, is rather uniformly distributed within neurons of the sensory ganglia. These data define a subpopulation of sensory neurons which appear to contain an elevated capacity to synthesize glutamate through the glutamine cycle and suggest that glutaminase immunoreactivity may be an indicator of glutamatergic function in some nerve cells.

Animals↗
PubMed1985

N-Acetylation of L-aspartate in the nervous system: differential distribution of a specific enzyme.

L-Aspartate N-acetyltransferase, a nervous system enzyme that mediates the synthesis of N-acetyl-L-aspartic acid, has been characterized. In the presence of acetyl-CoA, L-aspartate was acetylated 10-fold more efficiently than L-glutamate, and the acetylation of aspartylglutamate was not detectable. Within the nervous system, a 10-fold variation in the enzyme activity was observed, with the brainstem and spinal cord exhibiting the highest activity (10-15 pmol/min/mg tissue) and retina the lowest detectable activity (1-1.5 pmol/min/mg). No enzyme activity was detected in pituitary, heart, liver, or kidney. The enzyme activity was found to be membrane-associated and was solubilized by treatment with Triton X-100.

Acetyl Coenzyme A↗
PubMed1984

Morphine treatment increases clonidine binding in brain cell cultures.

Exposure of murine brain cells in culture to 75 microM morphine for 6 days produced an increase in the number of [3H]clonidine binding sites without affecting the apparent affinity. Similar treatment increased the binding of this alpha 2-adrenergic receptor agonist to cell cultures prepared from cerebral cortex but not to cultures of brain minus cortex or to neuroblastoma-glioma hybrid cells which possess both opiate and alpha 2-adrenergic receptors.

Animals↗
PubMed1984

Proteolytic degradation of neuronal benzodiazepine binding sites.

The pathway of breakdown of membrane-bound benzodiazepine binding sites has been examined with proteolytic enzymes. Photoaffinity labeled benzodiazepine receptors were degraded for varying amounts of time and at varying enzyme concentrations. The properties of fractions both remaining in the membrane and released into the supernatant were examined for their apparent molecular weight by SDS gel electrophoresis. Trypsin treatment converted the 46K subunits of the GABA/BDZ complex which bind 3H-flunitrazepam into 40K and 27.5K fragments which remained in the membrane and finally a small fragment which was released into the supernatant. An endogenous trypsin-like activity in the membrane fractions has similar proteolytic effects on the membrane bound receptor.

Animals↗
PubMed1983

Benzodiazepine receptor binding by membranes from brain cell cultures following chronic treatment with diazepam.

Murine brain cells differentiated for 19 days in culture before treatment for 5 days with 10 microM diazepam. Radioligand binding to the benzodiazepine receptors on membranes obtained from these cultures was determined by the filtration assay method. Decreased binding was observed in the membranes from treated cultures relative to untreated cells. However, this decrease appears to be due, at least in part, to competition from residual drug in the assay system despite extensive cell and membrane rinses. These data emphasize the difficulty in ascribing the mechanism of benzodiazepine tolerance observed clinically with chronic treatment to receptor down-regulation as determined by binding assays.

Animals↗
PubMed1982

Localization of immunoreactive dynorphin in neurons cultured from spinal cord and dorsal root ganglia.

Antisera specific for dynorphin were used to study the cellular distribution of opioid peptides in spinal cord and dorsal root ganglion neurons in dissociated cell culture. Radioimmunoassay of 4-wk-old cultures yielded levels of dynorphin immunoreactivity similar to those in adult rodent spinal cord. Immunohistochemistry showed staining confined to the perinuclear region of neuronal cell bodies. In contrast, enkephalin immunoreactivity was found in extensive neurite fields as well as in neuronal perikarya. Opioid peptide immunoreactivity was observed in approximately equal to 5% of the spinal cord neurons either with dynorphin or enkephalin antiserum. No substantial increase in the number of reactive cells was observed when the two sera were applied simultaneously. These results suggest that the perinuclear region of opioid spinal cord neurons in culture contains peptide with an amino acid sequence similar to that of the midportion of dynorphin, whereas the neurites appear to contain smaller peptides related to NH2-terminal fragments of dynorphin. By using simple morphological criteria, spinal sensory neurons can be identified in these cell cultures and in cultures prepared from dorsal root ganglia without spinal cord. Approximately 1-2% of these ganglion cells showed intense immunostaining with an affinity-purified dynorphin antiserum. An additional few percent of the sensory neurons showed less intense opioid immunoreactivity. This result extends the observations of opioid peptides one step further along the pathway that processes sensory information.

Animals↗
PubMed1980

Comparative analysis of rapidly transported axonal proteins in sensory neurons of the frog and rat.

35S-labeled proteins carried by fast axonal transport in sciatic sensory axons of bullfrog and rat were separated electrophoretically on discontinuous polyacrylamide gradient slab gels. In contrast to the previously reported similarity in the electrophoretic profiles of rapidly transported proteins from functionally different neurons, we have found that there is very little correspondence in the profiles of these proteins in functionally similar neurons from two widely studied species. We also found very little correspondence between the two species in the profiles of locally synthesized sciatic nerve protein. The results demonstrate the difficulty inherent in comparing the electrophoretic profiles obtained using these two model systems for the study of rapidly transported axonal proteins. In particular, relationships between the major rapidly transported proteins in the two species could not be analyzed with this technique.

Animals↗
PubMed1978

Enkephalin-containing neurons visualized in spinal cord cell cultures.

Neuronal cells, axons, and terminals containing immunoreactive enkephalin have been visualized in cultures of dissociated fetal spinal cord. These cultures may provide a valuable system in which to explore the effects of chronic drug treatment on the physiology of enkephalin-containing cells and their interactions with other cells.

Animals↗
PubMed1978

Opiate peptide modulation of amino acid responses suggests novel form of neuronal communication.

Mouse spinal neurons grown in tissue culture were used to study the electrophysiological pharmacology of the opiate peptide leucine-enkephalin. Enkephalin depressed glutamate-evoked responses in a noncompetitive manner independent of any other effects on membrane properties. The results demonstrate a neuromodulatory action of opiate peptide functionally distinct from the conventional neurotransmitter class of operation.

Cells, Cultured↗
PubMed1977

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium.

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, alpha-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.

Acetyltransferases↗