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G R Dutton

Publications and source records attributed to G R Dutton.

At least 19 recordsLinked to original sources
PubMed1992

Neuronal origins of K(+)-evoked amino acid release from cerebellar cultures.

Neuronal cultures from rat cerebellum consisting of approximately 90% glutamatergic granule neurons, 5-7% GABAergic inhibitory interneurons, and 3-5% glial cells, were treated for four days with 50 microM kainic acid (KA) to determine the cellular origin of released endogenous neuroactive substances. KA, known to be selectively toxic to GABAergic neurons, caused an estimated 80% decrease in glutamic acid decarboxylase (GAD) immunofluorescence. Furthermore, K(+)-stimulated release of GABA decreased to 20% of control values, and did not return to control levels in cultures "recovered" two days in KA-free media, suggesting the loss of inhibitory interneurons. Similarly, adenosine and taurine showed decreased K(+)-stimulated release, which was unrecoverable when KA was removed from the medium. K(+)-stimulated release of glutamate and aspartate also decreased by 50% and 70%, respectively, after chronic KA treatment. In contrast, however, this release returned to control levels in recovered cultures. All decreases in K(+)-stimulated release were prevented by concurrent treatment with KA and the KA antagonist 6-cyano-6-nitroquinoxaline-2,3-dione (CNQX), indicating that a receptor-mediated mechanism was involved. We conclude that, in these cultures, most of the K(+)-stimulated release of adenosine and taurine originates from the GABAergic interneurons, the basket and stellate cells, which are selectively killed by the KA treatment. The data also strongly suggest that glutamate and aspartate, the levels of which recover after KA treatment, originate mainly from the granule neurons.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1991

Aluminum chloride stimulates the release of endogenous glutamate, taurine and adenosine from cultured rat cortical astrocytes.

Primary astrocyte cultures derived from neonatal rat cerebral cortex were treated for 5 min with 0.5 mM or 5.0 mM AlCl3, and the incubation medium was analyzed by HPLC for the content of released glutamate (Glu), taurine (Tau), serine (Ser) and the nucleoside adenosine (Ade). At 0.5 mM, AlCl3 stimulated Tau release to about 170% of basal levels, but did not affect the release of the other compounds. Treatment with 5.0 mM AlCl3 enhanced the release of Tau, Glu and Ade, to 800%, 1000% and 250%, respectively, but decreased the release of Ser to 70% compared to basal levels. The enhanced release of these neuroactive compounds from astrocytes may contribute to changes in neural transmission known to accompany exposure to aluminum.

Adenosine↗
PubMed1991

K(+)-stimulated amino acid release from cultured cerebellar neurons: comparison of static and dynamic stimulation paradigms.

The release of several endogenous amino acids and adenosine from rat cerebellar neuronal cultures following elevated K+ exposure in the presence and absence of added Ca2+ was studied. The amino acids aspartate (ASP), glutamate (GLU) and GABA were released from the cultures in a dose- and Ca(2+)-dependent manner. Taurine (TAU) and the nucleoside adenosine (ADN) efflux rates were dose-dependent but Ca(2+)-independent, and basal levels increased in the absence of Ca2+. The K+ depolarization induced release of serine (SER), alanine (ALA) and proline (PRO), was not dose-dependent and in the absence of extracellular Ca2+ (with added Mg2+) higher basal release of SER and ALA, but not PRO, was noted. These findings demonstrate that in addition to known cerebellar neurotransmitters, other neuroactive and neutral amino acids are released from cultured cerebellar neurons in response to K+ depolarization. Their observed efflux suggests they may have as yet unidentified roles in neuronal function with different classes of efflux corresponding to: neurotransmitter-type release (ASP, GLU, GABA), an osmoregulatory, possibly neuromodulatory-type release (TAU), a Ca(2+)-insensitive, possibly neuromodulatory-type release (ADN), and a depolarization-sensitive release (SER, ALA, PRO) of which SER and ALA are partially Ca(2+)-sensitive.

Amino Acids↗
PubMed1991

Astrocytes, not neurons, produce docosahexaenoic acid (22:6 omega-3) and arachidonic acid (20:4 omega-6).

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.

Animals↗
PubMed1991

Astrocyte taurine.

The evidence presented, together with the lack of solid evidence for a specific receptor site, strongly suggests that taurine does not act as a traditional neurotransmitter in the CNS. In fact, the properties seen to be governing its efflux from both glial cells and neurons argue strongly in favor of a primary role in volume regulation. However, subsequent to its release into the extracellular space, it is possible that the inherent neuroactive properties (e.g., inhibitory neuromodulation and Ca(2+)-level modulation) may be important at the synapse, at the cell plasma membrane, and intracellularly in further directing the level of neuronal activity. Whether or not the levels of released taurine are great enough to sustain these effects has still to be determined.

Animals↗
PubMed1990

K(+)- and temperature-evoked taurine efflux from hypothalamic astrocytes.

Hypothalamic astrocytes in culture released taurine, a suspected inhibitory amino acid neurotransmitter/neuromodulator/osmoregulator, in response to isoosmotically increasing extracellular K+ in a dose-dependent fashion. In the absence of added Ca2+, basal release levels rose to approach those obtained after exposure to 60 mM K+ in the presence of 2.5 mM Ca2+, and were only partially lowered by the addition of 10 mM Mg2+. Stimulation with K+ (60 mM) did not further increase taurine efflux above the high basal levels seen in the absence of Ca2+. Under standard conditions complete replacement of Na+ with choline Cl had little effect on basal taurine release, but reduced K(+)-evoked (60 mM) efflux by 60%. The temperature dependence of the basal levels of taurine released from hypothalamic astrocytes was similar to that seen for cultured cerebellar astrocytes and neurons over the range 5-50 degrees C. Taurine release increased from 5 to 15 degrees C, remained constant between 15 and 33 degrees C, decreased between 33 and 37 degrees C and increased thereafter. The infection point of increased basal taurine release seen around 37 degrees C (most prominent in astrocytes), may be of physiological significance. Results presented also show that the ion (Na+, Ca2+ and K+) sensitivities of taurine efflux for cultured hypothalamic astrocytes are similar to those previously reported for cultured astrocytes from the cerebellum.

Animals↗
PubMed1990

Glutamate receptor agonists cause efflux of endogenous neuroactive amino acids from cerebellar neurons in culture.

Cultured neurons from rat cerebellum were used to examine the effects of glutamate receptor agonists on the release of endogenous amino acids and adenosine. Kainic acid exposure resulted in the release of glutamate, taurine, GABA and alanine in a dose- and calcium-dependent manner. Stimulation with quisqualic acid resulted in the dose- and calcium-dependent release of GABA. N-Methyl aspartic acid did not elicit the release of any neuroactive amino acids. These findings suggest that N-methyl aspartate receptors are not coupled to transmitter release in these cultures, and that kainate and quisqualate receptors may have different neuronal distributions.

Amino Acids↗
PubMed1989

Dihydropyridines modulate K+-evoked amino acid and adenosine release from cerebellar neuronal cultures.

Partial depolarization of primary cerebellar neuronal cultures with K+ evoked the release of aspartate, glutamate, adenosine, serine, taurine, gamma-aminobutyric acid (GABA), alanine and proline. The dihydropyridine calcium channel agonist, BAY K 8644, significantly augmented the K+-induced release of adenosine, aspartate, glutamate and GABA, but not that of serine, taurine, alanine or proline. However, in all cases the dihydropyridine antagonist nifedipine decreased this BAY K 8644-enhanced, K+-evoked efflux to below control levels. Neither BAY K 8644 nor nifedipine alone affected basal efflux levels. The phenylalkylamine calcium channel antagonist, verapamil, was ineffective in antagonizing K+-evoked amino acid release except at very high concentration (100 microM). These findings suggest that L-type Ca2+ channels are present in both excitatory (glutamatergic granule cells) and inhibitory (GABAergic stellate and basket cells) neurons in these cultures, and that they appear to be involved in regulating the release of not only neuroactive amino acids, but also some neutral amino acids and adenosine.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Stimulus-coupled taurine efflux from cerebellar neuronal cultures: on the roles of Ca++ and Na+.

Primary cultures of cerebellar neurons obtained from 7-9-day-old rats and grown 7-9 days in vitro (DIV) were used to study the effects of Na+ and Ca++ on K+-evoked taurine release. These cultures, made up largely of granule neurons (90%) and inhibitory interneurons (5-7%), produced a dose-dependent, depolarization-evoked taurine release that was Ca++-dependent at 40 mM K+, and Ca++-independent at K+ concentrations above 40 mM. The dihydropyridine Ca++ channel agonist BAY K 8644 (1 microM) augmented 30 mM K+-evoked release, while the antagonist nifedipine (5 microM) abolished both the BAY K 8644- and K+-enhanced release. Depolarization with the Na+ channel agonist veratridine (50 microM) stimulated taurine efflux, which was completely blocked by pretreatment with tetrodotoxin (2 microM). However, 50 mM K+-evoked taurine release was not affected by tetrodotoxin pretreatment. Substitution of choline Cl for NaCl partially antagonized 50 mM K+-evoked release, and by itself, the Na+ ionophore monensin (50 microM) stimulated release. These results suggest that both K+-evoked and basal taurine release from primary cerebellar neuronal cultures are sensitive to the levels of both intracellular and extracellular Na+ and Ca++. In contrast to previous findings using cerebellar astrocytes, neuronal L-type Ca++ channels, but not voltage-dependent Na+ channels, also appear to be necessary. The implications of these results on taurine's status as a putative neurotransmitter are discussed.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

K+-evoked taurine efflux from cerebellar astrocytes: on the roles of Ca2+ and Na+.

The ionic requirements for K+-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca2+ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K+-induced efflux. The Ca2+-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K+. In addition, verapamil did not antagonize 50 mM K+-evoked efflux except at high, non-pharmacological concentrations (greater than 100 microM), and preincubation with 2 microM omega-conotoxin had no effect on 50 mM K+-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K+ stimulation, but did accelerate the onset of efflux by 2-4 min. Although 2 microM tetrodotoxin had no effect on K+-evoked release, replacing Na+ with choline abolished the taurine efflux seen in response to K+ stimulation. Together, these findings suggest that neuronal N- and L-type Ca2+- and voltage-dependent Na+-channels are not involved in the influx of Ca2+ which appears to be necessary for K+-evoked taurine efflux, and that in addition to Ca2+, extracellular Na+ is also required.

Animals↗
PubMed1988

Phorbol ester and dibutyryl cyclic AMP reduce content and efflux of taurine in primary cerebellar astrocytes in culture.

In 16-18 days in vitro (DIV) primary astrocyte cultures prepared from 7- to 9-day-old rats, 48 h exposure to 12,13-phorbol dibutyrate (PDBU) (1 microM) or dibutyryl cAMP (dbcAMP) (1 mM) reduced cellular taurine content, and both basal and 50 mM K+-evoked taurine efflux, but did not alter cellular glutamate or total protein content. Decreases in cellular taurine content first became apparent between 1 and 6 h and were maximal after 24 h. Treatment also rapidly altered astrocyte morphology to a more process-bearing form within 1 h. In contrast, fibroblast growth factor (FGF), epidermal growth factor (EGF), dbcGMP and alpha-PDBU did not affect cellular morphology, amino acid content or taurine efflux at any time tested. These findings suggest that, while protein kinase C translocation and adenylate cyclase activation may be only indirectly involved in the regulation of astrocyte morphology, long-term decreases in cellular taurine content and efflux may be the more direct result of these second messenger systems.

Animals↗
PubMed1988

Dose-dependent, K+-stimulated efflux of endogenous taurine from primary astrocyte cultures is Ca2+-dependent.

The K+-stimulated efflux of endogenous taurine from primary rat cerebellar astrocyte cultures prepared from 7-9-day-old rats was studied at 16-18 days in vitro using HPLC analysis. Taurine efflux was dose-dependent at K+ concentrations between 10 mM and 80 mM, with an EC50 of approximately 50 mM. Maximum stimulation of efflux above basal levels ranged from 56% at 10 mM K+ (204 pmol/min/mg protein) to 470% at 80 mM K+ (960 pmol/min/mg protein). Removal of Ca2+ from the buffer and the addition of either 1 mM EGTA or 10 mM Mg2+ abolished K+-stimulated efflux. Taurine efflux peaked and fell in parallel with the K+ concentration, but with an approximate lag of 3-5 min. The time course and amount of preloaded [3H]taurine released did not differ significantly from that seen for endogenous efflux. Basal taurine efflux varied inversely with the extracellular concentration of Ca2+ over the concentration range 0-5.0 mM. The observed Ca2+ dependence is consistent with a role for Ca2+ in the regulation of taurine release. Furthermore, taurine release from astrocytes in response to elevated K+ may reflect a neuromodulatory role for this amino acid in the CNS.

Animals↗
PubMed1987

Myelin-specific domain on the plasmalemma of oligodendroglia: differential expression in the rat and hypomyelinating mouse mutants jimpy and quaking.

Monoclonal antibody, 1A9, prepared against bovine white matter, recognizes a proteinaceous, myelin-specific domain in the CNS that is restricted to the surface of oligodendroglia in primary dissociated cell cultures. The antigen is not detected in the PNS or non-neural tissues. Antibody binding is abolished by heating, exposure to SDS and delipidation, indicating that a conformationally sensitive epitope is recognized. The antigen is present in tracts of developing white matter in rat cerebellum beginning at 5 days postnatally. In developing cultures of fetal rat brain the period of rapid onset for the phenotypic expression of 1A9 antigen is similar to that of galactocerebroside, corresponding to 2-4 postnatal days of age. The 1A9 antigen is not observed in white matter or cultured oligodendroglia of the hypomyelinating jimpy mutant mouse, but its expression is qualitatively normal in the quaking mutant. The possibility is raised that 1A9 may be the primary target of the jimpy mutation.

Animals↗
PubMed1987

HPLC analysis of putative amino acid neurotransmitters released from primary cerebellar cultures.

An HPLC method is described that measures amino acids (putative neurotransmitters and/or neuromodulators) released from primary, dissociated cerebellar cells maintained in monolayer culture. Precolumn derivatization with phenylisothiocyanate, followed by reverse phase chromatography with UV detection was used to quantitate the phenylcarbamyl amino acid derivatives in a chemically defined medium. Quantitation was linear, reproducible and sensitive to one picomole. This method is useful for the measurement of putative neurotransmitters GABA, glutamate, aspartate, taurine and adenosine, and can easily be modified to analyze other amino acids in physiological samples.

Amino Acids↗
PubMed1986

A monoclonal antibody to the endothelium of rat brain microvessels.

A rat brain fraction enriched with microvessels was used as the immunogen to produce mouse hybridoma cell lines secreting monoclonal antibodies. One of these antibodies, selected from 156 supernatants by enzyme-linked immunosorbent and immunofluorescent assays, reacted only with the endothelium of microvessels in the brain. The endothelium-specific antibody labelled the cytoplasm of microvascular endothelial cells, their luminal membranes, and an extracellular layer, the endocapillary coat, which covered the luminal surface of these cells. In the kidney, the antibody specifically stained the brush border of the proximal tubuli, and in the liver, the antibody specifically stained bile canaliculi. This demonstrates that 3 morphological structures with important transport functions, cerebral microvascular endothelium, brush border of kidney proximal tubuli, and liver bile canaliculi, express the same epitope.

Animals↗
PubMed1985

Extra-neural glial fibrillary acidic protein (GFAP) immunoreactivity in perisinusoidal stellate cells of rat liver.

The dendritic processes and perinuclear cytoplasm of stellate-shaped perisinusoidal cells in frozen sections of rat liver were specifically labeled with antisera raised independently to glial fibrillary acidic protein (GFAP), the major component of intermediate filaments in astrocytes. A liver protein co-migrating with authentic GFAP and immunoreactive with GFAP antisera was demonstrated with immunoblots of brain and liver extracts enriched in intermediate filament proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study presents yet another example of immunoreactivity to GFAP, or a highly similar protein localized outside the CNS, in cells of mesenchymal origin exhibiting some morphological features common to astroglia.

Animals↗
PubMed1983

A solid-phase beta-galactosidase ELISA for detecting and quantifying monoclonal antibody binding to dissociated cell cultures of postnatal rodent cerebellum.

A solid-phase, indirect beta-galactosidase-linked immunoassay (ELISA) is described for screening large numbers of monoclonal antibodies that recognize cell surface antigens of primary monolayer cerebellar cultures. Target cultures were prepared from perikaryal suspensions of postnatal rodent cerebellum seeded into poly-L-lysine pre-coated, flat-bottom microtiter wells and fixed with glutaraldehyde after growth in vitro. Hybridoma supernatants were then incubated on these cultures. After the addition of beta-galactosidase-linked anti-mouse IgG F(ab')2 fragments, antigen-positive supernatants were detected with the enzyme substrate o-nitrophenyl-beta-D-galactopyranoside. Using a monoclonal antibody specific for rat brain Thy-1 glycoprotein, this solid-phase ELISA was found to be useful in quantifying changes in the developmental expression of cerebellar surface antigens in these cultures.

Animals↗