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R A Philibert

Publications and source records attributed to R A Philibert.

25 records · Page 2Linked to original sources
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

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↗
PubMedDate not supplied

The genomic structure and developmental expression patterns of the human OPA-containing gene (HOPA).

We determined the genomic organization of the human OPA-containing gene (HOPA) and characterized its developmental expression. The gene encoding HOPA, which contains a rare polymorphism tightly associated with non-specific mental retardation, is 25 kb in length and consists of 44 exons. A promoter scan analysis demonstrates two possible transcription initiation sites without TATA boxes upstream from the putative translation initiation start site. Several informative polymorphisms are evident in the sequence including a large pentanucleotide repeat. Northern blot analysis of the gene transcript and its murine orthologue, MOPA-1, demonstrates that only one transcript is expressed throughout the soma and the CNS, and that the transcript is highly expressed during early fetal development. We conclude that the delineation of the function of the HOPA gene locus merits further study.

Blotting, Northern↗