Javier Ortiz-Caro

Comparison of the effects of forskolin and dibutyryl cyclic AMP in neuroblastoma cells: evidence that some of the actions of dibutyryl cyclic AMP are mediated by butyrate.

Abstract: We have compared the effects of forskolin, N6,2′‐O‐dibutyryladenosine 3′:5′‐cyclic monophosphate (dibutyryl cyclic AMP, Bt2‐cAMP), and butyrate on several aspects of neuroblastoma cell physiology. The morphology of Neuro 2A cells was similar after incubation with forskolin and Bt2‐cAMP, which caused extensive neurite outgrowth, whereas in the presence of butyrate some rudimentary neurites were formed but they were not nearly as extensive. All compounds produced a dose‐dependent inhibition of cell proliferation, but the effect of Bt2‐cAMP was more marked than that caused by forskolin, thus showing that the effect of Bt2‐cAMP is due partially to the butyrate released. Acetylcholinesterase activity was lower in the cells incubated with butyrate or Bt2‐cAMP than in untreated cells or in forskolin‐treated cells. This suggests that cyclic AMP does not play a role in the regulation of this enzyme. Bt2‐cAMP produced histone acetylation, a well‐known effect of butyrate in cultured cells, whereas forskolin did not affect this modification. Consequently, the levels of thyroid hormone receptor, a nuclear protein whose concentration is regulated by butyrate through changes in acetylation of chromatin proteins, were decreased in cells incubated with Bt2‐cAMP or butyrate, but were unaffected by forskolin. Butyrate elevated the concentration of histone H1°, a protein that increases in neuroblastoma cells as a result of different treatments that block cell division. The concentration of H1° in the cells treated with Bt2‐cAMP was at a level intermediate between that found after treatment with butyrate and with forskolin. The present results clearly indicate that some of the effects of Bt2‐cAMP on neuroblastoma cells can be attributed to the butyryl moiety of this compound rather than to the cyclic nucleotide itself.

Down-regulation of thyroid hormone nuclear receptor levels by l-triiodothyronine in cultured glial C6 cells ☆

L-Triiodothyronine (T3) produced a time- and dose-dependent depletion of nuclear thyroid hormone receptor levels in C6 cells, a rat glioma cell line. Receptor number diminished by 30-40% after a 48 h incubation with concentrations of T3 that saturate the nuclear receptor. The nuclear binding curve obtained in cells incubated for 48 h with T3 was shifted leftward of the curve obtained after a 3 h incubation, which indicates an apparent increase in receptor affinity after long-term incubation with T3. However, this change probably represents a further equilibration of the hormone, since the dissociation rate from the nuclei was similar in C6 cells after long- and short-term incubation with T3. The effect of T3 was further demonstrated in C6 cells incubated with short-chain fatty acids. Butyrate and isobutyrate increased receptor levels, and T3 partially decreased the response to these compounds. These findings suggest the existence of a desensitization process by which C6 glial cells would be protected against an excess of thyroid hormone.

Mechanism of l-triiodothyronine (T3) uptake by glial C6 cells: regulation by butyrate

The mode of entry of triiodothyronine (T3) and its regulation by butyrate was studied in cultured glial C6 cells. Uptake of [125I]T3 increases for at least 60 min in C6 cells. The amount of cell-associated radioactivity is 2- to 4-fold higher during the entire time-course in cells previously exposed to 2 mM butyrate for 48 h. Uptake was non-saturable since uptake velocity was linearly related to the extracellular hormone concentration between 0.2 and 800 nM T3 in control and butyrate-treated cells. Uptake velocity increased by more than 3-fold in the cells incubated with the fatty acid. T3 uptake was temperature dependent and the effect of butyrate was observed at the different temperatures examined. Preincubation with metabolic inhibitors did not block [125I]T3 uptake in either group, and monodansylcadaverine was also ineffective. Present results suggest that in C6 cells T3 uptake proceeds by a passive, energy-independent, non-saturable process, that is markedly affected by short-chain fatty acids. Additionally, this is the first study documenting that a natural compound directly influences the entry of thyroid hormones into cells.

Regulation by n-Butyrate of Thyroid Hormone Receptor Levels in Cultured Cells of Glial Origin

Sodium butyrate has been reported to affect the differentiation, proliferation, and biochemical properties of different types of mammalian cells in culture, including neural cells (1).