Kuribara M et al. (2010), BDNF stimulates Ca2+ oscillation frequency in m...

XB-ART-41968

Gen Comp Endocrinol 2010 Nov 01;1692:123-9. doi: 10.1016/j.ygcen.2010.08.010.

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BDNF stimulates Ca2+ oscillation frequency in melanotrope cells of Xenopus laevis: contribution of IP3-receptor-mediated release of intracellular Ca2+ to gene expression.

Kuribara M , Eijsink VD , Roubos EW , Jenks BG , Scheenen WJ .

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Pituitary melanotrope cells of the amphibian Xenopus laevis are neuroendocrine cells regulating the animal's skin color adaptation through secretion of α-melanophore-stimulating hormone (α-MSH). To fulfill this function optimally, the melanotrope cell undergoes plastic changes in structure and secretory activity in response to changed background light conditions. Xenopus melanotrope cells display Ca(2+) oscillations that are thought to drive α-MSH secretion and gene expression. They also produce brain-derived neurotrophic factor (BDNF), which stimulates in an autocrine way the biosynthesis of the α-MSH precursor, pro-opiomelanocortin (POMC). We have used this physiological adaptation mechanism as a model to investigate the role of BDNF in the regulation of Ca(2+) kinetics and Ca(2+)-dependent gene expression. By dynamic video imaging of isolated cultured melanotropes we demonstrated that BDNF caused a dose-dependent increase in Ca(2+) oscillation frequency up to 64.7±2.3% of control level. BDNF also induced a transient Ca(2+) peak in Ca(2+)-free medium, which was absent when calcium stores were blocked by thapsigargin and 2-aminoethoxydiphenyl borate, indicating that BDNF stimulates acute release of Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores. Moreover, we show that thapsigargin inhibits the expression of BDNF transcript IV (by 61.1±28.8%) but does not affect POMC transcript. We conclude that BDNF mobilizes Ca(2+) from IP(3)-sensitive intracellular Ca(2+) stores and propose the possibility that the resulting Ca(2+) oscillations selectively stimulate expression of the BDNF gene.

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Species referenced: Xenopus laevis
Genes referenced: bdnf itpr1 pomc

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	Fig. 1. Digital imaging of Ca2+ oscillations in Xenopus melanotrope cells, expressed as the F340/F380 ratio of Fura-2 fluorescence. (a) Representative example of cell that increased oscillation frequency upon treatment with 25 ng/ml BDNF (shaded rectangle). (b) Dose–response relation between BDNF and percentage increase in Ca2+ oscillation frequency (relative to frequency before treatment), which was calculated by fast Fourier analysis. Only cells that reacted with an increase in frequency were taken into account. Letters on top of bar refer to groups that are significantly different (P < 0.05). n (number of independent trials) = 3 for 25 ng/ml, 5 for 50 ng/ml, and 4 for 0, 12.5 and 100 ng/ml BDNF treatment.
	Fig. 2. Representative examples of the responses of Ca2+ oscillations in single melanotropes to (a) 5 mM EGTA followed by 100 ng/ml BDNF, (b) 5 mM EGTA followed by 1 μM thapsigargin (Tg) and 100 ng/ml BDNF, and (c) 5 mM EGTA followed by 50 μM 2-APB and 100 ng/ml BDNF.
	Fig. 3. Stimulation of Ca2+ oscillation frequency (relative to frequency before treatment) by treatment with 1 μM thapsigargin (Tg), 1 μM Tg + 50 ng/ml BDNF, and 50 ng/ml BDNF alone. n = 3. Letters on top of error bar indicate groups that are significantly different (P < 0.05).
	Fig. 4. Quantification by Q-RT-PCR of the amount of BDNF transcript IV (a) and POMC mRNA (b), in NILs incubated for 3 days without treatment (control, normalized to 1) and with 1 μM thapsigargin (Tg), 1 μM Tg + 50 ng/ml BDNF or 50 ng/ml BDNF. Letters on top of bar refer to groups that are significantly different (P < 0.05). n (number of independent trials) = 3.