The effect of barium on [3H]noradrenalin release from the human neuroblastoma SH-SY5Y.

Replacement of Ca2+ with Ba2+ in HEPES-buffered saline stimulated [3H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba(2+)-evoked release was inhibited by 85% by 3 microM tetrodotoxin and 95% by 5 microM nifedipine. Ba2+ also increased the potency of K(+)-evoked release of [3H]noradrenalin, as maximal release was observed with 60 mM K+ compared with the 100 mM K+ necessary to achieve maximal release in the presence of Ca2+. In contrast, replacing Ca2+ with Ba2+ had little effect on carbachol- and bradykinin-evoked release of [3H]noradrenalin. No evidence was obtained from studies on changes in [Ca2+]i (in response to 100 microM carbachol) using fura-2 that Ba2+ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba2+ depolarizes SH-SY5Y cells as well as enhancing inward Ca2+ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba2+ blocks K+ channels, leading to depolarization followed by opening of voltage-sensitive Na+ channels. This in turn opens voltage-sensitive L-type Ca2+ channels, which are coupled to the release of [3H]noradrenalin in SH-SY5Y cells.

Bradykinin-evoked release of [3H]noradrenaline from the human neuroblastoma SH-SY5Y.

Bradykinin (BK) evoked [3H]noradrenaline ([3H]NA) release from the human neuroblastoma SH-SY5Y and this was enhanced by pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 8 min. This effect of BK was inhibited by 500 microM [D-Phe7]BK and 100 microM [Thi5,8,D-Phe7]BK but not by 500 microM [Des-Arg9,Leu8]BK. The BK (B1)-agonist [Des-Arg9]BK did not evoke [3H]NA release. This suggested that SH-SY5Y expressed BK (B2)-receptors coupled to the release of [3H]NA. BK acting at B2-receptors, also elevated intracellular calcium and depolarized SH-SY5Y cells. Although pre-treatment of SH-SY5Y cells with TPA enhanced BK-evoked [3H]NA release, the elevation of intracellular calcium [Ca2+]; was decreased by about 50%. BK-evoked release of [3H]NA in cells not pre-treated with phorbol ester was only 23% dependent on extracellular calcium. In comparison, following phorbol ester treatment approximately 40% of [3H]NA release was dependent on extracellular calcium. Nifedipine (5 microM), CoCl2 (1 mM) and NiCl2 (1 mM) inhibited NA release in SH-SY5Y cells pre-treated with TPA by 16.0, 47 and 44%, respectively. The results of this study showed that BK, acting at B2-receptors, activated [3H]NA release in SH-SY5Y. Part of this effect appeared to be due to activation of L-type calcium channels but the majority of BK-evoked [3H]NA release in SH-SY5Y cells appeared to depend on [Ca2+]i.

Nicotinic receptor-mediated release of noradrenaline in the human neuroblastoma SH-SY5Y.

Dimethylphenylpiperazinium iodide (a nicotinic agonist) evokes noradrenaline release from human neuroblastoma SH-SY5Y cells that have been pretreated with 12-O-tetradecanoylphorbol 13-acetate for 8 min. This effect of dimethylphenylpiperazinium iodide was inhibited by 1 microM mecamylamine but not by 1 microM atropine, which suggests that SH-SY5Y cells express nicotinic receptors coupled to the release of noradrenaline. Dimethylphenylpiperazinium iodide-evoked release was enhanced by 5 microM Bay K 8644 (an L-type calcium agonist) and inhibited by 1 microM nifedipine. Dimethylphenylpiperazinium iodide depolarised SH-SY5Y cells and enhanced the level of intracellular calcium in cells loaded with fura 2. The effects of dimethylphenylpiperazinium iodide on noradrenaline release, depolarisation, and intracellular calcium levels were all inhibited by 1 microM desmethylimipramine. The results of this study show that nicotinic receptors in SH-SY5Y cells stimulate noradrenaline release by activation of L-type calcium channels.