Regulation by L channels of Ca2+-evoked secretory responses in ouabain-treated chromaffin cells

• Published in: Pflügers Archiv 2016-10, Vol.468 (10), p.1779-1792
• Main Authors: De Pascual, Ricardo, Colmena, Inés, Ruiz-Pascual, Lucía, Baraibar, Andrés Mateo, Egea, Javier, Gandía, Luis, García, Antonio G.
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Biomedicine; Cell Biology; Human Physiology; Molecular Medicine; Neurosciences; Receptors; Signaling and Cell Physiology
• ISSN: 0031-6768; 1432-2013
• DOI: 10.1007/s00424-016-1866-x

It is known that the sustained depolarisation of adrenal medullary bovine chromaffin cells (BCCs) with high K + concentrations produces an initial sharp catecholamine release that subsequently fades off in spite depolarisation persists. Here, we have recreated a sustained depolarisation condition of BCCs by treating them with the Na + /K + ATPase blocker ouabain; in doing so, we searched experimental conditions that permitted the development of a sustained long-term catecholamine release response that could be relevant during prolonged stress. BCCs were perifused with nominal 0Ca 2+ solution, and secretion responses were elicited by intermittent application of short 2Ca 2+ pulses (Krebs-HEPES containing 2 mM Ca 2+ ). These pulses elicited a biphasic secretory pattern with an initial 30-min period with secretory responses of increasing amplitude and a second 30-min period with steady-state, non-inactivating responses. The initial phase was not due to gradual depolarisation neither to gradual increases of the cytosolic calcium transients ([Ca 2+ ] c ) elicited by 2Ca 2+ pulses in BBCs exposed to ouabain; both parameters increased soon after ouabain addition. Νifedipine blocked these responses, and FPL64176 potentiated them, suggesting that they were triggered by Ca 2+ entry through non-inactivating L-type calcium channels. This was corroborated by nifedipine-evoked blockade of the L-type Ca 2+ channel current and the [Ca 2+ ] c transients elicited by 2Ca 2+ pulses. Furthermore, the plasmalemmal Na + /Ca 2+ exchanger (NCX) blocker SEA0400 caused a mild inhibition followed by a large rebound increase of the steady-state secretory responses. We conclude that these two phases of secretion are mostly contributed by Ca 2+ entry through L calcium channels, with a minor contribution of Ca 2+ entry through the reverse mode of the NCX.