Regulation by L channels of Ca 2+ -evoked secretory responses in ouabain-treated chromaffin cells

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 0Ca2+ solution, and secretion responses were elicited by intermittent application of short 2Ca2+ pulses (Krebs-HEPES containing 2 mM Ca2+). 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 ([Ca2+]c) elicited by 2Ca2+ 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 Ca2+ entry through non-inactivating L-type calcium channels. This was corroborated by nifedipine-evoked blockade of the L-type Ca2+ channel current and the [Ca2+]c transients elicited by 2Ca2+ pulses. Furthermore, the plasmalemmal Na+/Ca2+ 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 Ca2+ entry through L calcium channels, with a minor contribution of Ca2+ entry through the reverse mode of the NCX.