Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells

Abstract

The effects of NH 4Cl on cytoplasmic free calcium concentration ([Ca 2+] i) and pH (pH i) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH 4Cl (10–40 mM) in the presence of 1 mM extracellular calcium ([Ca 2+] e) increased [Ca 2+] i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH 4Cl. In medium containing 0.1 μM [Ca 2+] e, or in 1 mM [Ca 2+] e medium containing 0.1 μM nitrendipine, the plateau was absent leaving only a transient [Ca 2+] i spike. NH 4Cl also increased pH i and this, like the [Ca 2+] i plateau, remained elevated during the continued presence of NH 4Cl. In medium containing only 0.1 μM [Ca 2+] e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH 4Cl induced repeated [Ca 2+] i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20–500 nM) caused a [Ca 2+] i rise but, after an additional exposure to NH 4Cl, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abollshed the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH 4Cl. Neither acetate removal nor methylamine increased [Ca 2+] i in medium containing 0.1 μM [Ca 2+] e, although in both cases pH i increased. We conclude that in bovine anterior pituitary cells NH 4Cl raises [Ca 2+] i by two independent pathways, increasing net calcium entry and mobilizing Ca 2+ from a TRH-insensitive calcium store.