The effect of extracellular polyvalent cations on bovine anterior pituitary cells Evidence for a Ca 2+-sensing receptor coupled to release of intracellular calcium stores

Abstract

We have investigated the effects of extracellular cations ([ION] ex) on cytosolic free calcium levels ([Ca 2+] i) in bovine anterior pituitary (bAP) cells, using single-cell microfluorimetry. Increasing the [Ca 2+] ex from 1 mM to 20 mM caused [Ca 2+] i to increase in 64 ± 14% of bAP cells. The [Ca 2+] ex-induced [Ca 2+] i increase was observed when cells were maintained in the presence of the voltage-gated-calcium-channel antagonist nitrendipine, but not when cells were treated with thapsigargin. Addition of [La 3+] ex (5–15 μM) decreased [Ca 2+] i, whereas 30 μM-1 mM caused a [Ca 2+] i rise in 60.9 ± 8.8% of bAP cells. [La 3+] ex induced [Ca 2+] i changes were abolished by treating bAP-cells with either thapsigargin or ionomycin, but not nitrendipine. [La 3+] ex at 15 μM did not increase [Ca 2+] i in any cells tested, but when cells were treated with thimerosal, [La 3+] ex (15 μM) caused a [Ca 2+] i increase in 62.5 ± 12.2% of bAP cells. In the presence of 1 mM [Ca 2+] ex, successive additions of La 3+ caused successive [Ca 2+] i, rises, but in nominally [Ca 2+] ex-free medium only the first addition of [La 3+] ex caused a [Ca 2+] i rise. Addition of thyroliberin (TRH) in the presence of 1 mM [Ca 2+] ex, caused [Ca 2+] i to increase in 70% of bAP cells; subsequent addition of [La 3+] ex (1 mM) only caused [Ca 2+] i increases in 75% of those cells which had already responded to TRH. However, all cells which responded to 1 mM [La 3+] ex also responded subsequently to TRH. After treatment with TRH in medium that was nominally [Ca 2+] ex free, addition of La 3+ (0.5–1 mM) did not increase [Ca 2+] i in any cells tested. The number of cells which showed [La 3+] ex-induced [Ca 2+] i increases decreased in culture: only 21.75 ± 2.2% cells responded after 7–11 days. When cells were cultured for 7–11 days in the presence of tunicamycin, [La 3+] ex failed to increase [Ca 2+] i in any cells tested. [Mn 2+] ex rapidly quenched the Fura-2 signal measured from all bAP cells, but at 10 mM it also triggered a [Ca 2+] i rise in about 60% of bAP cells. The Mn 2+-induced [Ca 2+] i rise was specifically abolished in cells cultured in the presence of tunicamycin although quenching was still observed. From these data we suggest that bAP cells may express a polyvalent cation receptor coupled to the release of calcium from intracellular stores.