THAPSIGARGIN STIMULATES INTRACELLULAR CALCIUM MOBILIZATION AND INHIBITS PARATHYROID-HORMONE RELEASE

Ca2+ and other divalent cations like Sr2+, Ba2+, and Mg2+ stimulate rapid and sustained increases in intracellular Ca2+ ([Ca2+](i)) and 1,4,5-inositol trisphosphate (1,4,5-InsP(3)) presumably by interacting with recently identified parathyroid cell membrane Ca2+ receptors. We used thapsigargin (THAPS), an inhibitor of the microsomal Ca2+-ATPase, to deplete InsP(3)-sensitive intracellular Ca2+ stores to determine whether sustained increases in [Ca2+](i) due to divalent cations require intact cytosolic Ca2+ pools. In Fura 2-loaded parathyroid cells, THAPS produced a gradual increase in [Ca2+](i) which reached a steady-state level by 2-3 minutes. The effect of THAPS (3 x 10(-6) M) was substantial with [Ca2+](i), rising from 281 +/- 27 nM at 0.5 mM Ca2+ to a peak value of 684 +/- 30 nM (p < 0.0001). The addition of Sr2+ to cells at 0.5 mM extracellular Ca2+ induced an immediate 2-to 3-fold increase in [Ca2+](i) which stabilized at a [Ca2+](i) above baseline for greater than or equal to 10 minutes. THAPS (3 x 10(-6) M) pretreatment for greater than or equal to 5 minutes blocked this sustained-phase increment in [Ca2+](i) due to Sr2+. In the absence of extracellular Ca2+, there was a slight but nonsignificant effect of THAPS on [Ca2+](i). Incubation of cells with THAPS did not change the levels of H-3-inositol phosphates (InsP(3), InsP(2), and InsP(1)) or alter Sr2+-induced accumulation of InsP(3), InsP(2), and InsP(1). THAPS substantially reduced parathyroid hormone secretion at 1.0 mM Ca2+ by 20 +/- 16, 57 +/- 8, 75 +/- 10, and 83 +/- 9% at 10(-7), 3 x 10(-7), 10(-6), and 3 x 10(-6) M THAPS, respectively. We conclude that depletion of intracellular Ca2+ stores by THAPS stimulates Ca2+ mobilization, presumably from extracellular sources, and that this agent and divalent cations such as Sr2+ activate the same pathway for sustained Ca2+ mobilization. The inhibition of secretion by THAPS supports the idea that increases in [Ca2+](i) play a suppressive role in the control of hormone release in the parathyroid.