Thapsigargin stimulates intracellular calcium mobilization and inhibits parathyroid hormone release

Abstract

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-InsP₃) presumably by interacting with recently identified parathyroid cell membrane Ca²⁺ receptors. We used thapsigargin (THAPS), an inhibitor of the microsomal Ca²⁺-ATPase, to deplete InsP₃-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 × 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 ≥10 minutes. THAPS (3 × 10⁻⁶ M) pretreatment for ≥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 ³H-inositol phosphates (InsP₃, InsP₂, and InsP₁) or alter Sr²⁺-induced accumulation of InsP₃, InsP₂, and InsP₁. 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 × 10⁻⁷, 10⁻⁶, and 3 × 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.