Parathyroid hormone (PTH) stimulates adenylyl cyclase and release of Ca²⁺ from intracellular stores; the mechanism underlying the latter response is not understood. We previously demonstrated that in HEK 293 cells stably expressing human type 1 PTH receptors (HEK/PR), PTH potentiates the Ca²⁺ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate (IP₃ ) formation by a mechanism that does not require activation of cAMP-dependent protein kinase (Short & Taylor, 2000). We suggested that PTH may increase either the sensitivity of IP₃ receptors to IP₃ or the amount of Ca²⁺ within the IP₃ -sensitive stores. Here, we attempt to distinguish between these possibilities and to resolve whether an inhibitory effect of PTH on Ca²⁺ extrusion from the cell might have contributed to the increased amplitude of the IP₃-evoked Ca²⁺ signals detected in fura 2-loaded cells.

We sought to establish whether the amount of IP₃ made in response to a maximal concentration of carbachol (CCh) was sufficient to maximally activate the IP₃ receptors. The concentrations of CCh that caused half-maximal ⁴⁵Ca²⁺ release (EC₅₀ = 36 ± 6 µM, n = 3) and [³H] IP₃ formation (41 ± 7 µM, n = 3) were similar. This approach cannot therefore establish whether IP₃ receptors were maximally activated by a maximal concentration of CCh and cannot therefore unequivocally establish whether PTH must increase the size of the IP₃-sensitive Ca²⁺ store.

To address the possible inhibitory effects of PTH on Ca²⁺ extrusion, the intracellular stores of intact HEK/PR cells were loaded with ⁴⁵Ca²⁺ by incubation in Hepes-buffered saline (HBS) containing ⁴⁵Ca²⁺ for 2 h. The cells were then stimulated in Ca²⁺-free HBS and the amount of ⁴⁵Ca²⁺ released into the medium during each 45 s interval was expressed as a fraction of the ⁴⁵Ca²⁺ remaining within the cells at the beginning of that interval (a fractional release rate). The fractional rate of ⁴⁵Ca²⁺ release evoked by the second of two sequential challenges with a maximal concentration of CCh (1 mM) was reduced to 56 ± 7.5 % (n = 4) of the first challenge. When the second challenge with CCh was combined with PTH (100 nM, which alone had no effect), the ⁴⁵Ca²⁺ release was increased to 184 ± 21 % (n = 5) of the first response to CCh. In fura 2-loaded HEK/PR cells treated with thapsigargin to increase the cytosolic [Ca²⁺], the rate of decline of the cytosolic [Ca²⁺] after removal of extracellular Ca²⁺ was similar in the presence (t_1/2 = 24 ± 4 s, n = 4) and absence (t_1/2 = 31 ± 2 s, n = 3) of PTH. The results from ⁴⁵Ca²⁺-loaded cells (where the assay requires Ca²⁺ extrusion) and fura-2-loaded cells establish that the ability of PTH to potentiate IP₃-evoked Ca²⁺ signals cannot be due to inhibition of Ca²⁺ extrusion.This work was supported by The Wellcome Trust, BBSRC and a studentship from the MRC.

Short, A.D. & Taylor, C.W. (2000). J. Biol. Chem. 275, 1807-1813.