In human embryonic kidney (HEK) 293 cells stably expressing human type 1 parathyroid hormone (PTH) receptors, PTH (residues 1Ð34) potentiates the Ca²⁺ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate (IP₃ ) formation by a mechanism that is independent of protein kinase A (Short & Taylor, 2000). We previously proposed that PTH increases either the sensitivity of IP₃ receptors or the size of the Ca²⁺ pool available to IP₃ . Here we attempt to discriminate between these possibilities.

Incubation of fura-2-loaded cells with a membrane-permeant analogue of IP₃ (IP₃-BM) stimulated a slow release of intracellular Ca²⁺ stores. The resulting Ca²⁺ signals were increased by addition of PTH, but only if it is added before IP₃-BM had completely emptied the intracellular Ca²⁺ stores. When added 5 min after addition of IP₃-BM, PTH increased the cytosolic [Ca²⁺] to 435 ± 46 nM (mean ± S.E.M., n = 3), but after 10 min it increased it to only 69 ± 24 nM (n = 5). The amount of Ca²⁺ remaining within intracellular stores was similar after prolonged stimulation with IP₃-BM alone (100 µM, 10 min) or after stimulation with carbachol (CCh) in combination with PTH. We conclude that PTH potentiates responses to IP₃ without increasing the size of the IP₃-sensitive Ca²⁺ store. Because carbachol alone stimulates formation of insufficient IP₃ to completely empty intracellular Ca²⁺ stores, PTH potentiates responses to even a maximal concentration of carbachol (Short & Taylor, 2000) .

Intracellular cAMP levels increased from a basal level of 5 ± 1 to 736 ± 52 pmol well^-1 (n = 6) after a 45 s incubation with 10 nM PTH and to 1016 ± 187 pmol well^-1 (n = 6) after stimulation with 100 nM PTH. Incubation with an inhibitor of adenylyl cyclase (SQ 22536, 1 mM) reduced the amount of cAMP formed in response to 10 or 10 nM PTH by about 80 %. In parallel experiments measuring the ability of PTH to potentiate carbachol-evoked ⁴⁵Ca²⁺ release from cells, SQ 22536 had no effect on responses to maximal or submaximal concentrations of PTH. We conclude that PTH sensitises IP₃ receptors to IP₃ without affecting the size of the IP₃-sensitive Ca²⁺ pool by a mechanism that is independent of cAMP.

This work was supported by The Wellcome Trust, BBSRC and a studentship from the MRC (T.A.G).