Endocrine Abstracts

The calcium-sensing receptor (CaR) is the key controller of parathyroid hormone (PTH) secretion and extracellular calcium homeostasis. Hyperphosphataemia increases PTH secretion and is associated with secondary hyperparathyroidism (SHPT). We reported recently that inorganic phosphate (Pi), and sulphate, can attenuate CaR activity directly (in CaR-transfected HEK-293 cells) and Pi can increase PTH secretion rapidly from human and murine parathyroid cells. To investigate this further, here we used a thyroid parafollicular C-cell model, TT cells, which express CaR endogenously. TT cells, which exhibit CaR-induced calcitonin (CT) secretion, were assayed by epifluorescence intracellular Ca²⁺ imaging and CT assay (with a gastrin-releasing peptide (GRP)-induced CT control). When co-stimulated with the CaR-activating calcimimetic R568 (1μM) and spermine (1mM), TT cells exhibited classic CaR-induced Ca²⁺_i mobilisation, which the G_q/11-specific inhibitor YM-254890 largely abolished (-93 ± 8%). Similar CaR-induced responses were also inhibited by increasing the buffer Pi concentration from 0.8 mm (physiological) to a pathophysiological 2 mm (-33 ± 4%; P<0.001). In contrast, raising Pi concentration was without effect on carbachol-induced Ca²⁺_i mobilisation (acting via muscarinic receptors). Finally, 1.2 mm sulphate (high) elicited a similar CaR inhibition as for Pi (-28 ± 16%; P<0.05; vs physiological 0.3 mm sulphate). Similar inhibitory effects were seen when the anions were used in combination; 2mM Pi (high) & 0.3mM sulphate (normal) elicited a 15 ± 12% reduction in CaR-induced Ca²⁺_i mobilisation, while 2mM sulphate (high) & 0.8mM Pi (normal) produced a 19 ±3% inhibition (P<0.05). Regarding CT secretion, we observed time-dependent release that was stimulated maximally 15-20 fold by increasing Ca²⁺ concentration from 0.5–3.0mM (EC₅₀ ~1.5 mm). Inorganic Pi (0.8-3.0 mm) inhibited CT release in a non-competitive manner, with 3mM Pi almost abolishing CT release at all Ca²⁺_o concentrations tested. Even raising Pi concentration from 0.8-1.4 mm (representing the physiological range) elicited a striking 50% reduction in CT release. In contrast, 2mM Pi had no effect on 1μM GRP-stimulated CT release. Sulphate was also a non-competitive inhibitor of CT release but was less potent than Pi. These results further support the idea that the CaR is a mineral sensor, at which Pi acts directly as a non-competitive antagonist to limit CaR-induced reductions in PTH secretion. Further, Pi may also limit CaR-induced CT secretion when its serum concentration is raised. Together, our studies provide important new information regarding the physiological control of PTH and CT secretion, and, the pathophysiology of SHPT.