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THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells

Disclosure: K.A. Alghamdi: None. D. Ward: None. Calcium-sensing receptor (CaR) is the key controller of parathyroid hormone (PTH) secretion and extracellular calcium homeostasis. Hyperphosphataemia increases PTH secretion and we reported recently that pathophysiologic phosphate (Pi) concentrations c...

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Autores principales: Alghamdi, Khaleda Ali, Ward, Donald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10554438/
http://dx.doi.org/10.1210/jendso/bvad114.358
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author Alghamdi, Khaleda Ali
Ward, Donald
author_facet Alghamdi, Khaleda Ali
Ward, Donald
author_sort Alghamdi, Khaleda Ali
collection PubMed
description Disclosure: K.A. Alghamdi: None. D. Ward: None. Calcium-sensing receptor (CaR) is the key controller of parathyroid hormone (PTH) secretion and extracellular calcium homeostasis. Hyperphosphataemia increases PTH secretion and we reported recently that pathophysiologic phosphate (Pi) concentrations can attenuate CaR activity directly in transfected HEK-293 cells and can increase PTH secretion from human and murine parathyroid cells (1). This was investigated further using thyroidal C cell-derived TT cells (ATCC), which express CaR endogenously. Intracellular Ca(2+) (Ca(2+)(i)) mobilisation was assayed by epifluorescence microscopy, calcitonin secretion by ELISA, protein expression by immunoblotting and CaR mRNA expression by qRT-PCR. Co-stimulation of TT cells with the (CaR-activating) calcimimetic R568 (1µM) and spermine (1mM) elicited robust Ca(2+)(i) mobilisation, that was inhibited (33±4%; P<0.001; N=4) by 2mM (pathophysiologic) Pi-containing buffer vs 0.8mM Pi control. In contrast, raising Pi concentration was without effect on carbachol-induced Ca(2+)(i) mobilisation (acting via muscarinic receptors) (P=0.89; N=3). Also, 1.2mM (high) sulphate elicited a similar CaR inhibition as for Pi vs 0.3mM control (-28±16%; P<0.05; N=4). Next, it was found that CaR-mediated stimulation of the TT cells with 1µM R568 & 1mM spermine (in 2mM Ca(2+) and 0.8mM Pi) increased calcitonin secretion, as expected, whereas the same stimulation in 2mM Pi (in the presence of 2.2mM Ca(2+) to correct for any reduction in free Ca(2+)) significantly blunted this response (-59.7±15%; P<0.05;N=4). Interestingly, CaR mRNA expression was significantly downregulated 45±13%; P<0.05;N=6) after 48 hours treatment with media containing 2mM Pi (vs 0.8mM control). However, while CaR protein expression in the TT cells was confirmed by immunoblotting, its abundance was unaffected by the same 2mM Pi cotreatment (P=0.79; N=8). Therefore, pathophysiologic Pi treatment inhibits endogenous CaR-induced signalling, calcitonin secretion and CaR mRNA expression in thyroidal TT cells but without affecting CaR abundance. These results further support the idea that the CaR represents a mineral sensor, at which Pi acts directly as a non-competitive antagonist to limit CaR-induced suppression of PTH secretion. Reference: (1) Centeno et al. Nat Commun. 2019;10:4693. Presentation: Thursday, June 15, 2023
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spelling pubmed-105544382023-10-06 THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells Alghamdi, Khaleda Ali Ward, Donald J Endocr Soc Bone And Mineral Metabolism Disclosure: K.A. Alghamdi: None. D. Ward: None. Calcium-sensing receptor (CaR) is the key controller of parathyroid hormone (PTH) secretion and extracellular calcium homeostasis. Hyperphosphataemia increases PTH secretion and we reported recently that pathophysiologic phosphate (Pi) concentrations can attenuate CaR activity directly in transfected HEK-293 cells and can increase PTH secretion from human and murine parathyroid cells (1). This was investigated further using thyroidal C cell-derived TT cells (ATCC), which express CaR endogenously. Intracellular Ca(2+) (Ca(2+)(i)) mobilisation was assayed by epifluorescence microscopy, calcitonin secretion by ELISA, protein expression by immunoblotting and CaR mRNA expression by qRT-PCR. Co-stimulation of TT cells with the (CaR-activating) calcimimetic R568 (1µM) and spermine (1mM) elicited robust Ca(2+)(i) mobilisation, that was inhibited (33±4%; P<0.001; N=4) by 2mM (pathophysiologic) Pi-containing buffer vs 0.8mM Pi control. In contrast, raising Pi concentration was without effect on carbachol-induced Ca(2+)(i) mobilisation (acting via muscarinic receptors) (P=0.89; N=3). Also, 1.2mM (high) sulphate elicited a similar CaR inhibition as for Pi vs 0.3mM control (-28±16%; P<0.05; N=4). Next, it was found that CaR-mediated stimulation of the TT cells with 1µM R568 & 1mM spermine (in 2mM Ca(2+) and 0.8mM Pi) increased calcitonin secretion, as expected, whereas the same stimulation in 2mM Pi (in the presence of 2.2mM Ca(2+) to correct for any reduction in free Ca(2+)) significantly blunted this response (-59.7±15%; P<0.05;N=4). Interestingly, CaR mRNA expression was significantly downregulated 45±13%; P<0.05;N=6) after 48 hours treatment with media containing 2mM Pi (vs 0.8mM control). However, while CaR protein expression in the TT cells was confirmed by immunoblotting, its abundance was unaffected by the same 2mM Pi cotreatment (P=0.79; N=8). Therefore, pathophysiologic Pi treatment inhibits endogenous CaR-induced signalling, calcitonin secretion and CaR mRNA expression in thyroidal TT cells but without affecting CaR abundance. These results further support the idea that the CaR represents a mineral sensor, at which Pi acts directly as a non-competitive antagonist to limit CaR-induced suppression of PTH secretion. Reference: (1) Centeno et al. Nat Commun. 2019;10:4693. Presentation: Thursday, June 15, 2023 Oxford University Press 2023-10-05 /pmc/articles/PMC10554438/ http://dx.doi.org/10.1210/jendso/bvad114.358 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Bone And Mineral Metabolism
Alghamdi, Khaleda Ali
Ward, Donald
THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells
title THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells
title_full THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells
title_fullStr THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells
title_full_unstemmed THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells
title_short THU395 Phosphate Inhibits Calcium-sensing Receptor Expressed Endogenously In TT Cells
title_sort thu395 phosphate inhibits calcium-sensing receptor expressed endogenously in tt cells
topic Bone And Mineral Metabolism
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10554438/
http://dx.doi.org/10.1210/jendso/bvad114.358
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