Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C141
Pathophysiological changes in extracellular pH regulate calcium-sensing receptor activity in HEK-293 and isolated parathyroid cells.
K. L. Campion1, W. D. McCormick1, R. Atkinson-Dell1, J. Warwicker1, D. T. Ward1
1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
Mammalian extracellular calcium (Ca2+o) concentration is maintained by parathyroid hormone (PTH) secretion and renal Ca2+ reabsorption under the control of the calcium-sensing receptor (CaR). Elevated PTH secretion and metabolic acidosis are both associated with chronic renal failure as well as ageing. Interestingly, acidosis is associated with increased PTH secretion in humans and dogs (1) while in CaR stably-transfected human embryonic kidney (CaR-HEK) cells, large changes in extracellular pH (pHo) alter CaR agonist sensitivity (2). To examine whether smaller, pathophysiological changes in pHo cause significant changes in calcium-sensing receptor activity in both heterologous and endogenous expression systems, the effect of 0.2 unit pH changes were tested on intracellular calcium (Ca2+i) mobilisation in CaR-HEK and isolated bovine parathyroid cells. Bovine superior parathyroid glands were digested with collagenase and viable parathyroid cells collected using a self-forming Percoll gradient. [Ca2+]i and pHi were measured by epifluorescence microscopy using Fura2 and BCECF respectively. Data shown are mean values ± SEM. CaR-HEK cells were stimulated with 2.5mM Ca2+o (pH 7.4) to elicit CaR-induced Ca2+i mobilisation and then switched to the same buffer at either pH 7.2 or 7.6 resulting in rapidly attenuated (-60 ± 6%; N=7; P<0.05 by paired t-test) or elevated (+31 ± 9%; P<0.05) responses respectively. Where extracellular signal-regulated kinase phosphorylation or actin polymerisation were used as alternative readouts of CaR activity, similar pHo-sensitivity was observed. Furthermore, acidosis (7.2) inhibited Ca2+i mobilisation in isolated parathyroid cells (-50 ± 11%; N=4; P<0.05) while alkalosis (7.6) increased it (+41 ± 15%; N=3; P<0.05). These pHo-dependent effects are unlikely to result from intracellular pHi changes as ± 0.4 unit pHochanges had little effect on pHi in either cell type. Acidosis could cause disassociation of Ca2+o from serum albumin in vivo thus antagonising this response, however, the pHo effects on Ca2+i mobilisation were observed in both cell types even in the presence of 5% (w/v) albumin. Therefore, modest changes in pHo can modulate the sensitivity of the Ca2+o-sensing mechanism in CaR-HEK and parathyroid cells. Specifically, extracellular alkalinisation potentiates CaR activity while extracellular acidification inhibits CaR activity in parathyroid cells. This may help explain why plasma acidification and increased PTH secretion are both observed in chronic renal disease and in ageing.
Where applicable, experiments conform with Society ethical requirements