EXPERIMENTAL PROCEDURESMaterials-Fura-2/AM was from Molecular Probes, Inc. (Invitrogen). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from Dako (Ely, Cambridgeshire, UK). Unless stated otherwise, all other chemicals were purchased from Sigma-Aldrich (Poole, Dorset, UK).Cell Culture-HEK-293 cells, stably transfected with human parathyroid CaR (11), were a gift from Dr. E. F. Nemeth (NPS Pharmaceuticals). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen) and 200 g/ml hygromycin B (Boehringer-Mannheim, Lewes, Sussex, UK).CaR Phosphorylation Assays-Cells were grown to 80 -90% confluence in 35 mm culture dishes, and CaR T888 phosphorylation was assayed as described previously (15)
The calcium-sensing receptor (CaR) modulates renal calcium reabsorption and parathyroid hormone (PTH) secretion and is involved in the etiology of secondary hyperparathyroidism in CKD. Supraphysiologic changes in extracellular pH (pH o ) modulate CaR responsiveness in HEK-293 (CaR-HEK) cells. Therefore, because acidosis and alkalosis are associated with altered PTH secretion in vivo, we examined whether pathophysiologic changes in pH o can significantly alter CaR responsiveness in both heterologous and endogenous expression systems and whether this affects PTH secretion. In both CaR-HEK and isolated bovine parathyroid cells, decreasing pH o from 7.4 to 7.2 rapidly inhibited CaR-induced intracellular calcium (Ca 2+ i ) mobilization, whereas raising pH o to 7.6 potentiated responsiveness to extracellular calcium (Ca i mobilization. Intracellular pH was unaffected by acute 0.4-unit pH o changes, and the presence of physiologic albumin concentrations failed to attenuate the pH o -mediated effects. None of the individual point mutations created at histidine or cysteine residues in the extracellular domain of CaR attenuated pH o sensitivity. Finally, pathophysiologic pH o elevation reversibly suppressed PTH secretion from perifused human parathyroid cells, and acidosis transiently increased PTH secretion. Therefore, pathophysiologic pH o changes can modulate CaR responsiveness in HEK-293 and parathyroid cells independently of extracellular histidine residues. Specifically, pathophysiologic acidification inhibits CaR activity, thus permitting PTH secretion, whereas alkalinization potentiates CaR activity to suppress PTH secretion. These findings suggest that acid-base disturbances may affect the CaR-mediated control of parathyroid function and calcium metabolism in vivo.
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Objective: The calcium-sensing receptor (CASR) is a key controller of calcium homeostasis by regulating parathyroid hormone (PTH) secretion and renal calcium reabsorption. CASR T888 is a protein kinase C (PKC) phosphorylation site in the receptor's intracellular domain that has previously been identified as a critical negative regulator of CASR downstream signaling in vitro, but whose importance in vivo is unknown. Case report: The proband presented with mild symptomatic hypocalcemia following treatment for nephrotic syndrome due to minimal change glomerulonephropathy. Laboratory tests revealed inappropriately normal PTH concentrations and relative hypercalciuria typical of autosomal dominant hypocalcemia. His asymptomatic father had similar laboratory test results. Design and methods: The CASR gene was sequenced. To investigate the molecular consequences of CASR T888M mutation, site-directed mutagenesis was used to modify the wild-type (wt)-CASR gene, with the resulting mutant being transfected transiently into HEK-293 cells. Results: A novel CASR missense mutation, T888M, was identified in both cases. The CASR T888M mutant exhibited enhanced sensitivity to extracellular calcium concentration, both for intracellular calcium ðCa 2C i Þ mobilization and for ERK phosphorylation, despite having unaltered levels of cell surface expression. Furthermore, CASR T888M elicited sustained Ca 2C i mobilization rather than high frequency Ca 2C i oscillations, and, unlike the wt-CASR, the response was resistant to acute inhibition by the PKC activator, phorbol 12-myristate 13-acetate. Conclusions: The clinical and functional data provide the first genotype-phenotype correlation for a mutation at T888, indicating its critical physiological importance in CASR signaling. Thus, CASR T888 represents a functionally important, inhibitory phosphorylation site that contributes to the control of PTH secretion.
The Race Equality Charter (REC) was introduced in 2014 as a national policy initiative that aims to support UK universities in developing cultural and systemic changes to promote race equality for Black and minority ethnic (BME) staff and students. Drawing on quantitative data, we locate the REC within a complex picture of undergraduate student diversity and significant attainment gaps between white students and Black and ethnic minority groups. Utilising qualitative interviews and observations to further explore the questions our quantitative analysis raises, we show that the REC is not perceived as a significant vehicle for progressing race equality work in award-holding institutions.Rather, it is mostly applied as an enhancement tool to help shape and sustain existing race equality initiatives that produce incremental change. This, we argue, suggests the REC's intention to inspire race equality approaches that favour institutional strategic planning at the highest level, is yet to be realised.
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