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Coupling of Calcium Receptors to Inositol Phosphate and Cyclic AMP Generation in Mammalian Cells and Xenopus laevis Oocytes and Immunodetection of Receptor Protein by Region‐Specific Antipeptide Antisera
Author(s) -
Chang Wenhan,
Pratt Stacy,
Chen TsuiHua,
Nemeth Edward,
Huang Zhengmin,
Shoback Dolores
Publication year - 1998
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.1998.13.4.570
Subject(s) - hek 293 cells , transfection , extracellular , receptor , xenopus , calcium sensing receptor , microbiology and biotechnology , immunocytochemistry , inositol phosphate , parathyroid hormone , biology , chemistry , calcium , inositol , cell culture , biochemistry , endocrinology , organic chemistry , gene , genetics
Ca 2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell‐surface Ca 2+ ‐sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca 2+ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in different expression systems. In Xenopus laevis oocytes injected with bovine parathyroid CaR cRNA, the addition of extracellular cations to 1.5 mM Ca 2+ , 5.5 mM Mg 2+ , or 10 μM Gd 3+ significantly increased 45 Ca efflux ( p < 0.01). InsP accumulation also increased dramatically when adding these cations to human embryonic kidney (HEK) 293 cells stably transfected with wild‐type bovine parathyroid CaR cDNA. Raising the extracellular [Ca 2+ ] ([Ca 2+ ] o ) from 0.1 to >1.4 mM in oocytes and to >1.0 mM in HEK 293 cells stimulated significant increments in 45 Ca efflux and InsP accumulation, respectively ( p < 0.05). In contrast, Ca 2+ and Mg 2+ increased InsPs to a lesser extent in COS 7 cells transiently transfected with CaR cDNA. In HEK 293 cells stably expressing CaR cDNA, there were significant reductions in cAMP content when adding high Ca 2+ , Mg 2+ , Gd 3+ , or the CaR modulator NPS R‐467. Three region‐specific anti‐CaR peptide antisera immunoblotted bands of ∼140 and 155 kDa in membranes from CaR‐transfected HEK 293 cells and bovine parathyroid tissue. Immunocytochemistry demonstrated strong cell‐surface staining in CaR‐transfected HEK 293 cells and parathyroid tissue, which was absent when antisera were preabsorbed with CaR peptides. These results indicate that the activation of the recombinant CaR by extracellular Ca 2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca 2+ ] o .