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

J Bone Miner Res. 1998 Apr;13(4):570-80. doi: 10.1359/jbmr.1998.13.4.570.

Abstract

Ca2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell-surface Ca2+-sensing receptors (CaRs). We assessed the ability of these receptors to couple to Ca2+ 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 Ca2+, 5.5 mM Mg2+, or 10 microM Gd3+ significantly increased 45Ca 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 [Ca2+] ([Ca2+]o) from 0.1 to > 1.4 mM in oocytes and to > 1.0 mM in HEK 293 cells stimulated significant increments in 45Ca efflux and InsP accumulation, respectively (p < 0.05). In contrast, Ca2+ and Mg2+ 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 Ca2+, Mg2+, Gd3+, or the CaR modulator NPS R-467. Three region-specific anti-CaR peptide antisera immunoblotted bands of approximately 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 Ca2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca2+]o.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Aniline Compounds / pharmacology
  • Animals
  • COS Cells
  • Calcium / agonists
  • Calcium / pharmacology
  • Calcium-Binding Proteins / analysis
  • Calcium-Binding Proteins / genetics
  • Calcium-Binding Proteins / metabolism*
  • Cattle
  • Cell Line
  • Cyclic AMP / biosynthesis*
  • Female
  • Humans
  • Immune Sera
  • Immunoblotting
  • Immunohistochemistry
  • Inositol Phosphates / metabolism*
  • Kidney / cytology
  • Kidney / drug effects*
  • Kidney / embryology
  • Magnesium / pharmacology
  • Molecular Sequence Data
  • Oocytes / metabolism
  • Peptide Biosynthesis
  • Transfection
  • Xenopus laevis

Substances

  • Aniline Compounds
  • Calcium-Binding Proteins
  • Immune Sera
  • Inositol Phosphates
  • NPS R-467
  • Cyclic AMP
  • Magnesium
  • Calcium