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

Ca2+ and other divalent cations modulate parathyroid hormone secretion by interacting with cell-surface Ca2+-sensing receptors (CaRs), We as sessed the ability of these receptors to couple to Ca2+ mobilization, inositol phosphate (InsP) accumulation, and cyclic AMP production in d ifferent expression systems, In Xenopus laevis oocytes injected with b ovine parathyroid CaR cRNA, the addition of extracellular cations to 1 .5 mM Ca2+, 5.5 mM Mg2+, Or 10 mu M Gd3+ significantly increased Ca-45 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 e xtracellular [Ca2+] ([Ca2+](0)) from 0.1 to >1.4 mM in oocytes and to >1.0 mM in HEK 293 tens stimulated significant increments in Ca-45 eff lux and InsP accumulation, respectively (p < 0.05), In contrast, Ca2and Mg2+ increased InsPs to a lesser extent in COS 7 cells transiently transfected with CaR cDNA, In HER 293 cells stably expressing CaR cDN A, there were significant reductions in cAMP content when adding high Ca2+, Mg2+, Gd3+, Or the CaR modulator NPS R-467, Three region-specide anti-CaR peptide antisera immunoblotted bands of similar to 140 and 1 55 kDa in membranes from CaR-transfected HEK 293 cells and bovine para thyroid tissue, Immunocytochemistry demonstrated strong cell-surface s taining in CaR-transfected HEK 293 cells and parathyroid tissue, which was absent when antisera were preabsorbed with CaR peptides. These re sults indicate that the activation of the recombinant CaR by extracell ular Ca2+ can couple negatively to adenylate cyclase but positively to phospholipase C (PLC), the latter at physiological [Ca2+](0).