title: Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888.
abstract: To clarify the mechanism(s) underlying intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations induced by an elevation in extracellular Ca(2+) concentration ([Ca(2+)]e) via the extracellular Ca(2+)-sensing receptor (CaR), we analyzed the pattern of [Ca(2+)]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca(2+)]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca(2+)]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca(2+)]i oscillations, cells were exposed to increasing concentrations (0.5-5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca(2+). Ro-31-8220 at 3-5 μM completely eliminated the [Ca(2+)]e-evoked [Ca(2+)]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca(2+)]e-evoked [Ca(2+)]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca(2+)]e-evoked [Ca(2+)]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr(888) was converted to alanine (CaRT888A) showed [Ca(2+)]i oscillations after CaR activation. Our results show that [Ca(2+)]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca(2+) or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr(888).
authors: Division of Digestive Diseases, Department of Medicine, Center for Ulcer Research and Education: Digestive Diseases Research Center David Geffen School of Medicine and Molecular Biology Institute, University of California, Los Angeles, California; and.
authors: Steven H
authors: SH
authors: Young
authors: Osvaldo
authors: O
authors: Rey
authors: James
authors: J
authors: Sinnett-Smith
authors: Enrique
authors: E
authors: Rozengurt
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D002118:Calcium / Q000819:agonists / Q000378:metabolism*; D020013:Calcium Signaling; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D017136:Ion Transport; D008301:Maleimides / Q000494:pharmacology; D010627:Phenethylamines; D010766:Phosphorylation; D011437:Propylamines; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*
chemical_list: C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D000814:Aniline Compounds; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D044169:Receptors, Calcium-Sensing; D051571:Protein Kinase C-alpha; D002118:Calcium; C064758:Ro 31-8220
keywords: G protein-coupled receptor; PKC downregulation; PKC inhibitor Ro-31-8220; mutant CaRt888a; single cell Ca2+ imaging