PMID- 26190181 OWN - NLM STAT- MEDLINE DCOM- 20160920 LR - 20200115 IS - 1872-9754 (Electronic) IS - 0197-0186 (Print) IS - 0197-0186 (Linking) VI - 90 DP - 2015 Nov TI - Galphai/o-dependent Ca(2+) mobilization and Galphaq-dependent PKCalpha regulation of Ca(2+)-sensing receptor-mediated responses in N18TG2 neuroblastoma cells. PG - 142-51 LID - S0197-0186(15)30010-3 [pii] LID - 10.1016/j.neuint.2015.07.008 [doi] AB - A functional Ca(2+)-sensing receptor (CaS) is expressed endogenously in mouse N18TG2 neuroblastoma cells, and sequence analysis of the cDNA indicates high homology with both rat and human parathyroid CaS cDNAs. The CaS in N18TG2 cells appears as a single immunoreactive protein band at about 150 kDa on Western blots, consistent with native CaS from dorsal root ganglia. Both wild type (WT) and Galphaq antisense knock-down (KD) cells responded to Ca(2+) and calindol, a positive allosteric modulator of the CaS, with a transient increase in intracellular Ca(2+) concentration ([Ca(2+)]i), which was larger in the Galphaq KD cells. Stimulation with 1 mM extracellular Ca(2+) (Ca(2+)e) increased [Ca(2+)]i in N18TG2 Galphaq KD compared to WT cells. Ca(2+) mobilization was dependent on pertussis toxin-sensitive Galphai/o proteins and reduced by 30 muM 2-amino-ethyldiphenyl borate and 50 muM nifedipine to the same plateau levels in both cell types. Membrane-associated PKCalpha and p-PKCalpha increased with increasing [Ca(2+)]e in WT cells, but decreased in Galphaq KD cells. Treatment of cells with 1 muM GÓ§ 6976, a Ca(2+)-specific PKC inhibitor reduced Ca(2+) mobilization and membrane-associated PKCalpha and p-PKCalpha in both cell types. The results indicate that the CaS-mediated increase in [Ca(2+)]i in N18TG2 cells is dependent on Galphai/o proteins via inositol-1,4,5-triphosphate (IP3) channels and store-operated Ca(2+) entry channels, whereas modulation of CaS responses involving PKCalpha phosphorylation and translocation to the plasma membrane occurs via a Galphaq mechanism. CI - Copyright (c) 2015 Elsevier Ltd. All rights reserved. FAU - Sesay, John S AU - Sesay JS AD - Cardiovascular Disease Research Program, Julius L Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA; Department of Biology, North Carolina Central University, Durham, NC 27707, USA; Department of Physiology and Pharmacology and Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. FAU - Gyapong, Reginald N K AU - Gyapong RN AD - Cardiovascular Disease Research Program, Julius L Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA. FAU - Najafi, Leila T AU - Najafi LT AD - Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, MO 63104, USA. FAU - Kabler, Sandra L AU - Kabler SL AD - Department of Physiology and Pharmacology and Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. FAU - Diz, Debra I AU - Diz DI AD - Department of Physiology and Pharmacology and Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA; Hypertension & Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. FAU - Howlett, Allyn C AU - Howlett AC AD - Department of Biology, North Carolina Central University, Durham, NC 27707, USA; Department of Pharmacological and Physiological Science, Saint Louis University, St. Louis, MO 63104, USA; Department of Physiology and Pharmacology and Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA; Hypertension & Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. FAU - Awumey, Emmanuel M AU - Awumey EM AD - Cardiovascular Disease Research Program, Julius L Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707, USA; Department of Biology, North Carolina Central University, Durham, NC 27707, USA; Department of Physiology and Pharmacology and Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA; Hypertension & Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. Electronic address: eawumey@nccu.edu. LA - eng GR - HL099139/HL/NHLBI NIH HHS/United States GR - HL059868/HL/NHLBI NIH HHS/United States GR - R01 HL064761/HL/NHLBI NIH HHS/United States GR - R01 DA003690/DA/NIDA NIH HHS/United States GR - SC1 HL099139/HL/NHLBI NIH HHS/United States GR - SC1 HL136278/HL/NHLBI NIH HHS/United States GR - R25 HL059868/HL/NHLBI NIH HHS/United States GR - HL064761,/HL/NHLBI NIH HHS/United States GR - P20 MD000175/MD/NIMHD NIH HHS/United States GR - DA03690/DA/NIDA NIH HHS/United States GR - MD000175/MD/NIMHD NIH HHS/United States GR - UH1 HL059868/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20150716 PL - England TA - Neurochem Int JT - Neurochemistry international JID - 8006959 RN - EC 2.4.2.31 (Pertussis Toxin) RN - EC 2.7.11.13 (PRKCA protein, human) RN - EC 2.7.11.13 (Protein Kinase C-alpha) RN - SY7Q814VUP (Calcium) SB - IM MH - Calcium/*metabolism MH - Calcium Signaling/*drug effects/physiology MH - Cell Line, Tumor MH - Cells, Cultured MH - Humans MH - Neuroblastoma/*metabolism MH - Pertussis Toxin/*pharmacology MH - Phosphorylation MH - Protein Kinase C-alpha/*metabolism MH - Sensory Receptor Cells/metabolism PMC - PMC4641771 MID - NIHMS717442 OTO - NOTNLM OT - Ca(2+) mobilization OT - CaS responses OT - Galpha(i/o) OT - Galpha(q) antisense knock-down OT - N18TG2 cells OT - Neuronal CaS OT - Protein kinase C EDAT- 2015/07/21 06:00 MHDA- 2016/09/22 06:00 PMCR- 2016/11/01 CRDT- 2015/07/21 06:00 PHST- 2014/10/01 00:00 [received] PHST- 2015/06/24 00:00 [revised] PHST- 2015/07/14 00:00 [accepted] PHST- 2015/07/21 06:00 [entrez] PHST- 2015/07/21 06:00 [pubmed] PHST- 2016/09/22 06:00 [medline] PHST- 2016/11/01 00:00 [pmc-release] AID - S0197-0186(15)30010-3 [pii] AID - 10.1016/j.neuint.2015.07.008 [doi] PST - ppublish SO - Neurochem Int. 2015 Nov;90:142-51. doi: 10.1016/j.neuint.2015.07.008. Epub 2015 Jul 16.