title: Conditionally immortalized human proximal tubular epithelial cells isolated from the urine of a healthy subject express functional calcium-sensing receptor.
abstract: The calcium-sensing receptor (CaSR) is a G protein-coupled receptor, which plays an essential role in regulating Ca(2+) homeostasis. Here we show that conditionally immortalized proximal tubular epithelial cell line (ciPTEC) obtained by immortalizing and subcloning cells exfoliated in the urine of a healthy subject expresses functional endogenous CaSR. Immunolocalization studies of polarized ciPTEC revealed the apical localization of the receptor. By Western blotting of ciPTEC lysates, both monomeric and dimeric forms of CaSR at 130 and ∼250 kDa, respectively, were detected. Functional studies indicated that both external calcium and the positive CaSR allosteric modulator, NPS-R568, induced a significant increase in cytosolic calcium, proving a high sensitivity of the endogenous receptor to its agonists. Calcium depletion from the endoplasmic reticulum using cyclopiazonic acid abolished the increase in cytosolic calcium elicited by NPS-R568, confirming calcium exit from intracellular stores. Activation of CaSR by NPS-R significantly reduced the increase in cAMP elicited by forskolin (FK), a direct activator of adenylate cyclase, further confirming the functional expression of the receptor in this cell line. CaSR expressed in ciPTEC was found to interact with Gq as a downstream effector, which in turn can cause release of calcium from intracellular stores via phospholipase C activation. We conclude that human proximal tubular ciPTEC express functional CaSR and respond to its activation with a release of calcium from intracellular stores. These cell lines represent a valuable tool for research into the disorder associated with gain or loss of function of the CaSR by producing cell lines from patients.
authors: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Aldo Moro, Italy;
authors: Annarita
authors: A
authors: Di Mise
authors: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Aldo Moro, Italy;
authors: Grazia
authors: G
authors: Tamma
authors: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Aldo Moro, Italy;
authors: Marianna
authors: M
authors: Ranieri
authors: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Aldo Moro, Italy; Center of Excellence in Comparative Genomics(CEGBA), Bari, Italy;
authors: Maria
authors: M
authors: Svelto
authors: Department of Pediatric Nephrology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; and.
authors: Bert van den
authors: Bv
authors: Heuvel
authors: Department of Pediatric Nephrology, University Hospital Gasthuisberg, Leuven, Belgium.
authors: Elena N
authors: EN
authors: Levtchenko
authors: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Aldo Moro, Italy; Center of Excellence in Comparative Genomics(CEGBA), Bari, Italy; giovanna.valenti@uniba.it.
authors: Giovanna
authors: G
authors: Valenti
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing / Q000032:analysis / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D010627:Phenethylamines; D011437:Propylamines; D044169:Receptors, Calcium-Sensing; D002118:Calcium
keywords: NPS-R568; calcium signaling; calcium-sensing receptor; conditionally immortalized proximal tubular epithelial cell line