title: Stimulation by parathyroid hormone of calcium absorption in confluent Madin-Darby canine kidney cells.
abstract: Parathyroid hormone (PTH) increases renal calcium absorption exclusively in cortical thick limbs and distal tubules. Lack of sufficient tissue has precluded detailed biochemical study of the mechanisms responsible for the hypercalcemic effect of PTH. Therefore, we assessed PTH action on calcium transport in Madin-Darby canine kidney (MDCK) cells, a cell line expressing distal characteristics, to determine its suitability as a model for analyzing PTH action. Calcium transport across MDCK cells grown to confluence on porous filters was measured at 37 degrees C in Ussing chambers. Mucosal-to-serosal calcium fluxes (JCa, mol/min cm-2 x 10(-9)) were measured with 45Ca at -3, -1, 5, 10, and 20 min; agonist was added at 0 min. Basal JCa averaged 0.98. PTH at 0.2 microM increased JCa by 12% (P less than 0.05) and 1 microM PTH by 70% (P less than 0.01). Calcitonin (1 microM) had no effect on JCa. The fact that high concentrations of dibutyryl cAMP (1 mM) and forskolin (10 microM) increased JCa by only 37% and 22%, respectively, suggested that cAMP-independent mechanisms may participate in PTH-stimulated JCa. Therefore we examined the effect of other putative second messengers. In the presence of 2 mM external [Ca], 10 nM A23187 increased JCa by 88%, and 10 microM A23187 increased JCa by 121%. Addition of 10 microM phorbol 12-myristate 13-acetate (PMA) increased JCa by 60%. We conclude that: 1) PTH specifically stimulates unidirectional calcium absorption in MDCK cells; 2) both adenylate cyclase-coupled and calcium-coupled receptors may participate in signaling the response to PTH; and 3) confluent MDCK cells represent a useful experimental model for elucidating the biochemical mechanisms involved in the renal hypercalcemic action of PTH.
authors.affiliation: Department of Pharmacology, Dartmouth Medical School, Hanover, New Hampshire 03756.
authors.forename: S M
authors.initials: SM
authors.lastname: Kennedy
authors.forename: J L
authors.initials: JL
authors.lastname: Flanagan
authors.forename: J W
authors.initials: JW
authors.lastname: Mills
authors.forename: P A
authors.initials: PA
authors.lastname: Friedman
mesh_terms: D000042:Absorption; D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics*; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D004285:Dogs; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D015290:Second Messenger Systems
chemical_list: D010281:Parathyroid Hormone; D000242:Cyclic AMP; D002118:Calcium