Technical Reference #216

216.

Urea signaling to ERK phosphorylation in renal medullary cells requires extracellular calcium but not calcium entry Xiao-Yan Yang; Hongyu Zhao; Zheng Zhang; Karin D Rodland; Jean-Baptiste Roullet; David M. Cohen, Oregon Health Sciences University, American Journal of Physiology: Renal Physiology, 280(216), (2001)
Link To Paper

Abstract:
The renal cell line mIMCD3 exhibits markedly upregulated phosphorylation of the extracellular signal-regulated kinase (ERK) 1 and 2 in response to urea treatment (200 mM for 5 min). Previous data have suggested the involvement of a classical protein kinas

Keywords:
fura 1; inner medullary collecting duct; Madin-Darby canine kidney; protein kinase C; hypotonicity

Materials & Methods:
Cell culture experimental manipulations and materials. Cells (mIMCD3) were grown in DMEM-F-12 in monolayers (32) and on achieving confluence they were serum deprived for 24 h before experimental manipulation unless otherwise noted. Cells were treated with the gentle dropwise addition of concentrated stocks of urea or NaCl to achieve the desired final osmolarity. Downregulation of classic PKC (cPKC) isoforms was achieved through 6- or 18-h pretreatment with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA). For depletion of intra- and extracellular calcium monolayers weretaken out of serum for 24 h and treated with 12-bis(2- aminophenoxy)ethane NNN9N9-tetraacetic acid-acetomethoxy ester (BAPTA-AM; 75 mM for 30 min) after which medium was washed twice with Hanks’ balanced salt solution (HBSS; 130 mM NaCl 5 mM KCl 1.5 mM CaCl2 1 mM MgCl2 20 mM HEPES pH 7.4 10 mM glucose) or calciumfree HBSS (130 mM NaCl 5 mM KCl 1 mM MgCl2 20 mM HEPES pH 7.4 10 mM glucose 10 mM EDTA) and cells were incubated in either HBSS or calcium-free HBSS for 30 min before treatment with urea (200 mM for 5 min) or TPA (100 nM for 5 min). Inhibitors and activators of calcium transport and action (obtained from Calbiochem unless otherwise indicated) were used as follows: KN-93 20 mM for 30 min; A-23187 10 mM for 5–30 min; thapsigargin 10–100 nM times indicated duration; epidermal growth factor (EGF Sigma) 100 nM times indicated duration; dantrolene 100 mM for 30 min; xestospongin C 10 mM for 30 min; U-73122 5–20 mM for 30 min; verapamil 10–100 mM for 30 min; nimodipine 10 mM for 30 min; NiCl2 (Sigma) 1–3 mM for 30 min; CdCl2 (Sigma) 1–3 mM for 30 min; carboxyamidotriazole (CAI) 10 mM for 30 min; NNN9N9-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN; Sigma) 2 mMfor 30 min; caffeine 10 mM for 5 min; calmidazolium 20 mM for 30 min; gadolinium chloride (Sigma) 20 mM-1 mM for 2–60 min; BAY K 8644 10–100 mMfor 30 min; ATP 10 mMfor indicated interval; and bradykinin 100 nM for indicated interval. For titration of extracellular calcium (see Fig. 11) BAPTA concentration ([BAPTA]) necessary to establish indicated free calcium concentration (1.44 2.33 and 5.43 mM BAPTA for 300 100 and 30 nM free calcium respectively) was estimated using MaxChelator (3) shareware (www.stanford.edu/ ;cpatton/maxc.html). Immunoblot experiments were performed at least twice with equivalent results; calcium ratiometry was performed at least three times per condition on 3 separate days with equivalent results. Because it was observed that 30 min of calcium depletion approached the threshold of cell tolerance in terms of adherence an alternative strategy was employed to partially dissociate the effects of adherence and calcium depletion. Cells were grown on collagen-treated dishes (BioCoat; Falcon/Becton- Dickinson 354400) which facilitate integrin-mediated binding to the epithelial cell surface. Confluent mIMCD3 or Madin-Darby canine kidney (MDCK) cells were washed three times with minimal essential medium (MEM) (calcium-free) medium (Life Technologies 11385) to deplete extracellular calcium and placed in MEM. Cell adhesion of mIMCD3 was excellent; at 30 min cells were identical in appearance to control (calcium-replete) cells. At 2 h nearly all cells remained adherent; however by 6 h a substantial percentage of cells (50%) became nonadherent. Similarly on collagen substrate MDCK monolayers remained completely attached beyond 6 h of calcium depletion.

Microscopic Technique
Fluorescence Microscopy, fluorescence ratiometric imaging

Cell Type(s)
MDCK