The extracellular Ca2+-sensing receptor (CaR) has been associated with alterations in cell fate, such as protection from apoptosis in ovarian cells and proliferation in fibroblasts and osteoblasts. We have recently reported that the proximal tubular opossum kidney (OK) cell line expresses CaR and responds to CaR agonists (extracellular Ca2+o and aminoglycoside antibiotics e.g. neomycin) with increased Ca2+i mobilisation and activation of the extracellular signal-regulated kinase, ERK (Ward et al. 2002).
To investigate the involvement of CaR in cell-fate signalling in renal cells we studied the effects of neomycin on the phosphorylation status of the PI3-kinase-dependent, anti-apoptotic kinase, Akt (also known as protein kinase B). Akt is activated upon the dual phosphorylation of residues Thr-308 and Ser-473, a mechanism that was studied by semi-quantitative immunoblotting using phospho-specific polyclonal antibodies to these two residues (pAkt T308 and pAkt S473).
Treatment of OK cells (ATCC, Rockville, MD, USA) at 37 °C for 5 min with either neomycin or gentamicin (300 µM) increased the phosphorylation of pAkt S473 (430 ± 117 and 486 ± 109 %, respectively, means ± S.E.M.). Neomycin-induced pAkt S473 phosphorylation was inhibited by the PI3-kinase inhibitors wortmannin (30 nM; Neo, 345 ± 109 % control; wortmannin and Neo, 13 ± 2% N = 3; P < 0.001 by Student’s unpaired t test) and LY294002 (30 mM; Neo, 510 ± 360 % control; LY294002 and Neo, 17 ± 8% N = 3; P < 0.001) and by the PLC inhibitor U73122 (5 mM; Neo, 259 ± 72 % control; U73122 and Neo, 6 ± 3% N = 3; P < 0.001). However, the neomycin responses were unaffected by the PKC inhibitor GF109203X (500 nM; Neo, 244 ± 79 % control; GF109203X and Neo, 277 ± 98% N = 3; N.S.) or by the MEK (ERK kinase) activation blocker PD98059 (10 mM; Neo, 177 ± 14 % control; PD98059 and Neo, 162 ± 9% N = 3; N.S.). Qualitatively similar observations were made in all of the above experiments using the antibody to pAkt T308.
The current data indicate that in CaR-expressing OK cells, aminoglycoside treatment causes a PLC-dependent and PI3-kinase-dependent stimulation of Akt/PKB that does not involve either PKC or the MEK/ERK pathway.
The Wellcome Trust supported this work. D.W. is an NKRF Training Fellow.