Altered Phosphorylation of RRXS*/T* Motif in Ouabain-Treated Renal Epithelial Cells is not Mediated by Inversion of the [Na]i/[K+]i Ratio.

Recently, we reported that the death of ouabain-treated C7-MDCK cells resembling principal cells from collecting ducts of the Madin-Darby canine kidney (MDCK) is caused by ouabain interaction with Na⁺,K⁺-ATPase but is not mediated by inversion of the [Na⁺]_i/[K⁺]_i ratio. The mechanism of this intriguing phenomenon remains unknown. We therefore examined the action of ouabain on serine/threonine phosphoproteins as possible intermediates of cell death signaling. The death of ouabain-treated C7-MDCK cells proceeded by altered phosphorylation of the RRXS*/T*-motif in 4 proteins with Mr from 80 to 25 kDa. Similarly to cell death, inversion of the [Na⁺]_i/[K⁺]_i ratio evoked by Na⁺,K⁺-ATPase inhibition in K⁺-free medium did not affect the phosphorylation of RRXS*/T*-proteins but increased their sensitivity to ouabain. The action of ouabain was preserved in the presence of activators of protein kinases A (forskolin), G (sodium nitroprusside) and C (PMA) as well as inhibitors of protein kinase C (Gö 6983, Gö 6976) and serine-threonine phosphatases (okadaic acid). Phosphorylation of RRXS*/T*-proteins was also noted in ouabain-sensitive C11-MDCK cells resembling intercalated cells from collecting ducts, but was absent in ouabain-resistant smooth muscle cells from the rat aorta. Our results show that altered phosphorylation of RRXS*/T*-proteins in ouabain-treated C7-MDCK cells is mediated by its interaction with Na⁺,K⁺-ATPase but is not caused by inversion of the [Na⁺]_i/[K⁺]_i ratio. The molecular origin of serine-threonine kinases and/or phosphatases involved in phosphorylation of ouabain-sensitive proteins and their role in cell death signaling should be examined further. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]