The expression of the gamma subunit of Na-K-ATPase is regulated by osmolality via C-terminal Jun kinase and phosphatidylinositol 3-kinase-dependent mechanisms

Abstract

The alpha and beta subunits of Na-K-ATPase are up-regulated by hypertonicity in inner-medullary collecting duct cells adapted to survive in hypertonic conditions. We examined the regulation of the gamma subunit by hypertonicity. Although cultured inner-medullary collecting duct cells lacked the gamma subunits, both variants gamma(a) and gamma(b) were expressed in cells adapted to 600 and 900 mosmol/KgH(2)O. This expression was reversible with a half-time of 17.2 +/- 0.5 h. The message of the gamma subunit was absent in isotonic conditions and increased with higher tonicity in adapted cells. In acute experiments the appearance of the gamma subunit was found to be both time-dependent (> or =24 h) and osmolality-dependent (> or =500 mosmol/KgH(2)O). No induction was noted with urea and only minimal induction with mannitol. Increasing concentrations of the phosphatidylinositol 3-kinase inhibitor LY294002 resulted in a dose-dependent decrement in the expression of the gamma subunit with total abolition at 10 microM. This was associated with a decrease in cell viability as <20% survived the treatment with 10 microM of LY294002. Neither inhibition of extracellular response kinase nor p38 mitogen-activated protein kinase inhibited osmotic induction of the gamma subunit. In contrast, cells transfected with a dominant negative c-Jun N-terminal kinase 2-APF construct displayed complete inhibition of the gamma subunit. Such cells have accelerated loss of viability in hypertonic conditions. This study describes the regulation of the gamma subunit of Na-K-ATPase by hypertonicity. This regulation is transcriptionally regulated and involves signaling mediated by phosphatidylinositol 3-kinase and c-Jun N-terminal kinase 2 pathways.

Figure 1

(A) Cell lysates (100 μg of protein per line) from IMCD3 control and adapted cells were analyzed by Western blot using γ subunit ATPase antibody. The identity of γ_b was corroborated with a specific antibody. (Upper) The mean and SEM from four different experiments at 300 and 600 mosmol/kgH₂O and three at 900 mosmol/kgH₂O. (Lower) A representative Western blot. (B) Cytosolic RNA was isolated from IMCD3 control and adapted cells as described. Aliquots of RNA (15 μg) were run in duplicate agarose gels, transferred to a nylon membrane, and probed with ³²P-labeled oligonucleotide mapping to the middle of γ ATPase mRNA (Upper) or stained with ethidium bromide reflecting equal loading (Lower). Arrows indicate M_r markers.

Figure 2

IMCD3 cells adapted to 600 mosmol/kgH₂O were transferred to 300 mosmol/kgH₂O medium, harvested at different time intervals, and analyzed by Western blot. Mean and SEM of γ variant band intensity at initial time and after 3 days under isotonicity (n = 3). (Inset) The γ_b band intensity data were subjected to best-fit analysis and determined to match one-component exponential decay kinetics with k = 0.0408 h⁻¹.

Figure 3

Effects of water hydration in mice on γ_a (solid bars) and γ_b (open bars) variants in kidney inner medulla. Note the different scales for the two variants. Mice tissues were harvested after 1 wk of treatment and homogenates analyzed by Western blot (n = 4). The expression of γ_a (*, P < 0.02) and γ_b (**, P < 0.002) Na-K-ATPase subunits were significantly decreased in the inner medulla of water diuresis mice drinking D5W (5% dextrose in water). A representative Western blot of the inner medulla γ Na-K-ATPase obtained from two control and four D5W groups of mice are shown.

Figure 4

IMCD3 cells were challenged with 200 mosmol/kgH₂O of NaCl (A) or 250 mosmol/kgH₂O of NaCl (B) for 24 h, 48 h, or adapted to such conditions. Cell lysates were analyzed by Western blot (n = 4). Notice that in B there is a break in the scale for γ_b in the adapted cells. (C) Cells were challenged with 250 mosmol/kgH₂O, mannitol, or urea for 48 h (n = 4) and analyzed as above. A representative blot shown of two of these experiments is shown.

Figure 5

(A) Confluent IMCD3 cell cultures were kept for 24 h in low serum medium, incubated for 2 h with different LY294002 concentrations, and challenged with 250 mosmol/kgH₂O of NaCl for 48 h. Cell lysates were prepared and analyzed by Western blot analysis (n = 4). A representative blot of two of these experiments is shown. *, P < 0.01 when compared to controls without LY294002. (B) IMCD3 cells were grown in 24-well plates to confluence and treated as above with or without 10 μM LY294002 and 250 mosmol/kgH₂O NaCl. Cell survival was measured by the formation of formazan as described in Materials and Methods. Results are the mean and SEM of six determinations.

Figure 6

(A) Confluent IMCD3 cell cultures were kept for 24 h in low serum medium, incubated for 2 h with either SB203580 (10 μM) or PD98059 (20 μM), and challenged with 250 mosmol/kgH₂O of NaCl for 48 h. Cell lysates were prepared and analyzed by Western blot analysis (n = 4). A representative blot of two experiments is shown. (B) Wild-type IMCD3 cells and the dominant negative transfected JNK1-APF clone no. 5 and JNK2-APF clone no. 10 were challenged with 250 mosmol/kgH₂O NaCl for 24 and 48 h before being analyzed by Western blot (n = 4). A representative blot of two experiments is shown.