Fig. 5. Sodium bicarbonate and hypertonicity or hyperosmolarity induce kAE1 phosphorylation. A range of treatments were added to MDCKI-kAE1 cells. Cells were then lysed and two immunoprecipitations conducted per 10 cm² dish using Bric170. The immunoprecipitates were run on duplicate 8% SDS-PAGE gels and blots probed with either anti-Y359-P or anti-Y904-P, stripped and probed with anti-AE1Ct. A 5 minute pervanadate-positive control is included, loaded at half an immunoprecipitation equivalent to reduce the signal strength. (A) Effect of 1 hour exposure to either acidic pH 6.8 or increasing pH with either NaOH or NaHCO₃. Only the pH 8.7 NaHCO₃ medium induced kAE1 phosphorylation (on both Y359 and Y904) in MDCKI cells. (B) Effect of 1 hour exposure to various hypertonic media on kAE1 phosphorylation. Hypertonic solutions induce phosphorylation of kAE1 on Y359 but not Y904. NaHCO₃ induced a consistently stronger response than hypertonicity alone and this was not inhibited by 1 mM DIDS (see supplementary material Fig. S6D for quantification). (C) Comparison of the effects of hypotonic medium (medium diluted 1:1 with water), cell swelling induced by 300 mM urea, 514 mM NaCl, 1 M sucrose or 1 M sorbitol. Hyperosmotic sucrose or sorbitol induced a greater phosphorylation of kAE1 than NaCl but this was still not as great as the effects of NaHCO₃. The application of a hypotonic solution or cell swelling induced by urea had no effect on kAE1 phosphorylation. (D) Effect of incubation with 1 mM 8-bromo-cAMP, 60 µM forskolin, 5 µM ionomycin, 5 µM A23787, 1 µM thapsigargin, 10 µM PMA, either 200 µM H₂O₂ or 1 mM H₂O₂ and 100 µM ATP for 1 hour on MDCKI-kAE1 cells. No kAE1 phosphorylation was observed under the conditions tested using any of these reagents. Results representative of three separate experiments. (E) Effect of 1 hour exposure to vasopressin, angiotensin II or aldosterone. None of these hormones induced kAE1 phosphorylation under the conditions tested. Representative of at least two separate experiments. PV, pervanadate.