To assess the activation of the charybdotoxin-insensitive K+ channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH (pH i ), intracellular calcium ([Ca2+] i ) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume changes. Fluorescence spectroscopy was used to measure pH i and [Ca2+] i of cells in suspension, loaded with 2,7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca2+] i to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification of ≃0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 μm), an inhibitor of Ca2+/calmodulin kinase II, blocked RVD. Gramicidin (0.5 μm) bypassed this inhibition suggesting that the K+ channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase; inhibition of RVD generated by increasing the K+ gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 μm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K+ channels utilized for RVD after substantial (>7%) `nonphysiological' volume increases requires phosphorylation mediated by Ca2+/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca2+] i are a critical determinant of this activation.
The Journal of Membrane Biology – Springer Journals
Published: Nov 1, 1999
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