We studied the relationship between changes in intracellular pH (pH i ), intracellular Ca2+([Ca2+] i ) and charybdotoxin sensitive (CTX) maxi-K+ channels occurring after modest `physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH i and [Ca2+] i by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 × isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 × isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 × isotonic dilution, the pH i alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this ΔpH i and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH4Cl permitted complete RVD which could be inhibited by CTX. The rate of 86Rb efflux which also increased after this 0.93 × isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na+-free medium. Modest swelling transiently increased [Ca2+] i and Ca2+-free medium or blocking alkalinization by MIA or Na+-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca2+-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca2+] i and volume changes became sensitive to CTX. We conclude that both alkalinization of pH i and increased [Ca2+] i observed with `physiological' volume increase are essential for the activation of CTX-sensitive maxi-K+ channels required for RVD.
The Journal of Membrane Biology – Springer Journals
Published: Nov 1, 1999
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