SURPLUS NA+ PUMPS - HOW LOW-K+-INCUBATED LLC-PK(1) CELLS RESPOND TO K+ RESTORATION

We have previously shown that a pig kidney cell line (LLC-PK1/Cl4) responds to chronic exposure to 0.25 mM extracellular K+ by increasing the beta-, not alpha-, subunit mRNA levels and both alpha- and beta-abundance twofold over control (15). Our objective in the present study was to determine how the LLC-PK1/Cl4 cells respond when returned to control (5.5 mM) medium. A 1.8-fold increase in ouabain binding established that the induced pumps were expressed at the cell surface following 24-h incubation in low K+. On restoration to 5.5 mM K+, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i, respectively) rapidly returned to control levels within 15 min. The doubled pool size of pumps in the chronic low K+ cells had no significant influence on the rate of ion restoration when compared with the rate in cells acutely exposed to low K+. Despite the rapid return of ions to control values, beta-mRNA levels remained elevated for 2 h, then sharply declined to control levels by 6 h of K+ restoration. From these data, we estimate that the half-life of beta-mRNA is 2-3 h during restoration. Alpha-Subunit mRNA remained essentially unchanged from control after return of K+ to the medium and restoration of intracellular ions. Both alpha- and beta-synthesis rates remained elevated at 2 h and returned to near control levels by 8 h of K+ restoration, parallel to the change in beta-mRNA. Both alpha- and beta-subunit abundance and Na+-K+-adenosinetriphosphatase (ATPase) activity also remained elevated for 2 h, then decreased sharply between 2 and 8 h of K+ restoration to their zero time point control levels. Beta-Activity and Na+-K+-ATPase activity remained slightly elevated over the time-paired controls until 24 h of restoration. The rates of return in alpha, beta, and activity predict an increase in Na+ pump subunit degradation rate in K+ restored cells. We conclude that, since [Na+]i and [K+]i return to control within 15 min, whereas beta-mRNA and alpha- and beta-synthesis rates remain elevated for 2 h after K+ restoration, these pathways do not quickly respond to these ionic signals in LLC-PK, cells and that it is likely that both decreased synthesis rates and increased degradation rates contribute to the eventual return of Na+-K+-ATPase activity and pool size to control in K+ restored cells.