Hormonal-dependent recruitment of Na+,K+-ATPase to the plasmalemma is mediated by PKC beta and modulated by [Na+]i

Abstract

1. The present study demonstrates that stimulation of hormonal receptors of proximal tubule cells with the serotonin-agonist 8-hydroxy-2-(di-n-propylamino) tetraline (8-OH-DPAT) induces an augmentation of Na(+),K(+)-ATPase activity that results from the recruitment of enzyme molecules to the plasmalemma. 2. Cells expressing the rodent wild-type Na(+),K(+)-ATPase alpha-subunit had the same basal Na(+),K(+)-ATPase activity as cells expressing the alpha-subunit S11A or S18A mutants, but stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. 3. 8-OH-DPAT treatment of OK cells led to PKC(beta)-dependent phosphorylation of the alpha-subunit Ser-11 and Ser-18 residues, and determination of enzyme activity with the S11A and S18A mutants indicated that both residues are essential for the agonist-dependent stimulation of Na(+),K(+)-ATPase activity. 4. When cells were treated with both dopamine and 8-OH-DPAT, an activation of Na(+),K(+)-ATPase was observed at basal intracellular sodium concentration (approximately 9 mM), and this activation was gradually reduced and became a significant inhibition as the concentration of intracellular sodium gradually increased from 9 to 19 mM. Thus, besides the antagonistic effects of dopamine and 8-OH-DPAT, intracellular sodium modulates whether an activation or an inhibition of Na(+),K(+)-ATPase is produced.

Figure 1

Effect of the PKC_β-inhibitor LY333531 on the 8-OH-DPAT-induced activation of Rb⁺-transport mediated by the Na⁺,K⁺-ATPase. Cells were treated with either 10 nM or 3 μM LY333531 for 30 min before treatment with various concentrations of 8-OH-DPAT for 10 min. Control Rb⁺-transport was measured in the absence of LY333531. The basal Na⁺,K⁺-ATPase activity was 8.3±0.7 nmol mg min⁻¹. *Indicates significant differences with respect to the basal values. #Indicates significant differences compared to the respective control. Data were analysed by one-way analysis of variance (P<0.01).

Figure 2

The stimulatory effects of PMA and 8-OH-DPAT on the Na⁺,K⁺-ATPase activity are not additive. Cells were treated with 1 μM PMA and/or 3 μM 8-OH-DPAT for 10 min before assay of Rb⁺-transport. When indicated, cells were treated with 0.1 μM staurosporine for 30 min. The percentage of change for each experimental condition was calculated with respect to a control in the absence of 8-OH-DPAT and/or PMA. *P<0.05 with respect to control.

Figure 3

Both Ser-11 and Ser-18 are essential for the stimulation of Na⁺,K⁺-ATPase activity induced PMA or 8-OH-DPAT. The Rb⁺-transport mediated by the Na⁺,K⁺-ATPase of cells expressing the wild-type rodent α1-subunit and three α1 mutants was determined. Δ(1–26) is the mutant in which amino acids 1–26 of the mature α1-subunit were deleted. S11A and S18A represent the activity in cells expressing the α1 mutants in which Ser-11 and Ser-18 have been substituted by alanine residues. The percentage of change for each condition was calculated with respect to a control in the absence of either 8-OH-DPAT or PMA. *P<0.05 with respect to control.

Figure 4

Phosphorylation of the α1-subunit Ser-11 and Ser-18 residues induced by 8-OH-DPAT. The effect of 8-OH-DPAT treatment on the phosphorylation of the rodent α1 subunit wild-type and the S11A and S18A mutants was determined. A representative autoradiogram and the corresponding Western blot are shown in the upper panel. The ratio of radioactivity to protein was calculated for each α1 subunit band. No significant change in the basal level of phosphorylation between mutants and wild-type α-subunit was observed. Data are presented as a percentage of 8-OH-DPAT-dependent phosphorylation with respect to the corresponding non-treated controls. *P<0.05 with respect to the maximal level of 8-OH-DPAT-dependent phosphorylation of wild-type α1.

Figure 5

Binding of biotin to the plasma membrane Na⁺,K⁺-ATPase of OK cells transfected with the rodent α1-subunit. Plasmalemma proteins of OK cells transfected with the rodent α1 subunit were labelled by treatment with NHS-biotin. A representative experiment of the quantitation of biotin is illustrated in the upper panel. Data are presented as the percentage of biotinylation induced by either PMA or 8-OH-DPAT with respect to a non-treated control.

Figure 6

The intracellular Na⁺ concentration modulates the effects induced by 8-OH-DPAT and dopamine on the Na⁺,K⁺-ATPase activity. Cells were incubated with the indicated concentrations of monensin for 30 min before treatment with 3 μM 8-OH-DPAT for 10 min and/or 1 μM dopamine for 5 min. The percentage of change for each concentration of monensin was calculated with respect to a control in the absence of 8-OH-DPAT and dopamine. *P<0.05 with respect to a control.