Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Abstract

Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na(+)/H(+) and K(+)/H(+) exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na(+)/H(+) exchange and swelling-induced K(+)/H(+) exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1-2 orders of magnitude increase in the activity of both K(+)/H(+) and Na(+)/H(+) exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na(+) uptake and K(+) loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na(+)/H(+) and K(+)/H(+) exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K(+)/H(+) or Na(+)/H(+) exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na(+)/H(+) and K(+)/H(+) exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control.

Fig. 1.

Changes in cell Na⁺ and K⁺ content following exposure to 1 μM calyculin A (CLA) in isotonic medium. Amphiuma red blood cells (RBCs) were preincubated in isotonic media in the presence of ouabain (1 mM) for 90 min. At time 0 the cells were transferred to isotonic medium + 1 μM CLA, samples were removed at the times indicated and analyzed for Na⁺ (squares) and K⁺ (circles) content. The data are from two independent experiments (solid and dashed lines), representing 4 similar results.

Fig. 2.

Cell water content in Amphiuma RBCs in control and experimental media. Amphiuma RBCs were transferred at time 0 from isotonic medium to either isotonic (▪), isotonic + 1 μM CLA (▵), hypertonic (○) or hypotonic medium (•). Aliquots were removed at specified times, and water content was determined gravimetrically, normalized to dried whole cell solids (dcs). Significant differences (***P < 0.001) were seen in water content for cells in hypertonic or hypotonic media over 30 min relative to the control condition. No significant difference in cell water content was seen in cells treated with CLA versus untreated cells in isosmotic media (n = 5 means ± SE).

Fig. 3.

Time course for CLA-induced unidirectional Na⁺ and Rb⁺ uptake by Amphiuma RBCs in isotonic media. To initiate the experiment, cells were transferred at time 0 to ²²Na⁺ or ⁸⁶Rb⁺ containing isotonic media + 1 μM CLA and sampled at 3-min intervals during the flux period to measure Na⁺ (○) and Rb⁺ (•) uptake. Similar results were obtained in 4 experiments.

Fig. 4.

CLA concentration dependence of unidirectional Na⁺ (○) and Rb⁺ (•) uptake in isotonic medium. To initiate flux, cells were transferred to ²²Na⁺- and ⁸⁶Rb⁺-containing medium at the CLA concentration indicated in the ordinate axis. Both Na⁺ and Rb⁺ unidirectional uptake rates were measured and expressed as mmol·kg dcs⁻¹·min⁻¹. The data were fit to a sigmoidal dose-response function by nonlinear regression to obtain values for the half-maximal stimulatory concentration (EC₅₀) of CLA for Na⁺ (dashed line) and K⁺ (solid line) flux. EC₅₀ values for CLA in this experiment were 42.3 nM for Na⁺ flux and 39.9 nM for K⁺ flux. Similar results were obtained in 3 experiments.

Fig. 5.

CLA-induced unidirectional ²²Na⁺ (○) and ⁸⁶Rb⁺ (•) uptake in nulled isotonic medium containing 0.5 μM CLA. Cells were preincubated in ouabain (1 mM)-containing isotonic media for 90 min. At time 0 cells were placed in nulled isotonic media + 500 nM CLA, and samples were removed at the times indicated to measure ²²Na⁺ and ⁸⁶Rb⁺ unidirectional uptake. Similar results were obtained in 3 experiments.

Fig. 6.

Inhibition of CLA-induced unidirectional ²²Na⁺ (○) and ⁸⁶Rb⁺ (•) uptake by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in nulled isotonic medium. Cells were preincubated in ouabain (1 mM)-containing nulled isotonic media + 500 nM CLA for 35 min. At time 0 cells were placed in isotope-containing nulled isotonic media and sampled over a 5-min interval to measure ²²Na⁺ (○) and ⁸⁶Rb⁺ (•) unidirectional uptake rates. The half-maximal inhibitory concentration (IC₅₀) for EIPA was 1.0 μM for ²²Na⁺ and 1.5 μM for ⁸⁶Rb⁺. Similar results were obtained in 3 experiments.

Fig. 7.

Effect of CLA on Na⁺ (○) and K⁺ (•) content of cells in isotonic media. Cells were incubated in normal isotonic media + 1 mM ouabain for 90 min (period 1) before suspension in thermodynamically nulled isotonic medium containing 1 mM ouabain and 1 μM CLA for 30 min (period 2). Subsequently, the cells were transferred to CLA-free normal isotonic medium at time = 30 min (period 3). Samples were removed at the times indicated and analyzed for Na⁺, K⁺, and water content. Similar results were obtained in 4 experiments.

Fig. 8.

Selective activation of net Na⁺ or K⁺ flux in Amphiuma RBCs by CLA in anisotonic media. Shown are the Na⁺ (○) and K⁺ (•) content of cells exposed to CLA in thermodynamically nulled hypotonic (A) or hypertonic (B) media for 30 min, then transferred to CLA-free, hypotonic (A) or hypertonic (B) media at time = 30 min. The data were generated from the same batch of cells as those in Fig. 7. Similar results were obtained in 3 experiments.

Fig. 9.

Potential model for the activation and deactivation of the volume-sensitive Na⁺/H⁺ and K⁺/H⁺ exchangers in Amphiuma RBCs. In this scheme, AM/H° represents the alkali metal/H⁺ exchanger at minimal activity. Na/H* and K/H* represent the alkali metal/H⁺ exchanger activated in the Na⁺/H⁺ and K⁺/H⁺ exchanger mode.