Coordinated control of volume regulatory Na+/H+ and K+/H+ exchange pathways in Amphiuma red blood cells

Abstract

The Na(+)/H(+) and K(+)/H(+) exchange pathways of Amphiuma tridactylum red blood cells (RBCs) are quiescent at normal resting cell volume yet are selectively activated in response to cell shrinkage and swelling, respectively. These alkali metal/H(+) exchangers are activated by net kinase activity and deactivated by net phosphatase activity. We employed relaxation kinetic analyses to gain insight into the basis for coordinated control of these volume regulatory ion flux pathways. This approach enabled us to develop a model explaining how phosphorylation/dephosphorylation-dependent events control and coordinate the activity of the Na(+)/H(+) and K(+)/H(+) exchangers around the cell volume set point. We found that the transition between initial and final steady state for both activation and deactivation of the volume-induced Na(+)/H(+) and K(+)/H(+) exchange pathways in Amphiuma RBCs proceed as a single exponential function of time. The rate of Na(+)/H(+) exchange activation increases with cell shrinkage, whereas the rate of Na(+)/H(+) exchange deactivation increases as preshrunken cells are progressively swollen. Similarly, the rate of K(+)/H(+) exchange activation increases with cell swelling, whereas the rate of K(+)/H(+) exchange deactivation increases as preswollen cells are progressively shrunken. We propose a model in which the activities of the controlling kinases and phosphatases are volume sensitive and reciprocally regulated. Briefly, the activity of each kinase-phosphatase pair is reciprocally related, as a function of volume, and the volume sensitivities of kinases and phosphatases controlling K(+)/H(+) exchange are reciprocally related to those controlling Na(+)/H(+) exchange.

Fig. 1.

Unidirectional Na⁺ (○) and K⁺ (●) uptake rates as a function of media osmolarity (cell volume) in Amphiuma RBCs. Cells were suspended in media across a broad range of osmolarity (abscissa), and Na⁺ (²²Na⁺) or K⁺ (⁸⁶Rb⁺) unidirectional uptake rates were measured periodically over a 30-min interval. The rates reported in this figure are the maximal rates of Na⁺ or K⁺ uptake observed during the 30-min flux interval. The data represent 3 similar results.

Fig. 2.

Shrinkage-induced unidirectional Na⁺ (²²Na⁺) uptake in hyperosmotic media as a function of time. Cells were incubated in isosmotic media for 1 h prior to transfer (at T = 0) to hyperosmotic ²²Na⁺-containing media at 1.2 (●; 288 mosM), 1.4 (■; 336 mosM) or 1.6 (▴; 384 mosM) times isosmotic osmolarity. The data shown are individual data points from a single representative experiment. Solid lines represent the best fit to Eq. 1 (see materials and methods), yielding final Na⁺ fluxes of 2.0 ± 0.1, 8.0 ± 0.2, and 11.5 ± 0.2 mmol Na⁺/kg dry cell solid (dcs) × min for 1.2, 1.4, and 1.6 times isosmotic osmolarity. The analysis also revealed relaxation times (τ) of 64 ± 5 min (R² = 0.96), 36 ± 1 min (R² = 0.997), and 23 ± 0.4 min (R² = 0.999) for media 1.2, 1.4, and 1.6 times isosmotic, respectively. The data represent 4 similar results.

Fig. 3.

Shrinkage-induced activation of unidirectional Na⁺ (²²Na⁺) uptake in thermodynamically nulled media of increasing hyperosmolarity. The data in this figure were obtained under conditions identical to those in Fig. 2 with the exception that media Na⁺ concentrations were selected to maintain the Na⁺/H⁺ exchanger at thermodynamic equilibrium (nulled media). Cells were incubated in isosmotic media for 1 h prior to osmotic shrinkage by transfer at T = 0, to nulled hyperosmotic media at 1.2 (●), 1.4 (■), and 1.6 (▴) times isosmotic medium osmolarity. The final Na⁺ flux for these conditions was 0.46 ± 0.01, 2.40 ± 0.04, and 2.80 ± 0.02 mmol Na⁺/kg dcs × min for 1.2, 1.4, and 1.6 times isosmotic osmolarity. The calculated τ values from fluxes of cells in nulled hyperosmotic media are 76 ± 1 (R² = 0.997), 47 ± 1 (R² = 0.997) and 22.0 ± 0.2 (R² = 0.999) min, for 1.2, 1.4, and 1.6 times isosmotic, respectively. The data represent 3 similar results.

Fig. 4.

Deactivation of shrinkage-induced unidirectional Na⁺ (²²Na⁺) uptake as a function of time following swelling of preshrunken cells. Cells were suspended for 30 min in nulled hyperosmotic (384 mosM) medium to activate Na⁺/H⁺ exchange yet prevent net Na⁺ uptake and cell volume recovery during the incubation period. At time zero, preshrunken cells were swollen to or above normal control volume by transfer to isosmotic (230 mosM; ▴) or hyposmotic (173 mosM; ●) media respectively, and unidirectional Na⁺ (²²Na⁺) uptake was measured as a function of time after cell swelling. Curve regressions were generated with Eq. 1 as described in Fig. 1. The final Na⁺ flux for these conditions was 0.31 ± 0.06 and 0.31 ± 0.05 mmol Na⁺/kg dcs × min for isosmotic and 0.55 times isosmotic osmolarity. The τ values for shrinkage-induced Na⁺/H⁺ exchange deactivation in isosmotic and hyposmotic media are 4.4 ± 0.04 (R² = 0.996) and 2.6 ± 0.03 (R² = 0.997) min, respectively. The data represent 3 similar results.

Fig. 5.

K⁺/H⁺ exchange activation as a function of time after swelling in hyposmotic media. At time zero, cells were transferred to media of 192 mosM (0.8 times isosmotic; A), 156 mosM (0.65 times isosmotic; B), or 132 mosM (0.55 times isosmotic; C), and unidirectional K⁺ (⁸⁶Rb⁺) uptake was measured as a function of time after cell swelling in hyposmotic media. The final ⁸⁶Rb⁺ flux for these conditions was 0.048 ± 0.002, 0.0420 ± 0.0004, and 0.059 ± 0.001 mmol ⁸⁶Rb⁺/kg dcs × min, respectively, for 0.8, 0.65, and 0.55 times isosmotic osmolarity. The calculated relaxation times for K⁺/H⁺ exchange activation in 0.8, 0.65 and 0.55RVD are 30 ± 2 (R² = 0.98), 13.0 ± 0.2 (R² = 0.99), and <1 min (0 ± 1 min), respectively. The data represent 4 similar results.

Fig. 6.

Deactivation of swelling-induced K⁺/H⁺ exchange following cell shrinkage in hyperosmotic media. Cells were incubated for 30 min in null hyposmotic (0.55 times isosmotic; 132 mosM) media to activate K⁺/H⁺ exchange yet prevent net K⁺ loss and therefore changes in cell volume. At time = 0, the preswollen cells were transferred to normal isosmotic medium (240 mosM; ●) or medium 1.6 times isosmotic (384 mosM; ▴), and K⁺ (⁸⁶Rb⁺) uptake was measured as a function of time. The final ⁸⁶Rb⁺ flux for these conditions was 0.012 ± 0.001 and 0.021 ± 0.001 mmol ⁸⁶Rb⁺/kg dcs × min for isosmotic and 1.6 times isosmotic osmolarity. The calculated relaxation times for K⁺/H⁺ exchange deactivation in isosmotic and hyperosmotic media are 6.0 ± 0.1 (R² = 0.99) and 2.7 ± 0.1 (R² = 0.99) min, respectively. The data represent 3 similar results.

Fig. 7.

Model depicting the relative rates of the forward and reverse rate constants (k₁₂ and k₂₁, respectively) for Na⁺/H⁺ and K⁺/H⁺ exchange in isosmotic medium (cells at normal control volume; A). In isosmotic media the Na⁺/H⁺ and K⁺/H⁺ exchangers are both inactive (I) because the rate constants for the activating kinases (k₁₂) are small relative to the rate constants of the deactivating phosphatases (k₂₁). The Na⁺/H⁺ exchanger is controlled by a shrinkage-activated (swelling-deactivated) kinase (k₁₂^sh) and a swelling-activated (shrinkage-deactivated) phosphatase (k₂₁^sw). In contrast, the K⁺/H⁺ exchanger is activated by a swelling-activated (shrinkage-deactivated) kinase (k₁₂^sw) and deactivated by a shrinkage-activated (swelling-deactivated) phosphatase (k₂₁^sh). In hyperosmotic media (shrunken cells; B), Na⁺/H⁺ exchange is activated in response both to increases in the activity of the shrinkage-activated kinase (k₁₂^sh) that activates Na⁺/H⁺ exchange and to decreases in the activity of the swelling-activated phosphatase (k₂₁^sw) that deactivates Na⁺/H⁺ exchange. At the same time, the activity of the swelling-activated kinase (k₁₂^sw) that activates K⁺/H⁺ exchange is decreased below the kinase activity in isosmotic medium, whereas the activity of the shrinkage-activated phosphatase (k₂₁^sh) that deactivates K⁺/H⁺ exchange is increased relative to phosphatase activity in isosmotic medium. In contrast to hyperosmotic or isosmotic media, in hyposmotic media (swollen cells; C) the activity of the swelling-activated kinase (k₁₂^sw) that activates K⁺/H⁺ exchange is increased, whereas the activity of the shrinkage-activated phosphatase (k₂₁^sh) that deactivates K⁺/H⁺ exchange is decreased. In addition, during swelling the shrinkage-activated kinase (k₁₂^sh) responsible for Na⁺/H⁺ exchange activation is decreased, whereas the swelling-activated phosphatase (k₁₂^sw) that deactivates Na⁺/H⁺ exchange is greatly increased.