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.

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

“…In unstimulated cells at normal volume, phosphatase activity is dominant and maintains NHE1 in a tonic inactivated state. NHE1-inactivating phosphatase activity decreases precipitously with cell shrinkage upon suspension of cells in hyperosmotic media [32] . In contrast, NHE1-activating kinase activity increases as a graded function of cell shrinkage in increasingly hyperosmotic media, imparting exquisite volume sensitivity to Na + /H + exchange activity.…”

“…In contrast to mammalian cells, NHE1 transport activity is virtually nonexistent in quiescent atRBCs. Following suspension in hyperosmotic media, at RBCs exhibit an increase in NHE1-mediated Na + flux activity that is nominally 2-orders of magnitude greater than that of cells at normal resting volume in isosmotic medium [31], [32], [33]. In addition, the at RBC NHE1 homolog ( at NHE1) is 79% identical to human NHE1 at the amino acid level, retains the hallmark housekeeping characteristics of mammalian NHE1: cell pH and volume regulation, and is expressed in abundance in at RBCs compared to cell types known to over-express NHE1 (e.g., tumor cells) [33], [34].…”

“…Recently we proposed a model in which OCS activates NHE1 via a rate-limiting phosphorylation-dependent signal transduction event consisting of forward (activating) kinase activity that is activated by OCS, and reverse (inactivating) phosphatase activity that is inactivated by OCS [32] . This model is based on analysis of relaxation kinetics describing activation and inactivation of NHE1 transport activity, in response to acute osmotic shrinkage or re-swelling after OCS, respectively.…”

Laser interference microscopy of amphibian erythrocytes: impact of cell volume and refractive index

“…Blood samples were collected from the caudal vein with a syringe containing 20 U/ml heparin and kept on ice for no more than 1–2 h. Then, 1 volume of blood was mixed with 10 volumes of test medium at room temperature, and 1 μl of suspension was placed on the mirror's object glass, pre‐conditioned with bovine serum albumin (1 ml/mg) and covered with a plastic slip. It is well documented that amphibian erythrocytes undergo RVI and RVD in 1–2 h of transfer to hyper‐ or hyposmotic media, mediated by Na + /H + and K + /H + exchangers, respectively (Ortiz‐Acevedo et al , 2010). Keeping this in mind, samples were viewed by LIM during the initial 5–10 min of their transfer to the test medium.…”

Cell volume homeostatic mechanisms: effectors and signalling pathways

“…Where this has been addressed, such systems appear to constitute shrinkage‐activated/swelling‐inhibited protein kinases and/or swelling‐activated, shrinkage‐inhibited protein phosphatases (Parker et al. 1991, Ortiz‐Acevedo et al. 2010).…”