Rapid restoration of intracellular pH in erythrocytes protects oxygen transport

In the current issue of Acta Physiologica, Harter et al.1 provide novel evidence for soluble adenylyl cyclase (sAC), a ubiquitous regulatory enzyme present in the cytosol of almost all cells, acting as a sensor for intracellular pH (pHi) in fish red blood cells. Harter and coworkers also propose an acid–base sensing mechanism that acts to protect adequate oxygen delivery in face of alkalosis or acidosis, and therefore may be of considerable functional importance. Erythrocytes are very specialized cells, packed with hemoglobin, that provide for oxygen delivery to the tissues. In humans and other mammals, the nucleus and mitochondria are lost as the erythrocytes mature, and ATP is mostly derived from glycolysis.2 This is not the case in other vertebrates where the red blood cells from birds, reptiles, amphibians, and fish retain their nucleus and capacity for mitochondrial respiration. In contrast to the other vertebrates, the mammalian red blood cell is therefore often viewed as a simple cell, a bag of hemoglobin. However, there is mounting evidence that mammalian red blood cells can rapidly restore pHi upon acid–base disturbances by activating the chloride-bicarbonate exchanger (or anion exchanger, AE1).3 In addition to safeguarding the metabolic processes within the erythrocyte, the protection of pHi has important implications for oxygen delivery as oxygen affinity of the hemoglobin is sensitive to pHi through the Bohr effect.4 This is the case for all vertebrates, but perhaps particularly so in fish. In addition to having rather large Bohr effects, fish are endowed with the so-called Root effect, named after its discoverer Raymond W. Root, who demonstrated that acidosis not only right-shifts the blood oxygen equilibrium curve, that is, lowers oxygen affinity, but also reduces the capacity for oxygen binding, such that part of the hemoglobin is unable to bind oxygen.5 The Root effect may, at least at first, seem maladaptive, but provides for the possibility of creating very high partial pressures of oxygen when local acidification forces the oxygen to separate from the hemoglobin. This is exploited in the swim bladder of many fish species where local acidification of the blood in a specialized gas gland with a rete mirabile elicits extraordinarily high partial pressures of oxygen that then fills the swim-bladder and enables exquisite control of buoyancy.6 Moreover, the Root effect also plays an important role in delivering oxygen to the eyes of numerous groups of fishes.6 Here, specialized mechanisms for acidification provide partial pressures of oxygen in excess of 500 mmHg that enable diffusion into the avascular retina.7 To allow fish to exploit the Root effect in these specialized structures, it is essential that the acidification of the blood is transmitted to hemoglobin in the red blood cell interior. On the other hand, convective oxygen transport by the cardiovascular system may be compromised during general acidosis, for example, in connection with intense exercise. As a counteractive measure that prevents the decrease in blood oxygen carrying capacity from decreasing arterial oxygen concentration during strenuous exercise, fish species with a strong Root effect hemoglobin express a special isoform of the sodium-hydrogen exchanger (beta-NHE) in their red blood cell membrane that can be activated by beta-adrenergic stimulation.8 Adrenergic activation of this NHE leads to extrusion of protons, and hence, an elevation of pHi while extracellular pH decreases even further.9 It is well-known that the adrenergic stimulation of the fish red blood cell NHE is exacerbated in acidosis, but the mechanism underlying this seemingly adaptive trait and the degree to which changes in AE1 activity may be involved have remained rather enigmatic. It is therefore of great interest that Harter et al.1 in the current issue of Acta Physiologica, provide evidence for an acid–base sensing mechanism through sAC in rainbow trout erythrocytes. Based on the observation that the sAC protein is broadly distributed in the tree of life, the authors first used immunocytochemistry and confocal super-resolution microscopy to demonstrate that the sCA protein is widely distributed within the cytosol of rainbow trout red blood cells. The sCA protein was also associated with the nucleus and the cell membrane and this subcellular localization could therefore enable the activation of ion exchangers relevant for acid–base regulation. Having provided compelling evidence for the presence of sCA in these fish red blood cells, Harter et al.1 then used the pH-sensitive fluorophore SNARF-1 to visualize the rate at which pHi was restored upon an acute intracellular acidosis. This was elicited by an ammonia pre-pulse where part of the NaCl in the saline in which the cells were suspended in vitro is replaced by an equal part of NH4Cl, which is then rapidly washed out again. The ammonia pre-pulses decreased pHi by around 0.5 pH units, and both the rate of pHi recovery and the calculated proton flux were reduced when the sCA protein function was inhibited by the specific inhibitors KH7 and LRE1. Inhibition of AE1 also reduced the rate of pHi recovery, whereas a general inhibitor of sodium-hydrogen exchange was without effect. It seems therefore that AE1 is the sole mechanism regulating pHi under these conditions. Based on spectrophotometric measurements of hemoglobin-oxygen during the disturbances of pHi, Harter et al.1 propose an interesting mechanism whereby the HCO3− sensitivity of sAC is envisioned to increase the intracellular concentrations of cyclic AMP. An increase in this second messenger is then suggested to activate the anion-exchanger AE1, which then would affect the oxygen affinity of hemoglobin by regulating the flux of acid–base equivalents across the red blood cell membrane. It may be worthwhile to caution as to whether the model proposed by Harter et al.1 should be considered a bona fide-regulated mechanism. After all, the activation of the AE1 will merely hasten the rate anions reach their new steady state as predicted by the Donnan equilibrium. Such temporal changes in kinetics may nevertheless be important as steady state conditions rarely exist in the capillaries where gas exchange occurs, and we are looking forward to seeing how the authors will address this difficult problem. While the proposed mechanism still needs to be corroborated, the authors have opened up new avenues of red blood cell research by tackling the technical challenges of fluorophore pHi measurements in cells packed with fluorescent light-absorbing hemoglobin, using state of the art immunocytochemistry, and choosing an animal model system with an exquisitely pH-sensitive hemoglobin oxygen affinity and strong capacity for red blood cell pHi regulation. Future questions include whether sAC is also involved in modulating beta NHE activity, how this then is coordinated with modulating AE1 activity, and finally, whether sAC-activated acid–base fluxes via AE1 are solely driven by a transmembrane electrochemical disequilibrium of acid–base equivalents or also involve (secondarily) active transport components. The authors contributed equally to this editorial. TW is supported by The Novo Nordisk Foundation. The authors have no conflict of interest to declare. Data sharing is not applicable to this article as no data were generated or analyzed during this study.