Root Effect Hemoglobin May Have Evolved to Enhance General Tissue Oxygen Delivery

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Science  14 Jun 2013:
Vol. 340, Issue 6138, pp. 1327-1329
DOI: 10.1126/science.1233692

Holding Your Breath

Hemoglobin and myoglobin are widely responsible for oxygen transport and storage (see the Perspective by Rezende). The ability of diving mammals to obtain enough oxygen to support extended dives and foraging is largely dependent on muscle myoglobin (Mb) content. Mirceta et al. (p. 1303) found that in mammalian lineages with an aquatic or semiaquatic lifestyle, Mb net charge increases, which may represent an adaptation to inhibit self-association of Mb at high intracellular concentrations. Epistasis results from nonadditive genetic interactions and can affect phenotypic evolution. Natarajan et al. (p. 1324) found that epistatic interactions were able to explain the increased hemoglobin oxygen-binding affinity observed in deer mice populations at high altitude. In mammals, the offloading of oxygen from hemoglobin is facilitated by a reduction in the blood's pH, driven by metabolically produced CO2. However, in fish, a reduction in blood pH reduces oxygen carrying capacity of hemoglobin. Rummer et al. (p. 1327) implanted fiber optic oxygen sensors within the muscles of rainbow trout and found that elevated CO2 levels in the water led to acidosis and elevated oxygen tensions.


The Root effect is a pH-dependent reduction in hemoglobin-O2 carrying capacity. Specific to ray-finned fishes, the Root effect has been ascribed specialized roles in retinal oxygenation and swimbladder inflation. We report that when rainbow trout are exposed to elevated water carbon dioxide (CO2), red muscle partial pressure of oxygen (PO2) increases by 65%—evidence that Root hemoglobins enhance general tissue O2 delivery during acidotic stress. Inhibiting carbonic anhydrase (CA) in the plasma abolished this effect. We argue that CA activity in muscle capillaries short-circuits red blood cell (RBC) pH regulation. This acidifies RBCs, unloads O2 from hemoglobin, and elevates tissue PO2, which could double O2 delivery with no change in perfusion. This previously undescribed mechanism to enhance O2 delivery during stress may represent the incipient function of Root hemoglobins in fishes.

In vertebrates, hemoglobin (Hb) plays a crucial role in optimizing tissue oxygen (O2) delivery by increasing blood O2-carrying capacity and regulating the partial pressure (PO2) at which O2 is delivered. Within tissues (such as muscle), metabolically produced carbon dioxide (CO2) reduces blood pH and thus Hb-O2 affinity, elevating blood PO2 and enhancing O2 delivery, which is collectively termed the Bohr effect (1). In mammals, this may elicit an increase in blood PO2 of up to 2 mmHg (2, 3) in vivo, providing some 5% increase in O2 delivery. Teleost fishes often have much more pH-sensitive Hbs, and a recent in vitro study indicates that this effect may be an order of magnitude greater (4). The current study confirms this in vivo. Enhanced O2 delivery may represent an important step in the extraordinary adaptive radiation of the teleost fishes (5), which make up almost half of all vertebrate species.

In teleosts, a reduction in blood pH reduces both Hb-O2 affinity and O2 carrying capacity, which is known as the Root effect (6) and has a well-studied role in securing O2 delivery to the retina and swimbladder (7, 8). These tissues possess specialized acid-producing cells in conjunction with a dense counter-current capillary network (rete) that localizes and magnifies a large acidosis, thus promoting O2-offloading via the Root effect (7, 8). This system is effective enough to generate O2 tensions exceeding 50 atm (~38,000 mmHg) within the gas-filled swimbladder (7).

The Root effect could, however, be a liability because it could severely limit O2 uptake at the gills if systemic blood becomes too acidic. Many teleosts release stress hormones (noradrenaline and adrenaline) that stimulate RBC Na+/H+ exchange (βNHE), which removes H+ in exchange for Na+, creating a pH disequilibrium across the RBC membrane (Fig. 1A) (9). This increases RBC pH and ensures O2 loading at the gill during acidotic stress associated with exhaustive exercise, hypoxia, or hypercarbia (Fig. 1A) (10, 11). Carbonic anhydrase (CA), which catalyzes the reversible conversion of HCO3 and H+ to CO2 for diffusive excretion, would rapidly short-circuit this process (Fig. 1B) (4, 12). Consequently, there is no plasma-accessible CA in the gills of teleosts (13), and this is understood to ensure O2-loading during stressful conditions. However, there is evidence for plasma-accessible CA in muscle capillaries (14, 15). This would short-circuit βNHE, reduce RBC pH and Hb-O2 affinity, and enhance O2-unloading and tissue delivery (Fig. 1B). A recent in vitro study provided proof of principle that this can occur; exposure of acidified and adrenergically stimulated rainbow trout blood to CA elevated PO2 by over 30 mmHg (4, 12). Thus, plasma-accessible CA may allow Root effect Hbs to enhance O2 delivery to aerobic tissues during acidotic stress (4, 12). The present study was designed specifically to determine whether plasma-accessible CA and the associated elimination of RBC pH disequilibrium states in capillary beds could markedly increase tissue PO2 and the driving force for O2 delivery in vivo.

Fig. 1 (A and B) Schematic representation of a catecholamine-activated RBC pH disequilibrium (A) short-circuited by plasma-accessible CA to elevate tissue PO2 (B).

AE, anion exchange; cAMP, adenylate cyclase and 3′,5′-cyclic monophosphate.

We implanted fiber optic O2 sensors into rainbow trout red muscle (RM) (fig. S1) to monitor RM PO2 in real-time. We then monitored changes in RM PO2 during a mild acidosis elicited by hypercarbia (elevated water CO2), which should enhance the RBC pH disequilibrium at the gills. We hypothesized that if present in the RM capillary endothelium, plasma-accessible CA would abolish the pH disequilibrium and increase RM PO2. To provide evidence for a role of plasma-accessible CA in this phenomenon, a new membrane-impermeant CA inhibitor, C18 [compound 18, laboratory-synthesized according to (16)] (fig. S2), was injected into the bloodstream so as to abolish the hypercarbic increase in RM PO2.

After anesthesia, dorsal aorta (DA) cannulation, and RM O2 sensor implantation (fig. S1) (17), rainbow trout recovered overnight in a black Perspex chamber while RM PO2 was continuously monitored. In the morning, blood was sampled, and fish were lightly anaesthetized and treated with a skeletal muscle relaxant (tubocurarine) so as to prevent movement of the fish and damage to the O2 sensor during hypercarbia. All blood variables recovered by 90 ± 21 min after tubocurarine, which represented “baseline” conditions against which subsequent responses were compared (Fig. 2 and fig. S3).

Fig. 2 The effect of hypercarbia, membrane-impermeant carbonic anhydrase inhibitor (C18) and combined hypercarbia and C18 on (A) pHi, (B) pHe, (C) PaO2, and (D) RM PO2.

Different lowercase letters demarcate significant differences between treatment groups within a panel. All data are presented as means ± SEM, n = 7 to 11 fish per treatment per treatment. Statistical significance (repeated measures analysis of variance) is indicated with P values (nonsignificance is indicated “NS”) and assessed using α < 0.05.

Fish were force-ventilated to ensure that arterial PO2 (PaO2; 111.1 ± 14.9 mmHg), Hb-O2 saturation (86.8 to 99.0%), and O2 content (7.1 to 7.7 ml O2 100 ml blood−1) remained constant (Fig. 2 and fig. S3). Plasma catecholamines (noradrenaline, NA; adrenaline, AD) were not significantly elevated over published resting values (≤12 and ≤9 nM, respectively) (fig. S4) (18).

Hypercarbia caused a significant increase in blood Pco2 from 1.7 ± 0.1 to 4.0 ± 0.3 mmHg [F(5, 10) = 8.20, P < 0.001] but no significant change in RBC or plasma total CO2 (Tco2) (table S1). The elevated Pco2 elicited a significant decrease in intracellular RBC pH (pHi) (7.23 ± 0.01 to 7.13 ±0.02) and extracellular (plasma) pH (pHe) (8.09 ± 0.04 to 7.79 ± 0.04) (Fig. 2 and fig. S3). There was a 65% increase in RM PO2 from 47.1 ± 6.4 to 75.2 ± 2.2 mmHg, a ΔPO2 of 30.9 ±7.6 mmHg (Figs. 2 and 3 and figs. S3 and S4). Upon return to normocarbia, all physiological variables returned to baseline (Fig. 2, figs. S3 and S4, and table S1). Intra-arterial C18 injection during normocarbia decreased [Hb] and Hct with no significant effects on blood, plasma, or RM variables (Fig. 2, figs. S3 and S5, and table S1). Upon reexposure to hypercarbia (C18+hypercarbia), blood Pco2 [F(5, 10) = 8.20, P < 0.001] and RBC Tco2 [F(5, 10) = 3.88, P < 0.001] both increased, with plasma Tco2 unaffected (table S1). pHi decreased to 7.14 ± 0.02 [F(5, 8) = 4.62, P < 0.01], but no changes in pHe were observed (Fig. 2 and fig. S3). After C18 treatment, however, hypercarbia did not elicit the significant change in RM PO2 that had previously been observed [ΔPO2 = 0.01 ± 2.96 mmHg; t(15) = 3.96, P = 0.001] (Fig. 2 and figs. S3 and S5).

Fig. 3 Representative trace illustrating on-line changes in rainbow trout RM PO2PO2) upon exposure to hypercarbia.

The y axis represents RM PO2 (mmHg), and the x axis indicates elapsed time (min). Vertical lines indicate experimental treatments.

Our findings support the hypothesis that in a teleost fish possessing a Root effect Hb and plasma-accessible CA, O2 delivery to tissues other than the retina and swimbladder—specifically, RM—can be greatly enhanced during acidotic stress. In RM, the proposed mechanism is a cascade by which plasma-accessible CA at the tissue eliminates an arterial RBC pH disequilibrium state to acidify RBCs. This causes Hb-O2 unloading and elevates RM PO2 (Fig. 1B). The level of hypercarbia we used caused only a moderate acidosis but was associated with a profound increase in RM PO2 over baseline. This effect on RM PO2 was abolished by the membrane-impermeant CA inhibitor (C18), highlighting the importance of plasma-accessible CA in the response. We estimate that a ΔPO2 of this magnitude could almost double tissue O2 delivery with no change in perfusion (supplementary text and fig. S8). Exposure to this level of hypercarbia did not significantly elevate catecholamine levels, indicating that the treatment was not overly stressful, and activation of RBC βNHE may not have been the prime source of the pH disequilibrium (4); other stimuli may activate the βNHE (19, 20), or additional RBC NHE isoforms may exist. Activation of the RBC βNHE under more stressful conditions, such as exercise or hypoxaemia, may have an even greater influence on tissue oxygenation. This may be particularly important to increase aerobic metabolism during environmental or exercise stress or to speed up recovery after an intense bout of exercise—for example, after a predator-prey encounter or during long, upstream migrations, as exhibited in Pacific salmon.

It has been suggested that the Bohr effect evolved independently three times in vertebrates, but only once was this associated with the Root effect (21, 22). According to the mechanism we propose [both here and in vitro (4)], the larger Bohr associated with the Root effect could actually promote O2 delivery in teleosts. Thus, in response to previous arguments (23), a large Bohr coefficient could have been selected for without compromising general O2 delivery. For this to be the case, however, three components are required: (i) pH-sensitive Hbs, such as Root effect Hbs; (ii) plasma-accessible CA in the respective capillary beds but not at the gill (4); and (iii) RBC βNHE or NHE activity to generate a pH disequilibrium in the absence of CA. All of these traits may depend on phylogeny, life history, and/or lifestyle of the species. With these components in place, however, a Root effect Hb can greatly enhance tissue O2 delivery during a relatively mild acidosis. Further studies are required to investigate the degree to which this occurs in other metabolically active tissues, other species, and ultimately, the role that this mechanism of enhanced oxygen delivery may have played in the extraordinary success of teleosts.

Root effect Hbs evolved in ray-finned fishes ~400 million years ago, at least 150 million years before the appearance of the choroid rete at the eye or rete mirabile at the swimbladder, the structures that are generally associated with this exceptional O2 delivery system (21, 22). Here, we provide in vivo evidence supporting the hypothesis that Root effect Hbs, in the presence of plasma-accessible CA, can greatly enhance systemic tissue O2 delivery during periods of acidotic stress. We estimate that the 65% elevation in RM PO2 would almost double tissue O2 delivery with no change in perfusion. The role of Root effect Hbs in the eye and swimbladder may, therefore, be an exaptation, as proposed by Berenbrink et al. (22). That is, an incipient function of Root Hbs—general O2 delivery—may have been co-opted to give rise to the complex physiological system at the eye and swimbladder 150 million to 270 million years later.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S8

Table S1

References (2477)

References and Notes

  1. Acknowledgments: This project was funded by a Natural Sciences and Engineering Research Council of Canada Discovery grant to C.J.B. and a University of British Columbia Graduate Fellowship and Australian Research Council grant to J.L.R. The authors wish to thank S. F. Perry for catecholamine analysis, W. K. Milsom for tubocurarine advice, and D. J. Randall, M. Regan, D. W. Baker, C. Ciuhandu, A. P. Farrell, K. Gilmour, A. Morash, P. Allen, N. Farrell, C. Fu, L. Hanson, K. Kamo, M. Roshan-Moniri, K. Suvadzjic, and the University of British Columbia Zoology workshop for interesting discussions and editorial and technical assistance. Additional information and all supporting data can be found in the supplementary materials.
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