Plasma-accessible Carbonic Anhydrase Research Articles

Developing rainbow trout (Oncorhynchus mykiss) lose branchial plasma accessible carbonic anhydrase expression with hatch and the transition to pH-sensitive, adult hemoglobin polymorphs.

Salmonids possess a unique respiratory system comprised of three major components: highly pH-sensitive hemoglobins, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma accessible carbonic anhydrase (paCA), specifically with absence of paCA at the gills. These characteristics are thought to have evolved to enhance oxygen unloading to the tissues while protecting uptake at the gills. Our knowledge of this system is detailed in adults, but little is known about it through development. Developing rainbow trout (Oncorhynchus mykiss) express embryonic RBCs containing hemoglobins that are relatively insensitive to pH; however, availability of gill paCA and RBC pHi protection is unknown. We show that pre-hatch rainbow trout express gill paCA, which is lost in correlation with the emergence of highly pH-sensitive adult hemoglobins and RBC pHi protection. Rainbow trout therefore exhibit a switch in respiratory strategy with hatch. We conclude that gill paCA likely represents an embryonic trait in rainbow trout and is constrained in adults due to their highly pH-sensitive hemoglobins.

An investigation of gill and blood carbonic anhydrase characteristics in three basal actinopterygian species: alligator gar (Atractosteus spatula), white sturgeon (Acipenser transmontanus) and Senegal bichir (Polypterus senegalus).

Many teleosts possess a unique set of respiratory characteristics allowing enhanced oxygen unloading to the tissues during stress. This system comprises three major components: highly pH sensitive haemoglobins (large Bohr and Root effects), rapid red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of membrane-bound plasma-accessible carbonic anhydrase (paCA; absence in the gills). The first two components have received considerable research effort; however, the evolutionary loss of branchial paCA has received little attention. In the current study, we investigated the availability of branchial membrane-bound CA, along with several other CA-related characteristics in species belonging to three basal actinopterygian groups: the Lepisosteiformes, Acipenseriformes and Polypteriformes to assess the earlier hypothesis that Root effect haemoglobins constrain branchial paCA availability. We present the first evidence suggesting branchial membrane-bound CA presence in a basal actinopterygian species: the Senegal bichir (Polypterus senegalus) and show that like the teleosts, white sturgeon (Acipenser transmontanus) and alligator gar (Atractosteus spatula) do not possess branchial membrane-bound CA. We discuss the varying respiratory strategies for these species and propose that branchial paCA may have been lost much earlier than previously thought, likely in relation to the changes in haemoglobin buffer capacity associated with the increasing magnitude of the Bohr effect. The findings described here represent an important advancement in our understanding of the evolution of the unique system of enhanced oxygen unloading thought to be present in most teleosts, a group that encompasses half of all vertebrates.

A novel perspective on the evolutionary loss of plasma-accessible carbonic anhydrase at the teleost gill.

The gills of most teleost fishes lack plasma-accessible carbonic anhydrase (paCA) that could participate in CO2 excretion. We tested the prevailing hypothesis that paCA would interfere with red blood cell (RBC) intracellular pH regulation by β-adrenergic sodium-proton exchangers (β-NHE) that protect pH-sensitive haemoglobin-oxygen (Hb-O2) binding during an acidosis. In an open system that mimics the gills, β-NHE activity increased Hb-O2 saturation during a respiratory acidosis in the presence or absence of paCA, whereas the effect was abolished by NHE inhibition. However, in a closed system that mimics the tissue capillaries, paCA disrupted the protective effects of β-NHE activity on Hb-O2 binding. The gills are an open system, where CO2 generated by paCA can diffuse out and is not available to acidifying the RBCs. Therefore, branchial paCA in teleosts may not interfere with RBC pH regulation by β-NHEs, and other explanations for the evolutionary loss of the enzyme must be considered.

ECR Spotlight – Till Harter

ECR Spotlight is a series of interviews with early-career authors from a selection of papers published in Journal of Experimental Biology and aims to promote not only the diversity of early-career researchers (ECRs) working in experimental biology during our centenary year, but also the huge variety of animals and physiological systems that are essential for the ‘comparative’ approach. Till Harter is an author on ‘ A novel perspective on the evolutionary loss of plasma-accessible carbonic anhydrase at the teleost gill’, published in JEB. Till is a Postdoc in the lab of Graham Scott at McMaster University, Hamilton, Canada, investigating comparative physiology, specifically, red blood cell function, and more broadly, cardio-respiratory physiology in vertebrates.

An atlas of plasma-accessible carbonic anhydrase availability in the model teleost, the rainbow trout.

The unique teleost oxygenation system that permits enhanced oxygen unloading during stress comprises three main characteristics: pH-sensitive haemoglobin, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma-accessible carbonic anhydrase (paCA). A heterogeneous distribution of paCA is essential; its presence permits enhanced oxygen unloading during stress, while its absence at the gills maintains conditions for oxygen uptake by pH-sensitive haemoglobins. We hypothesised that paCA would be absent in all four gill arches, as has been previously indicated for arch two, and that paCA would be present in all other tissues. Through a suite of biochemical and molecular methods, we confirmed the absence of paCA from all four arches. We also found evidence for paCA in nine other tissues and a lack of paCA availability in the stomach. Expression was highly variable between tissues and suggests these differences may be associated with their respective metabolic activities. Additionally, we analysed the specific CA-IV isoform expressed within each tissue and showed almost complete separation of expression between tissues; CA-IVa was detected in the heart, brain, anterior intestine, and liver, whereas CA-IVb was detected in all intestine sections, pyloric caeca, kidney, and white muscle. This adds to a growing collection of work suggesting CA-IVa and b play divergent roles in gas exchange and ion/acid-base balance, respectively. The current study represents the first comprehensive atlas of paCA availability within the circulatory system of the model teleost, rainbow trout, and fills important gaps in our knowledge of this unique oxygenation system.

The role of carbonic anhydrase-mediated tissue oxygen extraction in a marine teleost acclimated to hypoxia.

With the growing prevalence of hypoxia (O2 levels ≤2 mg l-1) in aquatic and marine ecosystems, there is increasing interest in the adaptive mechanisms fish may employ to better their performance in stressful environments. Here, we investigated the contribution of a proposed strategy for enhancing tissue O2 extraction - plasma-accessible carbonic anhydrase (CA-IV) - under hypoxia in a species of estuarine fish (red drum, Sciaenops ocellatus) that thrives in fluctuating habitats. We predicted that hypoxia-acclimated fish would increase the prevalence of CA-IV in aerobically demanding tissues to confer more efficient tissue O2 extraction. Furthermore, we predicted the phenotypic changes to tissue O2 extraction that occur with hypoxia acclimation may improve respiratory and swim performance under 100% O2 conditions (i.e. normoxia) when compared with performance in fish that have not been acclimated to hypoxia. Interestingly, there were no significant differences in relative CA-IV mRNA expression, protein abundance or enzyme activity between the two treatments, suggesting CA-IV function is maintained under hypoxia. Likewise, respiratory performance of hypoxia-acclimated fish was similar to that of control fish when tested in normoxia. Critical swim speed (Ucrit) was significantly higher in hypoxia-acclimated fish but translated to marginal ecological benefits with an increase of ∼0.3 body lengths per second. Instead, hypoxia-acclimated fish may have relied more heavily on anaerobic metabolism during their swim trials, utilizing burst swimming 1.5 times longer than control fish. While the maintenance of CA-IV may still be an important contributor for hypoxia tolerance, our evidence suggests hypoxia-acclimated red drum are using other mechanisms to cope in an O2-depleted environment.

The roles of plasma accessible and cytosolic carbonic anhydrases in bicarbonate (HCO3-) excretion in Pacific hagfish (Eptatretus stoutii).

Pacific hagfish (Eptatretus stoutii) are marine scavengers and feed on decaying animal carrion by burrowing their bodies inside rotten carcasses where they are exposed to several threatening environmental stressors, including hypercapnia (high partial pressures of CO2). Hagfish possess a remarkable capacity to tolerate hypercapnia, and their ability to recover from acid-base disturbances is well known. To deal with the metabolic acidosis resulting from exposure to high CO2, hagfish can mount a rapid elevation of plasma HCO3- concentration (hypercarbia). Once PCO2 is restored, hagfish quickly excrete their HCO3- load, a process that likely involves the enzyme carbonic anhydrase (CA), which catalyzes HCO3- dehydration into CO2 at the hagfish gills. We aimed to characterize the role of branchial CA in CO2/HCO3- clearance from the plasma at the gills of E. stoutii, under control and high PCO2 (hypercapnic) exposure conditions. We assessed the relative contributions of plasma accessible versus intracellular (cytosolic) CA to gill HCO3- excretion by measuring in situ [14C]-HCO3- fluxes. To accomplish this, we employed a novel surgical technique of individual gill pouch arterial perfusion combined with perifusion of the gill afferent to efferent water ducts. [14C]-HCO3- efflux was measured at the gills of fish exposed to control, hypercapnic (48h) and recovery from hypercapnia conditions (6h), in the presence of two well-known pharmacological inhibitors of CA, the membrane impermeant C18 (targets membrane bound, plasma accessible CA) and membrane-permeant acetazolamide, which targets all forms of CA, including extracellular and intracellular cytosolic CAs. C18 did not affect HCO3- flux in control fish, whereas acetazolamide resulted in a significant reduction of 72%. In hypercapnic fish, HCO3- fluxes were much higher and perfusion with acetazolamide caused a reduction of HCO3- flux by 38%. The same pattern was observed for fish in recovery, where in all three experimental conditions, there was no significant inhibition of plasma-accessible CA. We also observed no change in CA enzyme activity (measured in vitro) in any of the experimental PCO2 conditions. In summary, our data suggests that there are additional pathways for HCO3- excretion at the gills of hagfish that are independent of plasma-accessible CA.

Enhanced oxygen unloading in two marine percomorph teleosts

Teleost fishes are diverse and successful, comprising almost half of all extant vertebrate species. It has been suggested that their success as a group is related, in part, to their unique O2 transport system, which includes pH-sensitive hemoglobin, a red blood cell β-adrenergic Na+/H+ exchanger (RBC β-NHE) that protects red blood cell pH, and plasma accessible carbonic anhydrase which is absent at the gills but present in some tissues, that short-circuits the β-NHE to enhance O2 unloading during periods of stress. However, direct support for this has only been examined in a few species of salmonids. Here, we expand the knowledge of this system to two warm-water, highly active marine percomorph fish, cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus). We show evidence for RBC β-NHE activity in both species, and characterize the Hb-O2 transport system in one of those species, cobia. We found significant RBC swelling following β-adrenergic stimulation in both species, providing evidence for the presence of a rapid, active RBC β-NHE in both cobia and mahi-mahi, with a time-course similar to that of salmonids. We generated oxygen equilibrium curves (OECs) for cobia blood and determined the P50, Hill, and Bohr coefficients, and used these data to model the potential for enhanced O2 unloading. We determined that there was potential for up to a 61% increase in O2 unloading associated with RBC β-NHE short-circuiting, assuming a − 0.2 ∆pHa-v in the blood. Thus, despite phylogenetic and life history differences between cobia and the salmonids, we found few differences between their Hb-O2 transport systems, suggesting conservation of this physiological trait across diverse teleost taxa.

A novel acidification mechanism for greatly enhanced oxygen supply to the fish retina.

Previously, we showed that the evolution of high acuity vision in fishes was directly associated with their unique pH-sensitive hemoglobins that allow O2 to be delivered to the retina at PO2s more than ten-fold that of arterial blood (Damsgaard et al., 2019). Here, we show strong evidence that vacuolar-type H+-ATPase and plasma-accessible carbonic anhydrase in the vascular structure supplying the retina act together to acidify the red blood cell leading to O2 secretion. In vivo data indicate that this pathway primarily affects the oxygenation of the inner retina involved in signal processing and transduction, and that the evolution of this pathway was tightly associated with the morphological expansion of the inner retina. We conclude that this mechanism for retinal oxygenation played a vital role in the adaptive evolution of vision in teleost fishes.

Blood and Gill Carbonic Anhydrase in the Context of a Chondrichthyan Model of CO2 Excretion.

Pacific spiny dogfish (Squalus suckleyi) have been widely used as a representative species for chondrichthyan CO2 excretion. Pacific spiny dogfish have a slower red blood cell (RBC) carbonic anhydrase (CA) isoform than teleost fishes, extracellular CA activity, no endogenous plasma CA inhibitor, and plasma-accessible CA IV at the gills. Thus, both the RBC and plasma compartments contribute to bicarbonate ion () dehydration at the gills for CO2 excretion in contrast to teleost fishes, in which dehydration is restricted to RBCs. We compared CA activity levels, subcellular localization, and presence of plasma CA inhibitors in the blood and gills of 13 chondrichthyans to examine the hypothesis that the dogfish model of CO2 excretion applies broadly to chondrichthyans. In general, blood samples from the 12 other chondrichthyans examined had lower RBC CA activity than teleosts, some extracellular CA activity, and no endogenous plasma CA inhibitor. While type IV-like membrane-associated CA was found in the gills in all four of the chondrichthyans examined, S. suckleyi had three times more CA activity ( μmol CO2 min-1 mg protein-1) in the microsomal (membrane) fraction of gills than the other three. In addition, unexpected variation in CA characteristics was observed between chondrichthyan species. Thus, in general, it appears that the pattern of CA distribution in fishes can be generally categorized as either chondrichthyan or teleost models. However, further studies should examine the functional significance of the within-chondrichthyan differences we observed and investigate whether CO2 excretion patterns exist along a continuum or in discrete groups.

Regulation of erythrocyte function: Multiple evolutionary solutions for respiratory gas transport and its regulation in fish.

Gas transport concepts in vertebrates have naturally been formulated based on human blood. However, the first vertebrates were aquatic, and fish and tetrapods diverged hundreds of millions years ago. Water-breathing vertebrates live in an environment with low and variable O2 levels, making environmental O2 an important evolutionary selection pressure in fishes, and various features of their gas transport differ from humans. Erythrocyte function in fish is of current interest, because current environmental changes affect gas transport, and because especially zebrafish is used as a model in biomedical studies, making it important to understand the differences in gas transport between fish and mammals to be able to carry out meaningful studies. Of the close to thirty thousand fish species, teleosts are the most species-numerous group. However, two additional radiations are discussed: agnathans and elasmobranchs. The gas transport by elasmobranchs may be closest to the ancestors of tetrapods. The major difference in their haemoglobin (Hb) function to humans is their high urea tolerance. Agnathans differ from other vertebrates by having Hbs, where cooperativity is achieved by monomer-oligomer equilibria. Their erythrocytes also lack the anion exchange pathway with profound effects on CO2 transport. Teleosts are characterized by highly pH sensitive Hbs, which can fail to become fully O2 -saturated at low pH. An adrenergically stimulated Na+ /H+ exchanger has evolved in their erythrocyte membrane, and plasma-accessible carbonic anhydrase can be differentially distributed among their tissues. Together, and differing from other vertebrates, these features can maximize O2 unloading in muscle while ensuring O2 loading in gills.

Functional support for a novel mechanism that enhances tissue oxygen extraction in a teleost fish.

A successful spawning migration in salmon depends on their athletic ability, and thus on efficient cardiovascular oxygen (O2) transport. Most teleost fishes have highly pH-sensitive haemoglobins (Hb) that can release large amounts of O2 when the blood is acidified at the tissues. We hypothesized that plasma-accessible carbonic anhydrase (paCA; the enzyme that catalyses proton production from CO2) is required to acidify the blood at the tissues and promote tissue O2 extraction. Previous studies have reported an elevated tissue O2 extraction in hypoxia-acclimated teleosts that may also be facilitated by paCA. Thus, to create experimental contrasts in tissue O2 extraction, Atlantic salmon were acclimated to normoxia or hypoxia (40% air saturation for more than six weeks), and the role of paCA in enhancing tissue O2 extraction was tested by inhibiting paCA at rest and during submaximal exercise. Our results show that: (i) in both acclimation groups, the inhibition of paCA increased cardiac output by one-third, indicating a role of paCA in promoting tissue O2 extraction during exercise, recovery and at rest; (ii) the recruitment of paCA was plastic and increased following hypoxic acclimation; and (iii) maximal exercise performance in salmon, and thus a successful spawning migration, may not be possible without paCA.

Time course of red blood cell intracellular pH recovery following short-circuiting in relation to venous transit times in rainbow trout,Oncorhynchus mykiss

Accumulating evidence is highlighting the importance of a system of enhanced hemoglobin-oxygen (Hb-O2) unloading for cardiovascular O2 transport in teleosts. Adrenergically stimulated sodium-proton exchangers (β-NHE) create H+ gradients across the red blood cell (RBC) membrane that are short-circuited in the presence of plasma-accessible carbonic anhydrase (paCA) at the tissues; the result is a large arterial-venous pH shift that greatly enhances O2 unloading from pH-sensitive Hb. However, RBC intracellular pH (pHi) must recover during venous transit (31-90 s) to enable O2 loading at the gills. The halftimes ( t1/2) and magnitudes of RBC β-adrenergic stimulation, short-circuiting with paCA and recovery of RBC pHi, were assessed in vitro, on rainbow trout whole blood, and using changes in closed-system partial pressure of O2 as a sensitive indicator for changes in RBC pHi. In addition, the recovery rate of RBC pHi was assessed in a continuous-flow apparatus that more closely mimics RBC transit through the circulation. Results indicate that: 1) the t1/2 of β-NHE short-circuiting is likely within the residence time of blood in the capillaries, 2) the t1/2 of RBC pHi recovery is 17 s and within the time of RBC venous transit, and 3) after short-circuiting, RBCs reestablish the initial H+ gradient across the membrane and can potentially undergo repeated cycles of short-circuiting and recovery. Thus, teleosts have evolved a system that greatly enhances O2 unloading from pH-sensitive Hb at the tissues, while protecting O2 loading at the gills; the resulting increase in O2 transport per unit of blood flow may enable the tremendous athletic ability of salmonids.

Enhanced hemoglobin-oxygen unloading in migratory salmonids.

Recent findings indicate that some teleost fishes may be able to greatly enhance hemoglobin-oxygen (Hb-O2) unloading at the tissues under conditions that result in catecholamine release. The putative mechanism relies on the high pH sensitivity of teleost hemoglobin (Hb), intracellular red blood cell (RBC) pH regulation via β-adrenergic Na+/H+ exchanger (β-NHE) activity, and plasma-accessible carbonic anhydrase at the tissues that short-circuits RBC pH regulation. Previous studies have shown that in rainbow trout, this system may double Hb-O2 unloading to red muscle compared to a situation without short-circuiting. The present study determined that: (1) in rainbow trout this system may be functional even at low concentrations of circulating catecholamines, as shown by conducting a dose-response analysis; (2) Atlantic and coho salmon also possess β-NHE activity, as shown by changes in hematocrit in adrenergically stimulated cells; and (3) with β-NHE short-circuiting, Atlantic and coho salmon may be able to increase Hb-O2 unloading by up to 74 and 159%, respectively, as determined by modeling based on O2 equilibrium curves. Together, these results indicate that a system to enhance Hb-O2 unloading may be common among salmonids and may be operational even under routine conditions. In view of the life histories of Atlantic and coho salmon, a system to enhance Hb-O2 unloading during exercise may help determine a successful spawning migration and thus reproductive success.

Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium

Oxygen supply to the heart of most teleosts, including salmonids, relies in part or in whole on oxygen-depleted venous blood. Given that plasma-accessible carbonic anhydrase (CA) in red muscle of rainbow trout has recently been shown to facilitate oxygen unloading from arterial blood under certain physiological conditions, we tested the hypothesis that plasma-accessible CA is present in the lumen of coho salmon (Oncorhynchus kisutch) hearts, and may therefore assist in the luminal oxygen supply to the spongy myocardium, which has no coronary circulation. We demonstrate a widespread distribution of CA throughout the heart chambers, including lumen-facing cells in the atrium, and confirm that the membrane-bound isoform ca4 is expressed in the atrium and ventricle of the heart. Further, we confirm that CA catalytic activity is available to blood in the atrial lumen using a modified electrometric ΔpH assay in intact atria in combination with either a membrane-impermeable CA inhibitor or specific cleavage of the Ca4 membrane anchor. Combined, these results support our hypothesis of the presence of an enhanced oxygen delivery system in the lumen of a salmonid heart, which could help support oxygen delivery when the oxygen content of venous blood becomes greatly reduced, such as after burst exercise and during environmental hypoxia.

A unique mode of tissue oxygenation and the adaptive radiation of teleost fishes

Teleost fishes constitute 95% of extant aquatic vertebrates, and we suggest that this is related in part to their unique mode of tissue oxygenation. We propose the following sequence of events in the evolution of their oxygen delivery system. First, loss of plasma-accessible carbonic anhydrase (CA) in the gill and venous circulations slowed the Jacobs-Stewart cycle and the transfer of acid between the plasma and the red blood cells (RBCs). This ameliorated the effects of a generalised acidosis (associated with an increased capacity for burst swimming) on haemoglobin (Hb)-O2 binding. Because RBC pH was uncoupled from plasma pH, the importance of Hb as a buffer was reduced. The decrease in buffering was mediated by a reduction in the number of histidine residues on the Hb molecule and resulted in enhanced coupling of O2 and CO2 transfer through the RBCs. In the absence of plasma CA, nearly all plasma bicarbonate ultimately dehydrated to CO2 occurred via the RBCs, and chloride/bicarbonate exchange was the rate-limiting step in CO2 excretion. This pattern of CO2 excretion across the gills resulted in disequilibrium states for CO2 hydration/dehydration reactions and thus elevated arterial and venous plasma bicarbonate levels. Plasma-accessible CA embedded in arterial endothelia was retained, which eliminated the localized bicarbonate disequilibrium forming CO2 that then moved into the RBCs. Consequently, RBC pH decreased which, in conjunction with pH-sensitive Bohr/Root Hbs, elevated arterial oxygen tensions and thus enhanced tissue oxygenation. Counter-current arrangement of capillaries (retia) at the eye and later the swim bladder evolved along with the gas gland at the swim bladder. Both arrangements enhanced and magnified CO2 and acid production and, therefore, oxygen secretion to those specialised tissues. The evolution of β-adrenergically stimulated RBC Na(+)/H(+) exchange protected gill O2 uptake during stress and further augmented plasma disequilibrium states for CO2 hydration/dehydration. Finally, RBC organophosphates (e.g. NTP) could be reduced during hypoxia to further increase Hb-O2 affinity without compromising tissue O2 delivery because high-affinity Hbs could still adequately deliver O2 to the tissues via Bohr/Root shifts. We suggest that the evolution of this unique mode of tissue O2 transfer evolved in the Triassic/Jurassic Period, when O2 levels were low, ultimately giving rise to the most extensive adaptive radiation of extant vertebrates, the teleost fishes.

Plasma-accessible carbonic anhydrase at the tissue of a teleost fish may greatly enhance oxygen delivery:in vitroevidence in rainbow trout,Oncorhynchus mykiss

During a generalized acidosis in rainbow trout, catecholamines are released into the blood, activating red blood cell (RBC) Na(+)/H(+) exchange (βNHE), thus protecting RBC intracellular pH (pH(i)) and subsequent O(2) binding at the gill. Because of the presence of a Root effect (a reduction in oxygen carrying capacity of the blood with a reduction in pH), the latter could otherwise be impaired. However, plasma-accessible carbonic anhydrase (CA) at the tissues (and absence at the gills) may result in selective short-circuiting of RBC βNHE pH regulation. This would acidify the RBCs and greatly enhance O(2) delivery by exploitation of the combined Bohr-Root effect, a mechanism not previously proposed. As proof-of-principle, an in vitro closed system was developed to continuously monitor extracellular pH (pH(e)) and O(2) tension (P(O(2))) of rainbow trout blood. In this closed system, adding CA to acidified, adrenergically stimulated RBCs short-circuited βNHE pH regulation, resulting in an increase in P(O(2)) by >30 mmHg, depending on the starting Hb-O(2) saturation and degree of initial acidification. Interestingly, in the absence of adrenergic stimulation, addition of CA still elevated P(O(2)), albeit to a lesser extent, a response that was absent during general NHE inhibition. If plasma-accessible CA-mediated short-circuiting is operational in vivo, the combined Bohr-Root effect system unique to teleost fishes could markedly enhance tissue O(2) delivery far in excess of that in vertebrates possessing a Bohr effect alone and may lead to insights about the early evolution of the Root effect.

Membrane-associated carbonic anhydrase in the respiratory system of the Pacific hagfish (Eptatretus stouti)

Like other agnathans, the Pacific hagfish (Eptatretus stouti) lacks red blood cell (RBC) Cl(-)/HCO(3)(-) exchange. Despite this absence of anion exchange, the majority (86.7+/-1.4%) of the total CO(2) carried in the blood is found within the plasma as HCO(3)(-), and thus presumably is inaccessible to RBC carbonic anhydrase (CA). As such, a branchial plasma-accessible CA isozyme in hagfish would be beneficial for mobilizing the considerable plasma HCO(3)(-) stores for CO(2) excretion and blood acid-base balance. The current study used a combination of molecular and biochemical methods to identify two membrane-associated CA isozymes in the respiratory system of E. stouti. Using homology cloning methods, CA IV and XV-like isozymes were identified in the gill and RBC, respectively. Real-time PCR analysis of relative mRNA expression revealed that CA IV was specific to the gill, while CA XV was found in several tissues including the RBC, gill, liver, heart and muscle. Isolation of subcellular fractions of gill and RBC verified the presence of membrane-associated CA activity in each tissue that persisted after standard washing protocols. Unlike CA activity associated with the cytoplasmic fractions, the activity in gill membranes was not inhibited by sodium dodecyl sulphate, while RBC membrane activity was inhibited to a lesser degree than the cytoplasmic fraction. Additionally, incubation of gill membrane fractions with phosphatidylinositol-specific phospholipase C released significant CA activity into the supernatant indicating the presence of a glycophosphatidyl inositol-linkage to the membrane, as found with other CA IV and XV isozymes. These results demonstrate that Pacific hagfish possess gill and RBC plasma-accessible membrane-associated CA that may play important roles in respiratory gas exchange and acid-base balance.

Beyond buoyancy and vision: The potential for the Root effect to deliver oxygen to tissues other than the swim bladder and eye

Teleost fish possess a unique, pH-sensitive hemoglobin (Hb) that, in the presence of an acidosis, substantially reduces the affinity and carrying capacity for O₂ (Root effect). To date, this efficient O₂ delivery mechanism is only known for filling a swim bladder (SB) against huge pressure gradients (> 50 atm) associated with depth and for oxygenating the metabolically active, yet avascular retinal tissue of the eye. In spite of the clear benefits to O₂ delivery for buoyancy and vision, no study has been conducted to determine whether the Root effect may be important in optimizing O₂ delivery to other tissues such as muscle, which is the focus of this research.During environmental or exercise-induced stress, blood pH may fall; however, some fish regulate red blood cell (RBC) intracellular pH (pHi) by releasing catecholamines that activate the sodium/proton (Na⁺/H⁺) exchanger (βNHE) on the RBC membrane. The βNHE removes H⁺s from the RBC resulting in an intracellular alkalosis, an increase in Hb–O₂ affinity, and O₂ uptake at the respiratory surfaces is safeguarded, which is the ultimate goal of this mechanism. In our proposed model, when adrenergically stimulated blood encounters plasma-accessible carbonic anhydrase (CA), an enzyme found in the RBC but also membrane-bound and potentially plasma-accessible in select locations, it will catalyze H⁺s removed from the RBC to form CO₂. This CO₂ will back-diffuse into the RBC creating an intracellular acidosis (extracellular alkalosis), reducing Hb–O₂ affinity, and ultimately elevating PO₂ via the Root effect. We created an in vitro closed system using rainbow trout (Oncorhynchus mykiss) blood where we can (1) simulate an acid-induced Root effect, (2) adrenergically stimulate the RBCs, and finally (3) short-circuit the βNHE via CA (CA-mediated Root effect), all of which can be monitored in real-time ( Fig. 1). Data generated currently support our Hypothesis: adrenergic RBC pH regulation can be short-circuited in the presence of plasma-accessible CA, therefore generating a Root effect increase in PO₂. In fact, if this scenario also occurs in the tissues of O. mykiss, CA-mediated short-circuiting of adrenergic pH regulation can facilitate an increase in PO₂ over 30 times that which would be generated in vertebrates possessing only a Bohr shift! We are ready to test our model in vivo by implanting fiber-optic O₂ sensors in O. mykiss muscle while simulating environmental and exercise stress with and without CA blockers. Furthermore, even though CA is not found in general circulation, there are membrane-bound and potentially plasma-accessible isoforms in muscle endothelia, and research is underway to localize this enzyme to understand the relationship between location and function of the short-circuiting. Teleost fish, which are more numerous than all other vertebrates combined (terrestrial and aquatic), have evolved an extraordinary O₂ delivery mechanism, the Root effect, that allows O₂ delivery to the eye and to the SB, thus allowing efficient buoyancy regulation, which may be one of the most important factors responsible for the extensive adaptive radiation in teleost fishes. Therefore, it is particularly interesting that the Root effect has not yet been investigated for general O₂ delivery. If the Root effect can also facilitate general O₂ delivery in vivo, which our data currently support, this would help shed insight into how the Root effect was selected for prior to the evolution of the βNHE, choroid gland and retia of the eye, and the gas gland and rete mirabile associated with the SB.