Red Blood Cell pH Research Articles

Acute freshwater CO2 exposure does not impair seawater transfer in three different sizes of Atlantic salmon (Salmo salar) subjected to different photoperiod manipulations.

There is a growing interest in Atlantic salmon (Salmo salar) aquaculture to extend the time fish are reared in freshwater (FW) recirculating aquaculture systems (RAS), producing larger FW salmon that can then be induced to undergo smoltification before transfer into marine net pens for grow-out and harvest. Smolts can be produced by photoperiod (PT) manipulation in RASs, but little is known about how delaying smoltification to larger body sizes affects susceptibility to elevated CO2 levels (hypercapnia), which can occur at high stocking densities in FW RAS or during transport from FW RAS rearing facilities to marine net pens. To address this, Atlantic salmon were reared from hatch to one of three different sizes (~230, ~580, or ~1300 g) in FW (3 ppt) under continuous light (24:0, light:dark). Once fish reached the desired sizes, a group of salmon were maintained on continuous light 24L:0D to serve as a control salmon. A second group of salmon were exposed to 8 weeks of 12L:12D and then to 4 weeks of 24L:0D to serve as PT treatment salmon, which is the PT manipulation commonly used in Atlantic salmon aquaculture to induce smoltification. At the end of PT manipulation, both control and PT treatment salmon were exposed to 0% or 1.5% CO2 (30 mg/L) for 96 h in FW and then transferred to air-equilibrated seawater (SW, 35 ppt, normocapnia). Salmon were sampled at the end of the 96-h FW CO2 exposure and at 24 h and 7 days in SW for measurements of blood ion/acid-base status, muscle water content (MWC), and gill and kidney Na+/K+ ATPase (NKA) activity. Exposure to 96 h of CO2 in FW resulted in acid-base disturbances in fish from all three size classes, with decreases in blood pH and increases in blood PCO2 and plasma [HCO3 -] but no mortality. Despite these large acid-base disturbances in FW, after transfer to normocapnic SW, there were no significant effects of CO2 exposure on extracellular blood pH, intracellular red blood cell pH, or plasma osmoregulatory status for all three sizes of post-smolt salmon. In general, SW transfer was associated with significant increases in plasma ions and osmolality, as well as gill and kidney NKA activity after 24 h and 1 week in SW with no significant impacts between different sizes of salmon. Thus, exposure to 30 mg CO2/L that mimics levels experienced during transport from FW RAS to an SW transfer site may have minimal effects on Atlantic salmon smolts up to 1300 g.

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.

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.

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.

Beyond just hemoglobin: Red blood cell potentiation of hemoglobin-oxygen unloading in fish.

Teleosts comprise 95% of fish species, almost one-half of all vertebrate species, and represent one of the most successful adaptive radiation events among vertebrates. This is thought to be in part because of their unique oxygen (O2) transport system. In salmonids, recent in vitro and in vivo studies indicate that hemoglobin-oxygen (Hb-O2) unloading to tissues may be doubled or even tripled under some conditions without changes in perfusion. This is accomplished through the short circuiting of red blood cell (RBC) pH regulation, resulting in a large arterial-venous pH difference within the RBC and induced reduction in Hb-O2 affinity. This system has three prerequisites: 1) highly pH-sensitive hemoglobin, 2) rapid RBC pH regulation, and 3) a heterogeneous distribution of plasma-accessible CA in the cardiovascular system (presence in the tissues and absence at the gills). Although data are limited, these attributes may be general characteristics of teleosts. Although this system is not likely operational to the same degree in other vertebrates, some of these prerequisites do exist, and the generation and elimination of pH disequilibrium states at the RBC will likely enhance Hb-O2 unloading to some degree. In human disease states, there are conditions that may partly satisfy those for enhanced Hb-O2 unloading, tentatively an avenue for future work that may improve treatment efficacy.

Osmoregulatory bicarbonate secretion exploits H(+)-sensitive haemoglobins to autoregulate intestinal O2 delivery in euryhaline teleosts.

Marine teleost fish secrete bicarbonate (HCO3 −) into the intestine to aid osmoregulation and limit Ca2+ uptake by carbonate precipitation. Intestinal HCO3 − secretion is associated with an equimolar transport of protons (H+) into the blood, both being proportional to environmental salinity. We hypothesized that the H+-sensitive haemoglobin (Hb) system of seawater teleosts could be exploited via the Bohr and/or Root effects (reduced Hb-O2 affinity and/or capacity with decreasing pH) to improve O2 delivery to intestinal cells during high metabolic demand associated with osmoregulation. To test this, we characterized H+ equilibria and gas exchange properties of European flounder (Platichthys flesus) haemoglobin and constructed a model incorporating these values, intestinal blood flow rates and arterial–venous acidification at three different environmental salinities (33, 60 and 90). The model suggested red blood cell pH (pHi) during passage through intestinal capillaries could be reduced by 0.14–0.33 units (depending on external salinity) which is sufficient to activate the Bohr effect (Bohr coefficient of −0.63), and perhaps even the Root effect, and enhance tissue O2 delivery by up to 42 % without changing blood flow. In vivo measurements of intestinal venous blood pH were not possible in flounder but were in seawater-acclimated rainbow trout which confirmed a blood acidification of no less than 0.2 units (equivalent to −0.12 for pHi). When using trout-specific values for the model variables, predicted values were consistent with measured in vivo values, further supporting the model. Thus this system is an elegant example of autoregulation: as the need for costly osmoregulatory processes (including HCO3 − secretion) increases at higher environmental salinity, so does the enhancement of O2 delivery to the intestine via a localized acidosis and the Bohr (and possibly Root) effect.

Washing of banked blood by three different blood salvage devices

Storage lesions in red blood cells (RBCs) lead to an accumulation of soluble contaminants that can compromise the patient. Organ failures, coagulopathies, and cardiovascular events including lethal cardiac arrest have been reported, especially with massive transfusion or in pediatric patients. Washing improves the quality of stored RBCs, and autotransfusion devices have been proposed for intraoperative processing, but these devices were designed for diluted wound blood, and limited data on their performance with RBCs are available. Three autotransfusion devices (Electa, Sorin; CATS, Fresenius; OrthoPAT, Haemonetics) differing in function of their centrifugation chambers were evaluated with RBCs at the end of their shelf life and with dilutions thereof. Elimination rates of potassium, plasma free hemoglobin, total protein, citrate, acid equivalents, and iomeprol added as a marker substance were analyzed, in addition to RBC recoveries. Product hematocrit (Hct) levels ranged between 54.8 and 72.6%. RBC recovery rates were between 62.7 and 95.0%, the lowest being with the OrthoPAT processing of undiluted RBCs. Plasma elimination rates increased with predilution and ranged from 46.6% to 99.5%, the lowest being with the CATS and undiluted RBCs. Washing did not change pH and buffering capacity of RBCs. Autotransfusion devices offer a practical and obviously economical option to wash banked RBCs intraoperatively to prevent hyperkalemia and other disturbances in massive transfusion or pediatric patients. Predilution improves elimination rates, especially in devices that produce high product Hct levels. With a Y-tubing the RBCs should bypass reservoir and vacuum, and the procedure should be guarded by a policy and procedure manual and a quality management system.

Use it or lose it? Sablefish,Anoplopoma fimbria, a species representing a fifth teleostean group where the βNHE associated with the red blood cell adrenergic stress response has been secondarily lost

Like most teleosts, sablefish (Anoplopoma fimbria Pallas 1814) blood exhibits a moderate Root effect (~35% maximal desaturation), where a reduction in blood pH dramatically reduces O(2) carrying capacity, a mechanism important for oxygenating the eye and filling the swim bladder (SB) in teleosts. Although sablefish lack a SB, we observed a well-defined choroid rete at the eye. The adrenergically mediated cell swelling typically associated with a functional red blood cell (RBC) beta-adrenergic Na(+)/H(+) exchanger (betaNHE), which would normally protect RBC pH, and thus O(2) transport, during a generalized acidosis, was not observed in sablefish blood. Neither isoproterenol (a beta-agonist) nor 8-bromo cAMP could elicit this response. Furthermore, RBC osmotic shrinkage, known to stimulate NHEs in general and betaNHE in other teleosts such as trout and flounder, resulted in no significant regulatory volume increase (RVI), further supporting the absence of a functional RBC betaNHE. The onset of the Root effect occurs at a much lower RBC pH (6.83-6.92) than in other teleosts, and thus RBC betaNHE may not be required to protect O(2) transport during a generalized acidosis in vivo. Phylogenetically, sablefish may represent a fifth group of teleosts exhibiting a secondary reduction or loss of betaNHE activity. However, sablefish have not lost the choroid rete at the eye (unlike in the other four groups), which may still function with the Root effect to oxygenate the retina, but the low pH onset of the Root effect may ensure haemoglobin (Hb)-O(2) binding is not compromised at the respiratory surface during a general acidosis in the absence of RBC betaNHE. The sablefish may represent an anomaly within the framework of Root effect evolution, in that they possess a moderate Root effect and a choroid rete at the eye, but lack the RBC betaNHE and the SB system.

Red blood cell pH, the Bohr effect, and other oxygenation‐linked phenomena in blood O2 and CO2 transport

The discovery of the S-shaped O2 equilibrium curve and the Bohr effect in 1904 stimulated a fertile and continued research into respiratory functions of blood and allosteric mechanisms in haemoglobin (Hb). The Bohr effect (influence of pH/CO2 on Hb O2 affinity) and the reciprocal Haldane effect (influence of HbO2 saturation on H+/CO2 binding) originate in the Hb oxy-deoxy conformational change and allosteric interactions between O2 and H+/CO2 binding sites. In steady state, H+ is passively distributed across the vertebrate red blood cell (RBC) membrane, and intracellular pH (pHi) changes are related to changes in extracellular pH, Hb-O2 saturation and RBC organic phosphate content. As the Hb molecule shifts between the oxy and deoxy conformation in arterial-venous gas transport, it delivers O2 and takes up CO2 and H+ in tissue capillaries (elegantly aided by the Bohr effect). Concomitantly, the RBC may sense local O2 demand via the degree of Hb deoxygenation and release vasodilatory agents to match local blood flow with requirements. Three recent hypotheses suggest (1) release of NO from S-nitroso-Hb upon deoxygenation, (2) reduction of nitrite to vasoactive NO by deoxy haems, and (3) release of ATP. Inside RBCs, carbonic anhydrase (CA) provides fast hydration of metabolic CO2 and ensures that the Bohr shift occurs during capillary transit. The formed H+ is bound to Hb (Haldane effect) while HCO3- is shifted to plasma via the anion exchanger (AE1). The magnitude of the oxylabile H+ binding shows characteristic differences among vertebrates. Alternative strategies for CO2 transport include direct HCO3- binding to deoxyHb in crocodilians, and high intracellular free [HCO3-] (due to high pHi) in lampreys. At the RBC membrane, CA, AE1 and other proteins may associate into what appears to be an integrated gas exchange metabolon. Oxygenation-linked binding of Hb to the membrane may regulate glycolysis in mammals and perhaps also oxygen-sensitive ion transport involved in RBC volume and pHi regulation. Blood O2 transport shows several adaptive changes during exposure to environmental hypoxia. The Bohr effect is involved via the respiratory alkalosis induced by hyperventilation, and also via the pHi change that results from modulation of RBC organic phosphate content. In teleost fish, beta-adrenergic activation of Na+/H+ exchange rapidly elevates pHi and O2 affinity, particularly under low O2 conditions.

Hydrogen ion binding properties of tuna haemoglobins

Tunas are very active fish with a high aerobic capacity, but they also regularly perform burst swimming with massive production of lactic acid. The present study examines whether H + buffering by tuna haemoglobin (Hb) is elevated to cope with metabolic acidoses (by analogy with the high buffer capacity of tuna white musculature) or whether the Hb–H + binding properties resemble other teleosts that have low buffer values and high Haldane effects. H + titration of oxygenated and deoxygenated composite Hb from yellowfin tuna, skipjack tuna and bigeye tuna in 0.1 M KCl revealed low Hb-specific buffer values in all three tunas. Values at physiological pH were comparable to those reported in less active species such as carp and eel. The fixed acid Haldane effect was large (maximal uptakes of close to 4 mol H + per mol Hb tetramer upon deoxygenation). Thus, the Hb–H + binding properties of very active tuna species correspond to other teleosts. Low Hb buffer values may be a pre-requisite for the regulation of red blood cell pH via Na +/H + exchange. Approximately nine ‘neutral’ groups were titratable in tuna Hbs, suggesting that two α-amino groups and seven histidine residues are titrated within each tetramer.

Haemoglobin H+ equilibria in lamprey (Lampetra fluviatilis) and hagfish (Myxine glutinosa)

Agnathans, comprising lamprey and hagfish species, have been reported to be practically devoid of HCO3-/Cl- exchange across the red blood cell membrane. This suggests that the capacity of their haemoglobin (Hb) to remove H+ is essential for obtaining a high CO2-carrying capacity in the blood. Hydrogen ion titrations were performed on oxygenated and deoxygenated composite Hbs from river lamprey and from Atlantic hagfish at 15 degrees C and an ionic strength of 0.1 (0.1 mol l-1 KCl). Lamprey Hb was characterised by very low buffer values when the degree of oxygenation was constant, whereas the fixed-acid Haldane effect was large (uptake of approximately 0.9 H+ per monomer upon deoxygenation). Hagfish Hb, in contrast, had large buffer values and a moderate fixed-acid Haldane effect. In deoxygenated Hb, the low buffer values in lamprey correlated with the presence of only 1-1.5 titratable 'neutral' groups (normally histidines and &agr; -amino groups) per monomer, whereas there were 4-5 titratable 'neutral' groups per monomer in hagfish. The large differences in Hb/H+ equilibria between the two species reflect the early evolutionary divergence between lampreys and hagfish. With respect to CO2 transport, the special Hb/H+ equilibria and the high red blood cell pH in lamprey ensure a high concentration of free HCO3- inside the red cells in venous blood, which compensates for the absence of a shift of HCO3- to the plasma. The Hb/H+ equilibria in hagfish are less effective in ensuring a high CO2-carrying capacity given the virtual absence of a red blood cell HCO3-/Cl- exchange, and other adaptations may be involved.

Evaluation of canine red blood cells stored in a saline, adenine, and glucose solution for 35 days.

An additive solution for the storage of red blood cells was evaluated for use in dogs. Blood collected from 6 dogs was processed into packed red blood cells and stored for 35 days in the additive solution Nutricel (Miles, Inc, Pharmaceutical Division, West Haven, CT). Packed red blood cells stored in citrate-phosphate-dextrose-adenine (CPDA-1; Fenwal Laboratories, Baxter Health Care Corp, Deerfield, IL) also were evaluated for comparison. Red blood cell 2,3-diphosphoglycerate (2,3-DPG) concentration, adenosine triphosphate (ATP) concentration, percentage hemolysis, and pH were determined. The red blood cell post-transfusion viability (PTV) after 35 days of storage was assessed with both single-labeled chromium 51 (51Cr) and double-labeled technetium 99m/chromium 51 (99mTc/51Cr) techniques. Mean ATP concentration and percentage hemolysis of the cells stored in Nutricel were 1.1 mumol/g hemoglobin (Hb) and 0.28% respectively and did not differ significantly (P < .05) from the values of 1.0 mumol/g Hb and 0.33% from the CPDA.1-stored red blood cells. The mean pH of red blood cells stored in Nutricel was 6.19, which was significantly lower than the pH of 6.47 for cells stored in CPDA-1. The mean 2,3-DPG concentration of red blood cells stored in Nutricel was significantly higher at 10.1 mumol/g Hb than the 2,3-DPG concentration of 3.4 mumol/g Hb for cells stored in CPDA-1. The mean PTV of canine red blood cells stored in Nutricel for 35 days was 85% with 51Cr and 90% with 99mTc/51Cr. This was significantly higher than the mean PTVs of 38% and 36% for the CPDA-1 stored cells as assessed with 51Cr and 99mTc/51Cr techniques, respectively. It was concluded that 35-day-old canine red blood cells stored in Nutricel are of acceptable quality for transfusion purposes.

Adenosine receptor blockade and hypoxia-tolerance in rainbow trout and Pacific hagfish. II. Effects on plasma catecholamines and erythrocytes

The purpose of this study was to examine the role of adenosine receptors (ARs) in (1) the regulation of catecholamine secretion and (2) the modulation of blood oxygen capacitance by catecholamines. To this end, we assessed the response of rainbow trout and Pacific hagfish treated with either an AR blocker, theophylline, or saline under hypoxic and normoxic conditions. Compared with the control hypoxic rainbow trout, AR blockade resulted in a smaller increase in haematocrit and haemoglobin (Hb) concentration of the blood, smaller red blood cell transmembrane pH differences and mean cellular [Hb] (MCHC), as well as a 16-fold higher plasma adrenaline concentration after only 10 min of acute hypoxic exposure. In hypoxic hagfish, AR blockade had no effect on the [Hb] of the blood, and there was no regulation of red blood cell pH or changes in MCHC. However, whereas plasma [adrenaline] did not change following exposure to a PwO2 of 1.33 kPa in the hypoxic sham group, the concentration increased 3.8-fold within 10 min in the theophylline-injected group. These results suggest that adenosine modulates the circulating level of catecholamines in both hypoxic rainbow trout and hypoxic Pacific hagfish.

Effects of hypoxia on rainbow trout (Oncorhynchus mykiss): intraerythrocytic phosphates

Intraerythrocytic levels of guanosine and adenosine phosphates were estimated in normoxic and hypoxic rainbow trout after intra-arterial injection with either saline or carbonic anhydrase. A significant reduction of the total pool of adenosine and guanosine was observed in hypoxic animals. Similarly, a decrease in both ATP and GTP levels occurred in hypoxic animals injected either with saline or with carbonic anhydrase. Interestingly, there was a highly significant relationship between ATP and GTP levels, indicating that they are under similar control. In addition, a significant positive relationship between nucleoside triphosphate (NTP) and Mg2+ levels was observed. It is possible that the availability of ATP and GTP to hemoglobin (Hb) may be proportionally smaller than their intraerythrocytic absolute levels. No clear relationship between intraerythrocytic NTP levels and plasma carbonic anhydrase infusion was observed, despite the significant effect of carbonic anhydrase on acid&shy;base balance. There was a significant relationship between red blood cell pH and [NTP]:[Hb] in fish exposed to hypoxia for 48 h. No such relationship existed during the first 6 h of hypoxic exposure. Possible mechanisms accounting for the reduction in NTP levels during the initial phases of hypoxia are discussed.

In vitro analysis of volume and pH regulation in the red blood cells of a primitive air-breathing fish, the bowfin, Amia calva

In the bowfin (Amia calva), a decrease in extracellular pH in vitro was associated with an increase in the water content and chloride concentration in the red blood cells that could be inhibited by the anion-exchange blocker, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). After a step increase in CO2 tension, the extracellular total CO2 concentration was also significantly reduced by DIDS. Finally, over most of the experimental pH range, the red blood cell pH observed in the presence of DIDS was significantly elevated compared with that of controls. Taken together, these results indicate that as in most other fishes, chloride–bicarbonate exchange is clearly present and functional in bowfin red blood cells. Moreover, within the physiological pH range, ion movements across the anion exchanger have a marked influence on both the volume and the pH of bowfin red blood cells. In sharp contrast to the rainbow trout (Oncorhynchus mykiss), catecholamines had no effect on the volume, pH, or intracellular sodium concentration of red blood cells in the bowfin. Following osmotic swelling, rainbow trout red blood cells were able to regulate their volume back to control levels within 2 h. In the bowfin, however, there was no regulation of red blood cell volume after osmotic swelling. Thus, in contrast to many other fishes examined to date, it would appear that in the bowfin, the physiological mechanisms involved in the adrenergic response and in the regulatory volume decrease after osmotic swelling may be less active or possibly even absent in the red blood cells.

Relationship between blood buffering properties, erythrocyte pH and water content, in gray seals (Halichoerus grypus).

The separated plasma of gray seals (Halichoerus grypus) has an unusually high non-bicarbonate buffering capacity (beta NB = d[HCO3-]/dpH = -21 Slykes; Slyke = mmol l-1 pH unit-1). To examine whether this represents a trait of mammalian carnivora, or is related instead to deep diving, we examined blood from dog and killer whale, respectively. The beta NB of separated plasma of dog was typically low (-4 to -8 Slykes), as in other mammals, whereas that of the whale (-13.5 Slykes), while significantly higher than dog, was not nearly as high as in seal. As one would theoretically predict of a blood system with exceptionally high extracellular buffering and functional anion exchange across the erythrocyte membrane, there was little difference in the buffer slope between true plasma and whole blood of seal. This appears to be a novel pattern of CO2 carriage in mammals. Erythrocyte ion concentrations were similar to other mammalian carnivora, with intracellular sodium concentration being high and potassium low. The red cell water content was unaffected by changes in oxygen saturation and tended to decrease when extracellular pH decreased below 7.4. Apart from the comparatively elevated Bohr factor, -0.518 as a function of extracellular pH and -0.550 as a function of red blood cell pH, the blood oxygen binding properties were similar to terrestrial mammals of similar size.

In vitro Interactions Between Oxygen and Carbon Dioxide Transport in the Blood of the Sea Lamprey (Petromyzon Marinus)

ABSTRACT In vitro experiments were carried out to examine the interactions between oxygen and carbon dioxide transport in the blood of the sea lamprey. Oxygen dissociation curves for whole blood obtained from quiescent lampreys had Hill numbers (NH) ranging from 1.52 to 1.89. The Bohr coefficient for whole blood was −0.17 when extracellular pH (pHe) was considered, but was much greater (−0.63) when red blood cell pH (pHi) was considered. The pHi was largely dependent on haemoglobin oxygen-saturation and the pH gradient across the red blood cell membrane was often reversed when was increased and/or was lowered. The magnitude of the increase in pHi associated with the Haldane effect ranged from 0.169 pH units at 2.9 kPa to 0.453 pH units at a of 0.2 kPa. Deoxygenated red blood cells had a much greater total CO2concentration than oxygenated red blood cells, but the nonbicarbonate buffer value for the red blood cells was unaffected by oxygenation. Plasma was not significantly different under oxygenated or deoxygenated conditions. Partitioning of CO2 carriage in oxygenated and deoxygenated blood supports recent in vivo observations that red blood cell CO2 carriage can account for much of the difference between arterial and venous blood. Together, the results also suggest that oxygen and carbon dioxide transport may not be tightly coupled in the blood of these primitive vertebrates. Finally, red cell sodium concentrations were dependent on oxygen and carbon dioxide tensions in the blood, suggesting that sodium-dependent ion transport processes may contribute to the unique strategy for gas transport in sea lamprey blood.

The role of catecholamines in regulating arterial oxygen content during acute hypercapnic acidosis in rainbow trout ( Salmo gairdneri)

The involvement of catecholamines in regulating arterial oxygen content (Ca O 2 ) during hypercapnic acidosis in rainbow trout ( Salmo gairdneri) was investigated by comparing physiological responses during acute normoxic hypercapnia (a condition in which plasma epinephrine is elevated) and acute hyperoxic hypercapnia (a condition in which plasma epinephrine is not elevated). Red blood cell pH (rbc pH) was maintained significantly higher in the normoxic hypercapnic fish despite similar reductions in whole blood pH (pHe) in both groups. Elevation of rbc pH in the normoxic hypercanic fish was abolished by pre-treatment with the β-adrenoceptor antagonist, propranolol, whereas injection of epinephrine into hyperoxic hypercapnic fish significantly raised rbc pH. Arterial blood oxygen carrying capacity increased only in the normoxic hypercapnic fish due to significant increases in blood haemoglobin (Hb) levels. The ability in trout to elevate blood Hb was abolished by pre-treatment with the α-adrenoceptor antagonist, phentolamine. Injections of epinephrine into normacapnic fish caused increases in blood Hb and concomitant decreases in spleen wet weight and Hb content. Adrenergic elevation of blood Hb was not observed in splenectomized fish. Ca O 2 , although depressed during normoxic hypercapnia, was indeed regulated when compared to Ca O 2 values predicted from the in vitro Root effect. Pre-treatment with phentolamine, but not propranolol, abolished the ability of trout to regulate Ca O 2 .

Adrenergic involvement in blood oxygen transport and acid-base balance during hypercapnic acidosis in the Rainbow Trout,Salmo gairdneri

Rainbow trout (Salmo gairdneri) were subjected to 12 h of external hypercapnia (1% CO2 in air) during α- and/or β-adrenoceptor blockade in order to assess the importance of adrenergic responses in modulating blood oxygen transport and acid-base balance during an acute acidotic stress. External hypercapnia caused an elevation of blood carbon dioxide tension and a reciprocal decrease in whole blood pH. A gradual elevation of blood bicarbonate levels caused whole blood pH to increase toward pre-hypercapnic values throughout the hypercapnic period. Pre-treatment of fish with propranolol (a β-adrenoceptor antagonist) or phentolamine (an α-adrenoceptor antagonist) did not affect their ability to regulate extracellular acid-base status during hypercapnia. On the other hand, adrenergic responses were essential in the maintenance of arterial blood oxygen content during hypercapnia despite the severe extracellular acidosis and a marked Root effect in trout blood, in vitro. Important adrenergic responses included pronounced increases in haematocrit (an α-adrenergic effect) and arterial oxygen tension (α- and β-adrenergic effects) as well as partial regulation of red blood cell pH (a β-adrenergic effect). Although pre-treatment of fish with either propranolol or phentolamine caused a reduction in blood oxygen content during hypercapnia, fish died only during complete adrenoceptor blockade, presumably due to severe hypoxemia.

Effects of prolonged epinephrine infusion on blood respiratory and acid-base states in the rainbow trout: Alpha and beta effects

Rainbow trout were infused continuously for 12h with epinephrine in the presence or absence of alpha-and/or beta-adrenergic blockade to characterize the specific adrenergic mechanisms involved in the control of blood acid-base status and oxygen transport capacity. Infusion of epinephrine, alone, produced a transient respiratory acidosis, as indicated by an increase in carbon dioxide tension and a decrease in whole blood pH, yet arterial oxygen tension was elevated. Red blood cell pH increased by approximately 0.2 pH units during epinephrine infusion and this increase as well as the increase in oxygen tension were prevented by pretreatment with propranolol (a beta-adrenergic antagonist). Epinephrine infusion during alpha-adrenergic blockade caused a prolonged elevation of blood carbon dioxide tension and abolished the increases in hematocrit and hemoglobin concentrations observed during epinephrine infusion alone. Infusion of the alpha-adrenergic agonists phenylephrine (an alpha1 agonist) or clonidine (an alpha2 agonist) caused respiratory acidosis (decreased pH, increased CO2 tension) and a reduction in oxygen tension. Infusion of isoprenaline (a non-specific beta agonist) caused delayed increases in carbon dioxide and oxygen tensions. We speculate that the increased carbon dioxide tension observed during epinephrine infusion is a result of beta-adrenoceptor mediated inhibition of red blood cell bicarbonate dehydration and not branchial convective or diffusive adjustments. The effects of epinephrine on blood O2 tension, content and carrying capacity are discussed with reference to the participation of alpha- and beta-adrenergic mechanisms at the gill, spleen and red blood cell.