Analysis of the transport and interaction of oxygen and carbon dioxide in fish

Doctor of Philosoph

Gas transport and exchange: interaction between O2 and CO2 exchange

The interaction between O2 and CO2 in the general circulation of fish exists at the level of hemoglobin within the red blood cell, and is determined largely by the magnitude of the Bohr and Haldane effects. Assuming steady-state conditions, a Bohr–Haldane coefficient of 0.35–0.5 (0.5 × the respiratory quotient, RQ) is optimal for tissue O2 delivery (excluding the swimbladder and eye), and greater values may be important for CO2 excretion and acid–base homeostasis. Many teleosts possess a nonlinear Bohr–Haldane coefficient over the oxygen-equilibrium curve (OEC), which alters the nature of the interaction when different regions of the OEC are used for gas exchange. Recent in vivo experiments indicate that Bohr–Haldane coefficients close to RQ (typically 0.7–1.0) may play an important role in facilitating tissue O2 delivery in vivo likely due to the existence of large disequilibrium states in the blood

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.\ud \ud 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

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss)

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 °C to 25.5 °C in FW, held for 2 h, returned to 13.5 °C for 12 h, and then transferred to 32 ppt SW at 13.5 °C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na⁺ and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 °C were heat-shocked at 22 °C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na⁺/K⁺-ATPase activity or Na⁺/K⁺-ATPase α1a and α1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer

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)–O(2) 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 O(2) and CO(2) transfer through the RBCs. In the absence of plasma CA, nearly all plasma bicarbonate ultimately dehydrated to CO(2) occurred via the RBCs, and chloride/bicarbonate exchange was the rate-limiting step in CO(2) excretion. This pattern of CO(2) excretion across the gills resulted in disequilibrium states for CO(2) 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 CO(2) 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 CO(2) and acid production and, therefore, oxygen secretion to those specialised tissues. The evolution of β-adrenergically stimulated RBC Na^+/H^+ exchange protected gill O(2) uptake during stress and further augmented plasma disequilibrium states for CO(2) hydration/dehydration. Finally, RBC organophosphates (e.g. NTP) could be reduced during hypoxia to further increase Hb–O(2) affinity without compromising tissue O(2) delivery because high-affinity Hbs could still adequately deliver O(2) to the tissues via Bohr/Root shifts. We suggest that the evolution of this unique mode of tissue O(2) transfer evolved in the Triassic/Jurassic Period, when O(2) levels were low, ultimately giving rise to the most extensive adaptive radiation of extant vertebrates, the teleost fishes

American alligator (Alligator mississippiensis) embryos tightly regulate intracellular pH during a severe acidosis

Crocodilian nests naturally experience high CO2 (hypercarbia), which leads to increased blood PCO2 and reduced blood pH (pHe) in embryos; their response to acid-base challenges is not known. During acute hypercarbia, snapping turtle embryos preferentially regulate tissue pH (pHi) against pHe reductions. This is proposed to be associated with CO2 tolerance in reptilian embryos and is not found in adults. In the present study, we investigated pH regulation in American alligator Alligator mississippiensis (Daudin, 1802) embryos exposed to 1 h hypercarbia hypoxia (13 kPa PCO2, 9 kPa PO2). Hypercarbia hypoxia reduced pHe by 0.42 pH units while heart and brain pHi increased, with no change in pHi of other tissues. The results indicate American alligator embryos preferentially regulate pHi, similar to snapping turtle embryos, which represents a markedly different strategy of acid-base regulation than what is observed in adult reptiles. These findings suggest that preferential pHi regulation may be a strategy of acid-base regulation used by embryonic reptiles.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author