Molecular basis of a novel adaptation to hypoxic-hypercapnia in a strictly fossorial mole

Background  

Elevated blood O₂ affinity enhances survival at low O₂ pressures, and is perhaps the best known and most broadly accepted evolutionary adjustment of terrestrial vertebrates to environmental hypoxia. This phenotype arises by increasing the intrinsic O₂ affinity of the hemoglobin (Hb) molecule, by decreasing the intracellular concentration of allosteric effectors (e.g., 2,3-diphosphoglycerate; DPG), or by suppressing the sensitivity of Hb to these physiological cofactors.     

Root Effect Haemoglobins in Fish May Greatly Enhance General Oxygen Delivery Relative to Other Vertebrates

The teleost fishes represent over half of all extant vertebrates; they occupy nearly every body of water and in doing so, occupy a diverse array of environmental conditions. We propose that their success is related to a unique oxygen (O₂) transport system involving their extremely pH-sensitive haemoglobin (Hb). A reduction in pH reduces both Hb-O₂ affinity (Bohr effect) and carrying capacity (Root effect). This, combined with a large arterial-venous pH change (ΔpH_a-v) relative to other vertebrates, may greatly enhance tissue oxygen delivery in teleosts (e.g., rainbow trout) during stress, beyond that in mammals (e.g., human). We generated oxygen equilibrium curves (OECs) at five different CO₂ tensions for rainbow trout and determined that, when Hb-O₂ saturation is 50% or greater, the change in oxygen partial pressure (ΔPO₂) associated with ΔpH_a-v can exceed that of the mammalian Bohr effect by at least 3-fold, but as much as 21-fold. Using known ΔpH_a-v and assuming a constant arterial-venous PO₂ difference (P_a-vO₂), Root effect Hbs can enhance O₂ release to the tissues by 73.5% in trout; whereas, the Bohr effect alone is responsible for enhancing O₂ release by only 1.3% in humans. Disequilibrium states are likely operational in teleosts in vivo, and therefore the ΔpH_a-v, and thus enhancement of O₂ delivery, could be even larger. Modeling with known P_a-vO₂ in fish during exercise and hypoxia indicates that O₂ release from the Hb and therefore potentially tissue O₂ delivery may double during exercise and triple during some levels of hypoxia. These characteristics may be central to performance of athletic fish species such as salmonids, but may indicate that general tissue oxygen delivery may have been the incipient function of Root effect Hbs in fish, a trait strongly associated with the adaptive radiation of teleosts.     

The effect of isovolemic anaemia on blood O2 affinity and red cell triphosphate concentrations in the painted turtle (Chrysemys picta).

The blood oxygen affinity of vertebrates is regulated, in part, through changes in red cell phosphate levels and increased oxygen affinity during reductions in inspired oxygen and is a well-described and common feature. However, during anaemia, when oxygen delivery is compromised by a reduction in blood oxygen carrying capacity, a lowering of blood oxygen affinity will facilitate oxygen unloading in the tissues, while oxygen loading at the gas exchange organ is not impaired. The present study investigated the effects of artificially induced anaemia in vivo on the blood oxygen affinity and red cell nucleoside triphosphate (NTP) concentrations in the turtle, Chrysemys picta. Blood was obtained from conscious animals through an arterial catheter and oxygen equilibrium curves were determined using the Tucker method while NTP concentrations were analyzed spectrophotometrically. Before induction of anaemia haematocrit averaged 23% and P50 was 18.5 +/- 0.7 with a NTP/Hb of 0.20 +/- 0.01 (mmol/mmol). After the haematocrit had been reduced to approximately 10% by bleeding (48-96 h) (blood volume was maintained by re-infusion of plasma and Ringer) there were no effects on P50 or red cell NTP concentrations. Thus, in contrast to fish and mammals, turtles do not exhibit a change in blood oxygen affinity during anaemia.     

Oxygen Transport in the Avian Egg at High Altitude

Oxygen transport to avian embryo tissues occurs by three steps,two of which are driven by diffusion. This results in a seriesof stepwise decrements in PO₂ between atmosphere and tissue.The PO₂ decrements for embryos of the domestic fowl incubatedat different altitudes are used here to examine potential adaptationsto hypobanc hypoxia. With exposure to moderate hypoxia embryosof the domestic fowl appear to maintain adequate tissue oxygenation.Adaptive adjustments in the shell, shell membranes and chorioallantoiscomplex were not observed. However, hemoglobin O₂ affinity wasincreased and preliminary evidence suggests a redistributionof blood flow to maintain adequate oxygenation in higher priorityareas of embryonic tissue. At severe hypoxia, embryos of thedomestic fowl show decreased O₂ consumption, embryo mass andlengthened incubition period. Thus at severe hypoxin the embryoof the domestic fowl does not appear to provide a realisticmodel. Evidence from avian embryos of species native to highaltitude suggest that they are able to maintain adequate tissueoxygenation even at severe hypoxia. Preliminary evidence suggeststhat some of the blood vascular system and tissue level adaptationspresent in the chicken embryo are also present in species nativeto high altitude. One of these, an increase in embryonic hemoglobin-O₂affinity which is physiologically mediated in the chicken embryois genetically-based in the embryo of the native high-altitudespecies.     

Splenic contraction-induced reversible increase in hemoglobin concentration in intermittent hypoxia

The effect of intermittent hypoxia (IHx) on blood hemoglobinconcentration ([Hb]) and the underlying mechanisms werestudied in rats exposed to 10%O₂, 1 h/day, for up to 5 wk. IHxprotocols with longer daily hypoxic exposure show persistentpolycythemia; however, it is unknown whether [Hb] increasestransiently during hypoxia in protocols without polycythemia. Hypoxiaproduced a reversible [Hb] increase after 4 days of IHx butnot in normoxic controls (NxC) or after shorter period of IHx.Splenectomy abolished the phenomenon. Plasma epinephrine andnorepinephrine levels during hypoxia were comparable in IHx and NxCgroups, but the epinephrine-induced [Hb] increase waslarger in IHx. The ₁- and₂-adrenoreceptor blockade(phentolamine) and ₂-blockade(yohimbine) abolished the [Hb] increase of IHx rats.Conversely, ₂-receptorstimulation (oxymetazoline) increased [Hb] during normoxiain IHx but not in NxC. In conclusion, this IHx protocol results inreversible [Hb] increases during hypoxia via spleniccontraction mediated by increased₂-adrenoreceptor response. Thismay protect O₂ supply duringhypoxia without the cardiovascular burden of polycythemia during normoxia.

     

Time-course of the polycythemic response in normoxic and hypoxic mice with high blood oxygen affinity induced by cyanate administration

The Hb-O₂ affinity and the erythropoietic response as a function of time were studied in mice treated with sodium cyanate for up to 2 months. Cyanate increased the Hb-O₂ affinity in normoxic mice more than in chronically hypoxic mice. The hemoglobin concentration rose as a function of time both in normoxic and hypoxic conditions but reached higher levels in hypoxia. After 42 days of study (21 days of hypoxia) hemoglobin reached maximum levels and thereafter showed a plateau in both cyanate and control animals. It is concluded that a chronic left-shifted oxygen dissociation curve does not avoid the development of hypoxic polycythemia in mice. Moreover, prolonged cyanate administration potentiates the crythropoietic response to chronic hypoxia. Since polycythemia is an index of tissue hypoxia, the results show that the high hemoglobin affinity did not prevent tissue hypoxia in low PO₂ conditions. Results showing beneficial effects of high hemoglobin oxygen affinity induced by cyanate based on acute hypoxic expositions should be cautiously interpreted with regard to their adaptive value in animals chronically exposed to natural or simulated hypoxia.Abbreviations Hb hemoglobin - NaOCN sodium cyanate - ODC oxygen dissociation curve - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂     

Kinetics of the acclimational responses of tench to combined hypoxia and hypercapnia

Summary Exposing tench to environmental hypoxia-hypercapnia reduces routine O₂ consumption, sharply decreases arterial O₂ tension and the difference between the water and the blood, and results in marked swelling of the erythrocytes. These changes are rapidly reversed upon return to normoxia.Hypoxic-hypercapnic conditions lower the blood NTP/Hb ratio to a new steady state level within 24 h, by reducing GTP/Hb but not ATP/Hb. A similar selective reduction of eryhtrocytic GTP content forms the initial response of blood incubated in vitro to anoxic conditions.The swelling as well as the reduced GTP/Hb ratio in the erythrocytes appear to improve O₂ loading in the gills during environmental hypoxia-hypercapnia.Symbols and abbreviations a arterial - GTP guanosine triphosphate - Hct hematocrit - I inspired - NTP nucleoside triphosphate - w water     

Reactive oxygen species formation in the transition to hypoxia in skeletal muscle

Many tissues produce reactive oxygen species (ROS) during reoxygenation after hypoxia or ischemia; however, whether ROS are formed during hypoxia is controversial. We tested the hypothesis that ROS are generated in skeletal muscle during exposure to acute hypoxia before reoxygenation. Isolated rat diaphragm strips were loaded with dihydrofluorescein-DA (Hfluor-DA), a probe that is oxidized to fluorescein (Fluor) by intracellular ROS. Changes in fluorescence due to Fluor, NADH, and FAD were measured using a tissue fluorometer. The system had a detection limit of 1 µM H₂O₂ applied to the muscle superfusate. When the superfusion buffer was changed rapidly from 95% O₂ to 0%, 5%, 21%, or 40% O₂, transient elevations in Fluor were observed that were proportional to the rise in NADH fluorescence and inversely proportional to the level of O₂ exposure. This signal could be inhibited completely with 40 µM ebselen, a glutathione peroxidase mimic. After brief hypoxia exposure (10 min) or exposure to brief periods of H₂O₂, the fluorescence signal returned to baseline. Furthermore, tissues loaded with the oxidized form of the probe (Fluor-DA) showed a similar pattern of response that could be inhibited with ebselen. These results suggest that Fluor exists in a partially reversible redox state within the tissue. When Hfluor-loaded tissues were contracted with low-frequency twitches, Fluor emission and NADH emission were significantly elevated in a way that resembled the hypoxia-induced signal. We conclude that in the transition to low intracellular PO₂, a burst of intracellular ROS is formed that may have functional implications regarding skeletal muscle O₂-sensing systems and responses to acute metabolic stress. dihydrofluorescein; tissue fluorometer; ebselen; N-acetylcysteine; rat     

Physiological Adaptations of Crayfish to the Hypoxic Environment

SYNOPSIS. Crayfish routinely encounter waters of reduced oxygentension, resulting in a broad array of behavioral and physiologicalresponses. Many animals when faced with this stress will simplyremove themselves from the irritating environment through voluntarymigration. When an animal, either by choice or through physicalconstraints remains in a hypoxic environment it must compensatefor the reduction in O₂ availability. Many crayfish have theability to maintain oxygen consumption independent of waterPo₂ down to some critical level; below this the animal can nolonger maintain normoxic levels of aerobic metabolism. Regulationof oxygen uptake is thought to be due to a hypoxia-induced hyperventilationalong with an increase in hemocyanin O₂ affinity and an improvementin the ability of the respiratory surfaces to transfer O₂. Crayfishexposed to a reduction in water oxygen also show a strong bradycardia,which is compensated for by an increase in stroke volume, resultingin a maintenance of cardiac output. The adaptive advantage ofthis response is uncertain. As water Po₂ drops crayfish havebeen shown to redistribute cardiac output, presumably throughthe action of the cardioarterial valves. Hemolymph is shuntedto the anterior end of the animal, resulting in a greater perfusionof nervous tissue. The animals' ability to detect changes inwater Po₂ appear to result from O₂ sensitive receptors locatedon the gills or in the branchiocardiac veins. The integratedphysiological response toward environmental hypoxia allows thecrayfish to not only deal with the stress but to maintain activity.     

Respiratory Function of the Hemocyanins

Recent studies clearly demonstrate the respiratory importanceof the hemocyanins in each of the three animal phyla in whichthey occur. Despite their generally low oxygen affinity, hemocyaninscan be highly oxygenated at the site of gas exchange with themedium as well as deoxygenated at the tissues. The functionalrange of a hemocyanin oxygen transport system is severely limitedhowever by environmental change. These systems function underincipient hypoxia due largely to responses of blood pH whichare not fully understood a normal Bohr shift is accompaniedby a rise in blood pH and a reverse Bohr shift by a decreasein blood pH. In both instances blood oxygen affinity increasesand its oxygenation state at the gill remains high in spiteof its lower Po₂. Dilution of the blood at low salinity generallyalters its oxygenation properties both oxygen affinity and cooperativity.These properties may or may not be restored by concomitant changesin blood pH, which depend on the various mechanisms of osmoticadaptation. Within a homogeneous taxon the oxygenation properties of a hemocyaninappear to be highly conservative showing little interspecificadaptation except to extreme changes in the mode of gas exchange.Unlike that in vertebrates air-breathing in crustaceans is accompaniedby an increase in blood oxygen affinity. Similar oxygen affinitiesin latitudinally separated species result in optimal functioningof the system at the same temperature, corresponding to differentseasons. In eurythermal species a temperature acclimation ofoxygen affinity extends the operating range of the crustaceanhemocyanins but they cannot deoxygenate at very low temperatures. Unsolved problems of hemocyanin function include specific effectsof pH and CO₂ the basis of which is not entirely clear, andthe postulated occurrence in native blood of both dialyzableand non-dialyzable substances that modify oxygen affinity theidentity of which is unknown. With the exception of the crustacean oxygen carrier the hemocyaninsconfer a respiratory advantage over their predecessors. Butthe oxygen carrying capacity of crustacean blood never reachesthe levels found in the annelids and molluscs due to the colloidosmotic pressure of the relatively low molecular weight hemocyaninand to the drop in blood hydrostatic pressure accompanying theloss of a fluid skeleton. The selection of a blood oxygen carrierwith an apparently limiting combination of respiratory and osmoticproperties is obscured by the uncertain phylogenetic positionof the phylum.     

Ecological, Evolutionary, and Physical Factors Influencing Aquatic Animal Respiration

Compared to air-breathers, animals that respire aquaticallyhave limited access to O₂ and their habitats are more subjectto hypoxia. Because O₂ diffuses more slowly through water thanair, animals in water experience greater diffusion boundarylayer effects on respiratory gas diffusion. While ventilationand specialized exchange surfaces mitigate O₂ diffusion limitationson respiration, most animal phyla, particularly those confinedto aquatic habitats, lack these. Diffusion limitation influencesthe ontogeny of aquatic animals and may have also shaped Precambrianmetazoans. In spite of a more limited O₂ access, aquatic animalsdisplay a much greater spectrum of respiratory adaptation, rangingfrom the loss of Hb in icefishes to the independent evolution,invention, and acquisition of Hb in many invertebrates confinedto hypoxic habitats. Three features of aquatic respiratory systemsdistinguishing them from aerial systems are the widespread occurrenceof integumental respiration, the frequent presence of combinedrespiratory and feeding surfaces, and the profound effect ofhypoxia on shaping respiratory adaptation, both in shallow waterand in the deep sea.     

The evolution of Root effect hemoglobins in the absence of intracellular pH protection of the red blood cell: insights from primitive fishes

The Root effect, a reduction in blood oxygen (O₂) carrying capacity at low pH, is used by many fish species to maximize O₂ delivery to the eye and swimbladder. It is believed to have evolved in the basal actinopterygian lineage of fishes, species that lack the intracellular pH (pH_i) protection mechanism of more derived species’ red blood cells (i.e., adrenergically activated Na⁺/H⁺ exchangers; βNHE). These basal actinopterygians may consequently experience a reduction in blood O₂ carrying capacity, and thus O₂ uptake at the gills, during hypoxia- and exercise-induced generalized blood acidoses. We analyzed the hemoglobins (Hbs) of seven species within this group [American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), mooneye (Hiodon tergisus), and pirarucu (Arapaima gigas)] for their Root effect characteristics so as to test the hypothesis of the Root effect onset pH value being lower than those pH values expected during a generalized acidosis in vivo. Analysis of the haemolysates revealed that, although each of the seven species displayed Root effects (ranging from 7.3 to 40.5% desaturation of Hb with O₂, i.e., Hb O₂ desaturation), the Root effect onset pH values of all species are considerably lower (ranging from pH 5.94 to 7.04) than the maximum blood acidoses that would be expected following hypoxia or exercise (pH_i 7.15–7.3). Thus, although these primitive fishes possess Hbs with large Root effects and lack any significant red blood cell βNHE activity, it is unlikely that the possession of a Root effect would impair O₂ uptake at the gills following a generalized acidosis of the blood. As well, it was shown that both maximal Root effect and Root effect onset pH values increased significantly in bowfin over those of the more basal species, toward values of similar magnitude to those of most of the more derived teleosts studied to date. This is paralleled by the initial appearance of the choroid rete in bowfin, as well as a significant decrease in Hb buffer value and an increase in Bohr/Haldane effects, together suggesting bowfin as the most basal species capable of utilizing its Root effect to maximize O₂ delivery to the eye.     

Effects of hypoxic hypoxia on O2 uptake and heart rate kinetics during heavy exercise

Engelen, Marielle, Janos Porszasz, Marshall Riley, KarlmanWasserman, Kazuhira Maehara, and Thomas J. Barstow. Effects ofhypoxic hypoxia on O₂ uptake andheart rate kinetics during heavy exercise. J. Appl.Physiol. 81(6): 2500-2508, 1996.It is unclearwhether hypoxia alters the kinetics ofO₂ uptake(O₂) during heavy exercise[above the lactic acidosis threshold (LAT)] and how thesealterations might be linked to the rise in blood lactate. Eight healthyvolunteers performed transitions from unloaded cycling to the sameabsolute heavy work rate for 8 min while breathing one of threeinspired O₂ concentrations: 21%(room air), 15% (mild hypoxia), and 12% (moderate hypoxia). Breathing12% O₂ slowed the time constantbut did not affect the amplitude of the primary rise inO₂ (period of first2-3 min of exercise) and had no significant effect on either thetime constant or the amplitude of the slowO₂ component (beginning2-3 min into exercise). Baseline heart rate was elevated inproportion to the severity of the hypoxia, but the amplitude andkinetics of increase during exercise and in recovery were unaffected bylevel of inspired O₂.We conclude that the predominant effect of hypoxia during heavyexercise is on the early energetics as a slowed time constant forO₂ and an additionalanaerobic contribution. However, the sum total of the processesrepresenting the slow component of O₂ is unaffected.

     

Absence of adrenergic red cell pH and oxygen content regulation in American eel (Anguilla rostrata) during hypercapnic acidosis in vivo and in vitro

Summary American eels (Anguilla rostrata) were exposed to acute (30 min) external hypercapnia (1% CO₂ or 5% CO₂ in air) in order to assess the involvement of circulating catecholamines in regulating red blood cell (RBC) pH and oxygen content during whole blood acidosis. Plasma adrenaline levels increased approximately 5-fold during severe hypercapnia yet absolute levels remained below 1.0 nM; plasma noradrenaline levels were unchanged. Both RBC pH and oxygen bound to haemoglobin ([O₂]/[Hb]) conformed to in vitro relationships with whole blood pH (pHe) indicating absence of regulation during hypercapnia in vivo. Pre-treatment of eels with - or -adrenoceptor antagonists, phentolamine or propranolol was without effect on RBC pH or [O₂]/[Hb] during hypercapnia. Further, intra-arterial injection of adrenaline (final plasma concentration=134 nM) or noradrenaline (final plasma concentration = 34 nM) into hypercapnic eels 5 min prior to blood sampling did not modify any measured blood variable RBC nucleoside triphosphate (NTP) levels, RBC pH and [O₂]/[Hb]. In vitro, the application of adrenaline or noradrenaline to eel RBC's during graded normoxic hypercapnia or hypoxic hypercapnia (noradrenaline only) did not affect RBC pH significantly. RBC NTP levels were depressed by noradrenaline in vitro but only during hypoxic hypercapnia.The results demonstrate adrenergic insensitivity of eel RBC's in vivo even under conditions (acidosis, hypoxemia) known to enhance catecholamine-mediated RBC responses in other species. We conclude that the American eel has no capacity to regulate RBC pH during hypercapnia and consequently [O₂]/[Hb] is reduced in accordance with the in vitro Root effect.     

Increased hemoglobin O2 affinity protects during acute hypoxia

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O(2)) transport and O(2) utilization. Although decreasing hemoglobin (Hb) O(2) affinity would favor the release of O(2) to the tissues, increasing Hb O(2) affinity would augment arterial O(2) saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O(2) affinity will augment O(2) transport during severe hypoxia (10 and 5% inspired O(2)) compared with normal Hb O(2) affinity. RBC Hb O(2) affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O(2) affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O(2) (Po(2)). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po(2) at which the Hb is 50% saturated with O(2) by 12.6 mmHg. During 10 and 5% O(2) hypoxia, 5HMF increased arterial blood O(2) saturation by 35 and 48% from the vehicle group, respectively. During 5% O(2) hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po(2) was three times higher in the 5HMF group compared with the control group at 5% O(2) hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O(2) affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization.     

Depressed ventilatory response to hypoxia in hypothermic newborn piglets: role of glutamate

To evaluatewhether changes in extracellular glutamate (Glu) levels in the centralnervous system could explain the depressed hypoxic ventilatory responsein hypothermic neonates, 12 anesthetized, paralyzed, and mechanicallyventilated piglets <7 days old were studied. The Glu levels in thenucleus tractus solitarius obtained by microdialysis, minute phrenicoutput (MPO), O₂ consumption, arterial blood pressure, heart rate, and arterial blood gases weremeasured in room air and during 15 min of isocapnic hypoxia (inspiredO₂ fraction = 0.10) at braintemperatures of 39.0 ± 0.5°C [normothermia (NT)]and 35.0 ± 0.5°C [hypothermia (HT)]. During NT, MPO increased significantly during hypoxia and remained above baseline. However, during HT, there was a marked decrease in MPOduring hypoxia (NT vs. HT, P < 0.03). Glu levels increased significantly in hypoxia during NT;however, this increase was eliminated during HT(P < 0.02). A significant linearcorrelation was observed between the changes in MPO and Glu levelsduring hypoxia (r = 0.61, P < 0.0001). Changes in pH, arterialPO₂, O₂ consumption, arterial bloodpressure, and heart rate during hypoxia were not different between theNT and HT groups. These results suggest that the depressed ventilatoryresponse to hypoxia observed during HT is centrally mediated and inpart related to a decrease in Glu concentration in the nucleus tractussolitarius.

     

Adaptive responses during anemia and its correction in lambs.

There is limited information available on which to base decisions regarding red blood cell (RBC) transfusion treatment in anemic newborn infants. Using a conscious newborn lamb model of progressive anemia, we sought to identify accessible metabolic and cardiovascular measures of hypoxia that might provide guidance in the management of anemic infants. We hypothesized that severe phlebotomy-induced isovolemic anemia and its reversal after RBC transfusion result in a defined pattern of adaptive responses. Anemia was produced over 2 days by serial phlebotomy (with plasma replacement) to Hb levels of 30-40 g/l. During the ensuing 2 days, Hb was restored to pretransfusion baseline levels by repeated RBC transfusion. Area-under-the-curve methodology was utilized for defining the Hb level at which individual study variables demonstrated significant change. Significant reciprocal changes (P < 0.05) of equivalent magnitude were observed during the phlebotomy and transfusion phases for cardiac output, plasma erythropoietin (Epo) concentration, oxygen extraction ratio, oxygen delivery, venous oxygen saturation, and blood lactate concentration. No significant change was observed in resting oxygen consumption. Cardiac output and plasma Epo concentration increased at Hb levels <75 g/l, oxygen delivery and oxygen extraction ratio decreased at Hb levels <60 g/l, and venous oxygen saturation decreased and blood lactate concentration increased at Hb levels <55 g/l. We speculate that plasma Epo and blood lactate concentrations may be useful measures of clinically significant anemia in infants and may indicate when an infant might benefit from a RBC transfusion.     

Microvascular Regulation of Cutaneous Gas Exchange in Amphibians

SYNOPSIS. Gas exchange across amphibian skin is regulated bythe cutaneous microcirculation. Parameters involved in regulatinggas exchange are capillary density, radius and blood flow. Changesin capillary density and radius should affect gas exchange byaltering the cutaneous diffusing capacity (D₂) while changesin capillary blood flow affect the perfusive conductance ofthe skin. A simple model predicts that the effect of capillary densitychanges on D₂ will become more pronounced as capillary densityand epidermal thickness decrease. Changes in capillary radiusshould have only a minor effect on D₂ Previous analyses havesuggested that cutaneous gas exchange is not significantly affectedby the perfusive conductance of the skin. Consequently, it hasbeen thought that changes in total capillary blood flow havelittle impact on cutaneous gas exchange. Earlier analyses, however,may have underestimated the importance of perfusive conductancein amphibian skin, primarily because functional heterogeneitiesin the microcirculation were not considered. The density of perfused capillaries is regulated in the footweb of Rana esculenta by environmental Po₂ and PCO₂, and alsoby lung ventilation. In Rana catesbeiana, capillary densityin the web decreases during air exposure. Chronic exposure toenvironmental hypoxia increases total capillary density in bullfrogtadpole skin. In Rana pipiens, regulation of cutaneous gas exchangeby environmental and pulmonary O₂ probably involves changesin total capillary blood flow.     

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 (pH_i) 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 pH_i). 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.     

Oxygen sensitivity of mitochondrial metabolic state in isolated skeletal and cardiac myocytes

In striatedmuscle the coupling of blood flow to changes in tissue metabolism ishypothesized to be dependent in part on release of vasodilatingmetabolic by-products generated when mitochondrial metabolism becomesO₂ limited. Cytochrome oxidase,the terminal step in oxidative phosphorylation, is half-maximallysaturated at <1 mmHg PO₂ inisolated mitochondria. However, blood flow is regulated at tissuePO₂ of ~20 mmHg. If the affinity ofmitochondrial respiration for O₂were higher in vivo than in vitro,O₂ limitation of mitochondrialmetabolism near mean tissue levels could occur. In the present studythe PO₂ at which mitochondrialmetabolism becomes inhibited (criticalPO₂) was measured for cardiacmyocytes in suspension (1.1 ± 0.15 mmHg) and single cells (1.0 ± 0.22 and 1.25 ± 0.22 mmHg in cardiac myocytes and ratspinotrapezius cells, respectively). These measurements are consistentwith those from isolated mitochondria, indicating that vasodilatorsproduced when oxidative phosphorylation becomes inhibited may beimportant for regulating blood flow only in highly glycolytic musclesor under conditions of severe O₂limitation.