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.     

Ventilatory responses to exercise and carbon dioxide in elderly and younger humans

The present investigation examined the relationship between CO₂ sensitivity [at rest (S _R) and during exercise (S _E)] and the ventilatory response to exercise in ten elderly (61–79 years) and ten younger (17–26 years) subjects. The gradient of the relationship between minute ventilation and CO₂ production ( _E/ CO₂) of the elderly subjects was greater than that of the younger subjects [mean (SEM); 32.8 (1.6) vs 27.3 (0.4); P<0.01]. At rest, S _R was lower for the elderly than for the younger group [10.77 (1.72) vs 16.95 (2.13) 1 · min^–1 · kPa^–1; 1.44 (0.23) vs 2.26 (0.28) 1 · min^–1 · mmHg^–1; P<0.05], but S _E was not significantly different between the two groups [17.85 (2.49) vs 19.17 (1.62) l · min^–1 · kPa^–1; 2.38 (0.33) vs 2.56 (0.21) 1 · min^–1 · mmHg^–1]. There were significant correlations between both S _R and S _E, and _E/ CO₂ (P<0.05; P<0.001) for the younger group, bot none for the elderly. The absence of a correlation for the elderly supports the suggestion that _E/ CO₂ is not an appropriate index of the ventilatory response to exercise for elderly humans.     

Unexpected expression of carbonic anhydrase I and selenium-binding protein as the only major non-heme proteins in erythrocytes of the subterranean mole rat (Spalax ehrenbergi)

Chromatographic separation of the non-heme proteins from the erythrocytes of the subterranean mole rat belonging to the superspecies Spalax ehrenbergi from Israel revealed two major peaks. On sequence analyses, the larger peak corresponded to a 56 kDa selenium-binding protein (SeBP) previously characterized from mouse and human liver, and the second peak to the low-activity carbonic anhydrase (CA) isozyme, CA I. There was no evidence of the high-activity CA II isozyme normally found in the red cells of all amniotes tested to date. Thus, the mole rat appears to be the first mammalian species to express both a SeBP and the low-activity CA I isozyme, as the major non-heme proteins in its red blood cells. It is possible that the absence of the high-activity CA II isozyme may be advantageous to the mole rat in adapting to the low O₂ and high CO₂ environment of its underground burrows. It is also likely that the 56 kDa SeBP may play an important adaptive role in the physiology of the red cell.     

Regulation of ventilation in the caiman (Caiman latirostris): effects of inspired CO2 on pulmonary and upper airway chemoreceptors

In order to study the relative roles of receptors in the upper airways, lungs and systemic circulation in modulating the ventilatory response of caiman (Caiman latirostris) to inhaled CO₂, gas mixtures of varying concentrations of CO₂ were administered to animals breathing through an intact respiratory system, via a tracheal cannula by-passing the upper airways (before and after vagotomy), or via a cannula delivering gas to the upper airways alone. While increasing levels of hypercarbia led to a progressive increase in tidal volume in animals with intact respiratory systems (Series I), breathing frequency did not change until the CO₂ level reached 7%, at which time it decreased. Despite this, at the higher levels of hypercarbia, the net effect was a large and progressive increase in total ventilation. There were no associated changes in heart rate or arterial blood pressure. On return to air, there was an immediate change in breathing pattern; breathing frequency increased above air-breathing values, roughly to the same maximum level regardless of the level of CO₂ the animal had been previously breathing, and tidal volume returned rapidly toward resting (baseline) values. Total ventilation slowly returned to air breathing values. Administration of CO₂ via different routes indicated that inhaled CO₂ acted at both upper airway and pulmonary CO₂-sensitive receptors to modify breathing pattern without inhibiting breathing overall. Our data suggest that in caiman, high levels of inspired CO₂ promote slow, deep breathing. This will decrease dead-space ventilation and may reduce stratification in the saccular portions of the lung.     

Effects of environmental hypercapnia on animal physiology: a 13C NMR study of protein synthesis rates in the marine invertebrate Sipunculus nudus

Global climate change is associated with a progressive rise in ocean CO(2) concentrations (hypercapnia) and, consequently, a drop in seawater pH. However, a comprehensive picture of the physiological mechanisms affected by chronic CO(2) stress in marine biota is still lacking. Here we present an analysis of protein biosynthesis rates in isolated muscle of the marine invertebrate Sipunculus nudus, a sediment dwelling worm living at various water depths. We followed the incorporation of (13)C-labelled phenylalanine into muscular protein via high-resolution NMR spectroscopy. Protein synthesis decreased by about 60% at a medium pH of 6.70 and a consequently lowered intracellular pH (pHi). The decrease in protein synthesis rates is much stronger than the concomitant suppression of protein degradation (60% versus 10-15%) possibly posing a threat to the cellular homeostasis of structural as well as functional proteins. Considering the progressive rise in ocean CO(2) concentrations, permanent disturbances of cellular protein turnover might seriously affect growth and reproductive performance in many marine organisms with as yet unexplored impacts on species density and composition in marine ecosystems.     

In Vivo Microdialysis of 2-Deoxyglucose 6-Phosphate into Brain

Abstract : A unique method for simultaneously measuring interstitial (pH_e) as well as intracellular (pH_i) pH in the brains of lightly anesthetized rats is described. A 4-mm microdialysis probe was inserted acutely into the right frontal lobe in the center of the area sampled by a surface coil tuned for the collection of ³¹P-NMR spectra. 2-Deoxyglucose 6-phosphate (2-DG-6-P) was microdialyzed into the rat until a single NMR peak was detected in the phosphomonoester region of the ³¹P spectrum. pH_e and pH_i values were calculated from the chemical shift of 2-DG-6-P and inorganic phosphate, respectively, relative to the phosphocreatine peak. The average in vivo pH_e was 7.24 ± 0.01, whereas the average pH_i was 7.05 ± 0.01 (n = 7). The average pH_e value and the average CSF bicarbonate value (23.5 ± 0.1 mEq/L) were used to calculate an interstitial Pco₂ of 55 mm Hg. Rats were then subjected to a 15-min period of either hypercapnia, by addition of CO₂ (2.5, 5, or 10%) to the ventilator gases, or hypocapnia (Pco₂ < 30 mm Hg), by increasing the ventilation rate and volume. pH_e responded inversely to arterial Pco₂ and was well described (r² = 0.91) by the Henderson-Hassel-balch equation, assuming a pK_a for the bicarbonate buffer system of 6.1 and a solubility coefficient for CO₂ of 0.031. This confirms the view that the bicarbonate buffer system is dominant in the interstitial space. pH_i responded inversely and linearly to arterial Pco₂. The intracellular effect was muted as compared with pH_e (slope = -0.0025, r² = 0.60). pH_e and pH_i values were also monitored during the first 12 min of ischemia produced by cardiac arrest. pH_e decreases more rapidly than pH_i during the first 5 min of ischemia. After 12 min of ischemia, pH_e and pH_i values were not significantly different (6.44 ± 0.02 and 6.44 ± 0.03, respectively). The limitations, advantages, and future uses of the combined microdialysis/³¹P-NMR method for measurement of pH_e and pH_i are discussed.     

Carbon dioxide storage and nonbicarbonate buffering in the human body before and after an Himalayan expedition

Before and 7-12 days after an Himalayan expedition CO2 equilibration curves were determined in the blood plasma of 12 mountaineers by in vitro and in vivo CO2 titration; in vivo osmolality changes (delta Osm x deltaPCO2(-1), deltaOsm x delta pH(-1), where PCO2 is the partial pressure of CO2) during the latter experiments yielded estimates of whole body CO2 storage. In vitro -delta[HCO3-] x delta pH(-1) [nonbicarbonate buffer capacity (beta) of blood] was increased 7 days after descent [before 31.3 (SEM 0.4) mmol x kgH2O(-1), after 38.3 (SEM 3.9) mmol x kgH2O(-1); P<0.05] resulting from an increased proportion of young erythrocytes; in additional experiments an augmented beta was found in young (low density cells) compared to old cells [<1.097 g x ml(-1): 0.216 (SEM 0.028) mmol x gHb(-1), >1.100 g x ml(-1): 0.145 (SEM 0.013) mmol x gHb(-1), where Hb is haemoglobin; P < 0.02]. In spite of increased Hb mass in vivo delta[CO2total] x deltaPCO2(-1) [0.192 (SEM 0.010) mmol x kgH2O(-1) x mmHg(-1)] and -delta[HCO3-] x delta pH(-1) [17.9 (SEM 1.0) mmol x kgH2O(-1)] as indicators of extracellular beta rose only slightly after altitude (7 days +16%, P<0.02; +7%, NS) because of haemodilution. The deltaOsm x deltaPCO2(-1) [0.230 (SEM 0.015) mosmol x kgH2O(-1) x mmHg(-1)] remained unchanged. Prealtitude differences in deltaOsm x delta pH(-1) between hypercapnia [-41 (SEM 5) mosmol x kgH2O(-1)] and hypocapnia [-20 (SEM 3) mosmol x kgH2O(-1); P<0.01] disappeared temporarily after return since the former slope was reduced. The high value during hypercapnia before ascent probably resulted from mechanisms stabilizing intracellular pH during moderate hypercapnia which were attenuated after descent.     

Control of breathing in the echidna (Tachyglossus aculeatus) during hibernation

Resting non-hibernating echidnas are characterised by low metabolic rates, but also have a very low respiratory frequency and a variable respiratory minute volume, often resulting in low levels of arterial O(2) and high CO(2). As the echidna lies at one physiological extreme among the hibernators, in terms of its large size and low metabolism and ventilatory requirement when not hibernating, a study of control of breathing during hibernation in echidnas should provide a useful test of the generality of various models. We used non-invasive techniques to study breathing patterns and the control of ventilation in 6 echidnas. Hibernating echidnas (T(b) range 7-10 degrees C) showed episodic breathing with bursts of breaths (average 36+/-16 breaths in 24+/-5 min) followed by a period of apnea (76+/-17 min) then a series (8+/-4) of slow breaths at 14+/-1 min intervals leading up to the next burst. Increasing CO(2) levels in the inspired air increased the number of breaths in a burst, eventually leading to continuous breathing. Inter burst breaths were controlled by O(2): hypoxia increased inter burst breaths, and decreased burst length, while hyperoxia abolished inter burst breaths and increased the apneic period. Overall, while CO(2) was a strong respiratory stimulus in hibernating echidnas, O(2) had little effect on total ventilation, but did have a strong effect on the breathing pattern.     

Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus)

Summary Exposure of adult brown bullheads Ictalurus nebulosus (120–450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (J _net ^Na+ ) and net chloride flux (J _net ^Cl- ) resulting largely from changes in whole body sodium influx (J _in ^Na+ ) and chloride influx (J _in ^Cl- ). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in J _in ^Cl- during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in J _in ^Na+ did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the post-hypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl^-/HCO ₃ ^- exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.     

Monoamine Synthesis and Concentration in Neonatal Rat Brain During Hypercapnia and Recovery

One-day-old rats were exposed to a gas mixture of 15% CO2-21% O2-64% N2 for a 30-min period. Monoamine synthesis in whole brain was measured during, and at various intervals after, hypercapnia by estimating the accumulation of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) after inhibition of aromatic L-amino-acid decarboxylase with NSD 1015. Endogenous concentrations of tyrosine, dopamine (DA), noradrenaline (NA), tryptophan, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured at the same intervals. Exposure to CO2 induced an increased synthesis of catecholamines and 5-HT. Further, an increase in DA concentration was seen during hypercapnia, while NA and 5-HT were unchanged. After the CO2 exposure the increased in vivo synthesis rates of catecholamines and 5-HT were rapidly normalized, as was the endogenous DA concentration. A slight increase in 5-HT and 5-HIAA concentrations was seen immediately after CO2 exposure. These results indicate that in neonatal animals, hypercapnia induces changes in central monoamine neurons, primarily an increased synthesis. These alterations may be relevant to some physiological changes seen during CO2 exposure, such as the alteration in central respiratory performance.