Physiology and evolution of oxygen secreting mechanism in the fisheye (2023)

Nitrobindins (Nbs) are all-β-barrel heme-proteins present in all the living kingdoms. Nbs inactivate reactive nitrogen species by sequestering NO, converting NO to HNO₂, and isomerizing peroxynitrite to NO₃⁻ and NO₂⁻. Here, the spectroscopic characterization of ferric Danio rerio Nb (Dr-Nb(III)) and NO scavenging through the reductive nitrosylation of the metal center are reported, both processes being relevant for the regulation of blood flow in fishes through poorly oxygenated tissues, such as retina. Both UV–Vis and resonance Raman spectroscopies indicate that Dr-Nb(III) is a mixture of a six-coordinated aquo- and a five-coordinated species, whose relative abundancies depend on pH. At pH≤7.0, Dr-Nb(III) binds reversibly NO, whereas at pH≥7.8 NO induces the conversion of Dr-Nb(III) to Dr-Nb(II)-NO. The conversion of Dr-Nb(III) to Dr-Nb(II)-NO is a monophasic process, suggesting that the formation of the transient Dr-Nb(III)-NO species is lost in the mixing time of the rapid-mixing stopped-flow apparatus (∼ 1.5ms). The pseudo-first-order rate constant for the reductive nitrosylation of Dr-Nb(III) is not linearly dependent on the NO concentration but tends to level off. Values of the rate-limiting constant (i.e., k_lim) increase linearly with the OH⁻ concentration, indicating that the conversion of Dr-Nb(III) to Dr-Nb(II)-NO is limited by the OH⁻-based catalysis. From the dependence of k_lim on [OH⁻], the value of the second-order rate constant k_OH− was obtained (5.2×10³M⁻¹s⁻¹). Reductive nitrosylation of Dr-Nb(III) leads to the inactivation of two NO molecules: one being converted to HNO_2, and the other being tightly bound to the heme-Fe(II) atom.

Nitrobindins (Nbs) are all-β-barrel heme-proteins present in prokaryotes and eukaryotes. Although the physiological role(s) of Nbs are still unclear, it has been postulated that they are involved in the NO/O₂ metabolism, which is particularly relevant in fishes for the oxygen supply. Here, the reactivity of ferrous Danio rerio Nb (Dr-Nb(II)) towards NO has been investigated from the spectroscopic and kinetic viewpoints and compared with those of Mycobacterium tuberculosis Nb, Arabidopsis thaliana Nb, Homo sapiens Nb, and Equus ferus caballus myoglobin. Between pH 5.5 and 9.1 at 22.0 °C, Dr-Nb(II) nitrosylation is a monophasic process; values of the second-order rate constant for Dr-Nb(II) nitrosylation and of the first-order rate constant for Dr-Nb(II)-NO denitrosylation are pH-independent ranging between 1.6 × 10⁶ M⁻¹ s⁻¹ and 2.3 × 10⁶ M⁻¹ s⁻¹ and between 5.3 × 10⁻² s⁻¹ and 8.2 × 10⁻² s⁻¹, respectively. Interestingly, both UV–Vis and EPR spectroscopies indicate that the heme-Fe(II) atom of Dr-Nb(II)-NO is five-coordinated. Kinetics of Dr-Nb(II) nitrosylation may reflect the ligand accessibility to the metal center, which is likely impaired by the crowded network of water molecules which shields the heme pocket from the bulk solvent. On the other hand, kinetics of Dr-Nb(II)-NO denitrosylation may reflect an easy pathway for the ligand escape into the outer solvent.

The molecular mechanism of O₂ binding to hemoglobin (Hb) has been critically reviewed on the basis of the information built up in the last decades. It allows to describe in detail from the kinetic and thermodynamic viewpoint the process of O₂ uptake in the lungs and release to the tissues, casting some light on the physiological and pathological aspects of this process. The relevance of structural-functional relationships for O₂ binding is particularly outlined in the case of poorly vascularized tissues, such as retina, briefly discussing of strategies employed for optimization of oxygen supply to this type of tissues.

In the early 20th century, August and Marie Krogh settled one of the most controversial questions in physiology, showing through elegant experiments that oxygen (O₂) uptake at the lung is driven by passive diffusion alone. Krogh's later work, on the regulation of local blood flow and capillary recruitment at the tissues, was awarded with the Nobel Prize in 1920. A century later it is still undisputed that O₂ moves across tissues by diffusion, however, animals use active mechanisms to regulate and facilitate the passive process. Teleost fishes have evolved a mechanism by which adrenergic sodium-proton-exchangers (β-NHEs) on the red blood cell (RBC) membrane actively create H⁺ gradients that are short-circuited in the presence of plasma-accessible carbonic anhydrase (CA) at the tissue capillaries. The rapid acidification of the RBC reduces the O₂ affinity of pH-sensitive haemoglobin, which increases the O₂ diffusion gradient to the tissues. When RBCs leave the site of plasma-accessible CA, β-NHE activity recovers RBC pH during venous transit, to promote renewed O₂ loading at the gills. This mechanism allows teleosts to unload more O₂ at their tissues without compromising O₂ diffusion gradients and therefore, to use the available O₂ carrying capacity of the blood to a greater degree. In Atlantic salmon, β-NHE short-circuiting reduces the requirements on the heart by up to 30% during moderate exercise and even at rest, with important ecological implications. Thus, in some teleosts, the RBCs participate in regulating the systemic O₂ flux by actively altering the passive diffusion of O₂ that Krogh discovered.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, Volume 252, 2021, Article 110850

Cold acclimation increases cold tolerance of chill-susceptible insects and the acclimation response often involves improved organismal ion balance and osmoregulatory function at low temperature. However, the physiological mechanisms underlying plasticity of ion regulatory capacity are largely unresolved. Here we used Ussing chambers to explore the effects of cold exposure on hindgut KCl reabsorption in cold- (11°C) and warm-acclimated (30°C) Locusta migratoria. Cooling (from 30 to 10°C) reduced active reabsorption across recta from warm-acclimated locusts, while recta from cold-acclimated locusts maintained reabsorption at 10°C. The differences in transport capacity were not linked to major rearrangements of membrane phospholipid profiles. Yet, the stimulatory effect of two signal transduction pathways were altered by temperature and/or acclimation. cAMP-stimulation increased reabsorption in both acclimation groups, with a strong stimulatory effect at 30°C and a moderate stimulatory effect at 10°C. cGMP-stimulation also increased reabsorption in both acclimation groups at 30°C, but their response to cGMP differed at 10°C. Recta from warm-acclimated locusts, characterised by reduced reabsorption at 10°C, recovered reabsorption capacity following cGMP-stimulation at 10°C. In contrast, recta from cold-acclimated locusts, characterised by sustained reabsorption at 10°C, were unaffected by cGMP-stimulation. Furthermore, cold-exposed recta from warm-acclimated locusts were insensitive to bafilomycin-α1, a V-type H⁺-ATPase inhibitor, whereas this blocker reduced reabsorption across cold-exposed recta from cold-acclimated animals. In conclusion, bafilomycin-sensitive and cGMP-dependent transport mechanism(s) are likely blocked during cold exposure in warm-acclimated animals while preserved in cold-acclimated animals. These may in part explain the large differences in rectal ion transport capacity between acclimation groups at low temperature.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, Volume 252, 2021, Article 110823

Satellite cell (SCs), the main progenitors for post-hatch poultry muscle growth, has maximal mitotic activity and sensitivity to temperature during the first week after hatch. The objective of the present study was to determine the effect of hot and cold temperatures on the proliferation and differentiation of SCs from pectoralis major (P. major) muscle of fast-growing 1-week-old Nicholas commercial (NC) turkeys compared to Randombred Control Line 2 (RBC2) turkeys representing commercial turkeys from 1966. Three temperature regimens were used: SCs proliferation at 38°C (control) with differentiation at 43° or 33°C; proliferation at 43° or 33 °C with differentiation at 38°C; or both proliferation and differentiation at 43°, 38°, or 33°C. Satellite cell proliferation and differentiation increased at 43°C and decreased at 33°C in both lines. When a thermal challenge was administered during proliferation, greater stimulatory or suppressive effects on differentiation were observed compared to if the thermal challenge was applied only during differentiation in both lines. Expression of myoblast determination protein 1 during proliferation showed a higher increase in the NC line compared to the RBC2 line at 43°C. Increased myogenin expression was observed in all hot treatment groups in the NC line but was only observed in the RBC2 line if the hot treatment was administered throughout proliferation and differentiation. Cold treatment suppressed myogenin expression independent of line. These results suggest turkey P. major muscle SCs are more sensitive to environmental temperatures during proliferation, and SCs from growth-selected NC turkeys are more sensitive to thermal stress compared to the RBC2 turkeys.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, Volume 252, 2021, Article 110839

Angiotensin II (ANG II) is part of the renin-angiotensin system (RAS) in vertebrates and exert vasoconstriction in all species studied. The present study examines the vasopressor effect of ANG II in the ball python (Python regius), and examines whether ANG II exert its effect through direct angiotensin receptors or through an activation of α-adrenergic receptors. The studies were conducted in snakes with chronic arterial catheters that had recovered from anesthesia. In addition to demonstrating a clear and pronounced dose-dependent rise in arterial blood pressure upon repeated injections of boluses with ANG II (0.001–1μg/kg), we demonstrate that the pressor response persisted following α-adrenergic blockade using the α-adrenergic antagonist phentolamine (2.5mg/kg). Unfortunately, it proved impossible to block the ANG receptors using losartan (1, 3 or even 10mg/kg). The pressor response to ANG II was associated with a significant rise in heart rate at the higher dosages, pointing to a resetting of the barostatic mechanism for heart rate regulation. The responses were similar in fasting and digesting pythons despite the expected rise in baseline values for blood pressure and heart rate of the digesting snakes.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, Volume 253, 2021, Article 110857

In contrast to most vertebrates, freshwater turtles of the genera Trachemys and Chrysemys survive total oxygen deprivation for long periods of time. This remarkable tolerance makes them ideal August Krogh's model animals to study adaptions to survive oxygen deprivation. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H₂S) and their metabolic derivatives are central in regulating the physiological responses to oxygen deprivation. Here, we explore the role of these signaling molecules in the anoxia tolerance of the freshwater turtle, including metabolic suppression and protection against oxidative damage with oxygen deprivation. We describe the interaction of NO and H₂S with protein thiols and specifically how this regulates the function of central metabolic enzymes. These interactions contribute both to metabolic suppression and to prevent oxidative damage with oxygen deprivation. Furthermore, NO and H₂S interact with ferrous and ferric heme iron, respectively, which affects the activity of central heme proteins. In turtles, these interactions contribute to regulate oxygen consumption in the mitochondria, as well as vascular tone and blood flow during oxygen deprivation. The versatile biological effects of NO and H₂S underscore the importance of these volatile signaling molecules in the remarkable tolerance of freshwater turtles to oxygen deprivation.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, Volume 252, 2021, Article 110836

While evidence that telomere length is associated with health and mortality in humans and birds is accumulating, a large body of research is currently seeking to identify factors that modulate telomere dynamics. We tested the hypothesis that high levels of glucocorticoids in individuals under environmental stress should accelerate telomere shortening in two wild populations of roe deer (Capreolus capreolus) living in different ecological contexts. From two consecutive annual sampling sessions, we found that individuals with faster rates of telomere shortening had higher concentrations of fecal glucocorticoid metabolites, suggesting a functional link between glucocorticoid levels and telomere attrition rate. This relationship was consistent for both sexes and populations. This finding paves the way for further studies of the fitness consequences of exposure to environmental stressors in wild vertebrates.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, Volume 252, 2021, Article 110835

Climate changes affecting aquatic environments are increasing, and the resultant environmental challenges require animals to adopt alternative compensatory behavioral and physiological strategies. In particular, low levels of dissolved O₂ are a regular problem for estuarine animals, leading to activation of a series of behavioral and physiological responses. This study on the semi-terrestrial crab Neohelice granulata examined patterns of emersion behavior under different levels of dissolved O₂ availability and the role of lactate in this behavior. Emersion behavior was recorded for 4.5h for crabs in water at four different levels of dissolved O₂ (6, 3, 2, and 1mg O₂/L) and with free access to air. Oxygen consumption and hemolymphatic lactate levels were measured using the same experimental design. Emersion behavior was also recorded for 70min in normoxic water after lactate or saline injections. Crabs increased their emersion behavior only in severe hypoxia (1mg O₂/L), and O₂ consumption decreased under more severe hypoxic conditions. Despite the increase in emersion behavior, which leads to higher O₂ availability, an increase in hemolymphatic lactate levels indicates that the animals still need to resort to anaerobic pathways to fulfill their metabolic demand. Furthermore, animals injected with lactate showed higher emersion behaviors than animals injected with a saline solution even in normoxia. These results suggest that the increase in hemolymphatic lactate can act directly or indirectly as a trigger for the increase in emersion behavior in the semi-terrestrial crab N. granulata.