Michael Berenbrink

Nitrite Regulates Hypoxic Vasodilation via Myoglobin-Dependent Nitric Oxide Generation

Background— Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·. Methods and Results— We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO·, and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice. Conclusions— Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO· via the heme globin myoglobin enhances blood flow and matches O2 supply to increased metabolic demands under hypoxic conditions.

Evolution of Mammalian Diving Capacity Traced by Myoglobin Net Surface Charge

Introduction Evolution of extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and morphological and physiological adaptations that promote their economical use. High muscle myoglobin concentrations in particular are mechanistically linked with an extended dive capacity phenotype, yet little is known regarding the molecular and biochemical underpinnings of this key specialization. We modeled the evolutionary history of this respiratory pigment over 200 million years of mammalian evolution to elucidate the development of maximal diving capacity during the major mammalian land-to-water transitions. Evolutionary reconstruction of myoglobin net surface charge in terrestrial and aquatic mammals. The figure reveals a molecular signature of elevated myoglobin net surface charge in all lineages of living elite mammalian divers with an extended aquatic history (upper silhouettes). This signature is used here to infer the diving capacity of extinct species representing stages during mammalian land-to-water transitions (†). Methods We first determined the relationship between maximum myoglobin concentration and its sequence-derived net surface charge across living mammalian taxa. By using ancestral sequence reconstruction we then traced myoglobin net surface charge across a 130-species phylogeny to infer ancestral myoglobin muscle concentrations. Last, we estimated maximum dive time in extinct transitional species on the basis of the relationship of this variable with muscle myoglobin concentration and body mass in extant diving mammals. Results We reveal an adaptive molecular signature of elevated myoglobin net surface charge in all lineages of mammalian divers with an extended aquatic history—from 16-g water shrews to 80,000-kg whales—that correlates with exponential increases in muscle myoglobin concentrations. Integration of this data with body mass predicts 82% of maximal dive-time variation across all degrees of diving ability in living mammals. Discussion We suggest that the convergent evolution of high myoglobin net surface charge in mammalian divers increases intermolecular electrostatic repulsion, permitting higher muscle oxygen storage capacities without potentially deleterious self-association of the protein. Together with fossil body-mass estimates, our evolutionary reconstruction permits detailed assessments of maximal submergence times and potential foraging ecologies of early transitional ancestors of cetaceans, pinnipeds, and sea cows. Our findings support amphibious ancestries for echidnas, talpid moles, hyraxes, and elephants, thereby not only establishing the earliest land-to-water transition among placental mammals but also providing a new perspective on the evolution of myoglobin, arguably the best-known protein. Holding Your Breath Hemoglobin and myoglobin are widely responsible for oxygen transport and storage (see the Perspective by Rezende). The ability of diving mammals to obtain enough oxygen to support extended dives and foraging is largely dependent on muscle myoglobin (Mb) content. Mirceta et al. (p. 1234192) found that in mammalian lineages with an aquatic or semiaquatic lifestyle, Mb net charge increases, which may represent an adaptation to inhibit self-association of Mb at high intracellular concentrations. Epistasis results from nonadditive genetic interactions and can affect phenotypic evolution. Natarajan et al. (p. 1324) found that epistatic interactions were able to explain the increased hemoglobin oxygen-binding affinity observed in deer mice populations at high altitude. In mammals, the offloading of oxygen from hemoglobin is facilitated by a reduction in the bloods pH, driven by metabolically produced CO2. However, in fish, a reduction in blood pH reduces oxygen carrying capacity of hemoglobin. Rummer et al. (p. 1327) implanted fiber optic oxygen sensors within the muscles of rainbow trout and found that elevated CO2 levels in the water led to acidosis and elevated oxygen tensions. Increasing the number of charged amino acids allows for higher myoglobin concentrations in the muscles of diving mammals. [Also see Perspective by Rezende] Extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and adaptations that promote their economical use. However, little is known regarding the molecular and evolutionary underpinnings of the high muscle myoglobin concentration phenotype of divers. We used ancestral sequence reconstruction to trace the evolution of this oxygen-storing protein across a 130-species mammalian phylogeny and reveal an adaptive molecular signature of elevated myoglobin net surface charge in diving species that is mechanistically linked with maximal myoglobin concentration. This observation provides insights into the tempo and routes to enhanced dive capacity evolution within the ancestors of each major mammalian aquatic lineage and infers amphibious ancestries of echidnas, moles, hyraxes, and elephants, offering a fresh perspective on the evolution of this iconic respiratory pigment.

Evolution of vertebrate haemoglobins: Histidine side chains, specific buffer value and Bohr effect

This review highlights the use of analytical tools, recently developed in the comparative method of evolutionary biology, for the study of haemoglobin (Hb) adaptation. It focuses on the functional consequences of a previously largely ignored structural feature of Hb, namely the degree and positional specificity of histidine (His) substitution in Hb chains. The importance of His side chains for hydrogen ion buffering, blood CO(2) transport capacity and the molecular mechanism of the Bohr effect in vertebrate Hbs is discussed. Using phylogenetically independent contrasts, a significant correlation between the specific buffer value of Hb and the number of predicted physiological buffer groups from Hb sequence data is shown. In a new result, the evolution of the number of physiological buffer groups in 77 vertebrate species is reconstructed on a phylogenetic tree. The analysis predicts that teleost fishes, passeriform birds and some snakes have independently evolved a much-reduced specific buffer value of Hb, possibly for enhancing the efficiency of an acid load to change oxygen affinity via the Bohr effect. This analysis demonstrates how in comparative physiology analysis of genetic databases in an evolutionary framework can identify candidate species for further experimental in vitro and whole animal studies.

Diverse cell-specific expression of myoglobin isoforms in brain, kidney, gill and liver of the hypoxia-tolerant carp and zebrafish

SUMMARY Myoglobin (Mb) is famous as a muscle-specific protein – yet the common carp expresses the gene (cMb1) encoding this protein in a range of non-muscle tissues and also expresses a novel isoform (cMb2) in the brain. Using a homologous antibody and riboprobes, we have established the relative amounts and cellular sites of non-muscle Mb expression in different tissues. The amounts of carp myoglobin (cMb) in supernatants of different tissues were just 0.4–0.7% relative to that of heart supernatants and were upregulated by two-to-four fold in liver, gill and brain following 5 days of hypoxic treatment. Brain exhibited both cMb proteins in western analysis, whereas all other tissues had only cMb1. We have also identified cells expressing cMb protein and cMb mRNA using immunohistology and RNA in situ hybridisation (RNA-ISH), respectively. Mb was strongly expressed throughout all cardiac myocytes and a subset of skeletal muscle fibres, whereas it was restricted to a small range of specific cell types in each of the non-muscle tissues. These include pillar and epithelial cells in secondary gill lamellae, hepatocytes, some neurones, and tubular epithelial cells in the kidney. Capillaries and small blood vessels in all tissues exhibited Mb expression within vascular endothelial cells. The cMb2 riboprobe located expression to a subset of neurones but not to endothelial cells. In zebrafish, which possesses only one Mb gene, a similar expression pattern of Mb protein and mRNA was observed. This establishes a surprisingly cell-specific distribution of Mb within non-muscle tissues in both carp and zebrafish, where it probably plays an important role in the regulation of microvascular, renal and brain function.

PHYSIOLOGY AND BIOCHEMISTRY OF THE PSEUDOBRANCH : AN UNANSWERED QUESTION?

The structure and function of the pseudobranch has long interested scientists, but its overall role has remained a mystery. Previous studies have attributed respiratory, endocrine, osmoregulatory and sensory roles to the pseudobranch, and the present review concentrates on new findings. Perfusion experiments on the pseudobranch of the rainbow trout (Oncorhynchus mykiss) using both erythrocyte suspensions and Ringer solution have shown that this organ is able to generate values for the respiratory quotient (RQ) greater than 1.0. The release of carbon dioxide into the perfusate was found to be largely independent of flow between perfusion rates of 120-190 microl/min and could be inhibited by acetazolamide (10(-5) M), indicating a role for carbonic anhydrase. Noradrenaline (10(-5) M) had no effect on oxygen consumption or carbon dioxide release of the pseudobranch. The rate of carbon dioxide release was also dependent on the pH of the pre-pseudobranch perfusate, carbon dioxide release being reduced at lower perfusate pH values. Based on the glucose balance of the isolated saline-perfused rainbow trout pseudobranch and on the enzyme profiles for the rainbow trout, cod, swordfish and deep-water grenadier pseudobranch, it is suggested that the pentose phosphate shunt might be a source of carbon dioxide, yielding the high RQ values found for this organ. Most evidence now available indicates that the pseudobranch is integrally linked with the choroid rete and the supply of oxygen to the retina of the fish eye.

Physiology: Myoglobin's new clothes

Nitric oxide generated from the nitrite ion limits the tissue damage caused by restricted blood flow. Gene knockout experiments in mice now reveal that myoglobin is the mediator of this effect.

O2‐dependent K+ fluxes in trout red blood cells: the nature of O2 sensing revealed by the O2 affinity, cooperativity and pH dependence of transport

1 The effects of pH and O2 tension on the isotonic ouabain‐resistant K+ (Rb+) flux pathway and on haemoglobin O2 binding were studied in trout red blood cells (RBCs) in order to test for a direct effect of haemoglobin O2 saturation on K+ transport across the RBC membrane. 2 At pH values corresponding to in vivo control arterial plasma pH and higher, elevation of the O2 partial pressure (PO2) from 7.8 to 157 mmHg increased unidirectional K+ influx across the RBC membrane several‐fold. At lower extracellular pH values, stimulation of K+ influx by O2 was depressed, exhibiting an apparent pKa (pK′a) for the process of 8.0. Under similar conditions the pK′a for acid‐induced deoxygenation of haemoglobin (Hb) was 7.3. 3 When trout RBCs were exposed to PO2 values between 0 and 747 mmHg, O2 equilibrium curves typical of Hb O2 saturation were also obtained for K+ influx and efflux. However, at pH 7.9, the PO2 for half‐maximal K+ efflux and K+ influx (P50) was about 8‐ to 12‐fold higher than the P50 for Hb‐O2 binding. While K+ influx and efflux stimulation by O2 was essentially non‐cooperative, Hb‐O2 equilibrium curves were distinctly sigmoidal (Hill parameters close to 1 and 3, respectively). 4 O2‐stimulated K+ influx and efflux were strongly pH dependent. When the definition of the Bohr factor for respiratory pigments (Φ=ΔlogP50×ΔpH−1) was extended to the effect of pH on O2‐dependent K+ influx and efflux, extracellular Bohr factors (Φo) of ‐2.00 and ‐2.06 were obtained, values much higher than that for Hb (Φo= ‐0.49). The results of this study are consistent with an O2 sensing mechanism differing markedly in affinity and cooperativity of O2 binding, as well as in pH sensitivity, from bulk Hb.

Functional differentiation of myoglobin isoforms in hypoxia-tolerant carp indicates tissue-specific protective roles

Because of a recent whole genome duplication, the hypoxia-tolerant common carp and goldfish are the only vertebrates known to possess two myoglobin (Mb) paralogs. One of these, Mb1, occurs in oxidative muscle but also in several other tissues, including capillary endothelial cells, whereas the other, Mb2, is a unique isoform specific to brain neurons. To help understand the functional roles of these diverged isoforms in the tolerance to severe hypoxia in the carp, we have compared their O(2) equilibria, carbon monoxide (CO) and O(2) binding kinetics, thiol S-nitrosation, nitrite reductase activities, and peroxidase activities. Mb1 has O(2) affinity and nitrite reductase activity comparable to most vertebrate muscle Mbs, consistent with established roles for Mbs in O(2) storage/delivery and in maintaining nitric oxide (NO) homeostasis during hypoxia. Both Mb1 and Mb2 can be S-nitrosated to similar extent, but without oxygenation-linked allosteric control. When compared with Mb1, Mb2 displays faster O(2) and CO kinetics, a lower O(2) affinity, and is slower at converting nitrite into NO. Mb2 is therefore unlikely to be primarily involved in either O(2) supply to mitochondria or the generation of NO from nitrite during hypoxia. However, Mb2 proved to be significantly faster at eliminating H(2)O(2,) a major in vivo reactive oxygen species (ROS), suggesting that this diverged Mb isoform may have a specific protective role against H(2)O(2) in the carp brain. This property might be of particular significance during reoxygenation following extended periods of hypoxia, when production of H(2)O(2) and other ROS is highest.

Ancient and modern duplication events and the evolution of stearoyl-CoA desaturases in teleost fishes

Stearoyl-CoA desaturases (SCDs) are key enzymes of fatty acid biosynthesis whose regulation underpins responses to dietary, thermal, and hormonal treatment. Although two isoforms are known to exist in the common carp and human and four in mouse, there is no coherent view on how this gene family evolved to generate functionally diverse members. Here we identify numerous new SCD homologs in teleost fishes, using sequence data from expressed sequence tag (EST) and cDNA collections and genomic model species. Phylogenetic analyses of the deduced coding sequences produced only partially resolved molecular trees. The multiple SCD isoforms were, however, consistent with having arisen by an ancient gene duplication event in teleost fishes together with a more recent duplication in the tetraploid carp and possibly also salmonid lineages. Critical support for this interpretation comes from comparison across all vertebrate groups of the gene order in the genomic environments of the SCD isoforms. Using syntenically aligned chromosomal fragments from large-insert clones of common carp and grass carp together with those from genomically sequenced model species, we show that the ancient and modern SCD duplication events in the carp lineage were each associated with large chromosomal segment duplications, both possibly linked to whole genome duplications. By contrast, the four mouse isoforms likely arose by tandem duplications. Each duplication in the carp lineage gave rise to differentially expressed SCD isoforms, either induced by cold or diet as previously shown for the recent duplicated carp isoforms or tissue specific as demonstrated here for the ancient duplicate zebrafish isoforms.

Historical reconstructions of evolving physiological complexity: O2 secretion in the eye and swimbladder of fishes

SUMMARY The ability of some fishes to inflate their compressible swimbladder with almost pure oxygen to maintain neutral buoyancy, even against the high hydrostatic pressure several thousand metres below the water surface, has fascinated physiologists for more than 200 years. This review shows how evolutionary reconstruction of the components of such a complex physiological system on a phylogenetic tree can generate new and important insights into the origin of complex phenotypes that are difficult to obtain with a purely mechanistic approach alone. Thus, it is shown that oxygen secretion first evolved in the eyes of fishes, presumably for improved oxygen supply to an avascular, metabolically active retina. Evolution of this system was facilitated by prior changes in the pH dependence of oxygen-binding characteristics of haemoglobin (the Root effect) and in the specific buffer value of haemoglobin. These changes predisposed teleost fishes for the later evolution of swimbladder oxygen secretion, which occurred at least four times independently and can be associated with increased auditory sensitivity and invasion of the deep sea in some groups. It is proposed that the increasing availability of molecular phylogenetic trees for evolutionary reconstructions may be as important for understanding physiological diversity in the postgenomic era as the increase of genomic sequence information in single model species.