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• W2143149723 abstract "The visual function of the vertebrate retina relies on sufficient supply with oxygen. Neuroglobin is a respiratory protein thought to play an essential role in oxygen homeostasis of neuronal cells. For further understanding of its function, we compared the distribution of neuroglobin and mitochondria in both vascular and avascular mammalian retinae. In the vascular retinae of mouse and rat, oxygen is supplied by the outer choroidal, deep retinal, and inner capillaries. We show that in this type of retina, mitochondria are concentrated in the inner segments of photoreceptor cells, the outer and the inner plexiform layers, and the ganglion cell layer. These are the same regions in which oxygen consumption takes place and in which neuroglobin is present at high levels. In the avascular retina of guinea pig the deep retinal and inner capillaries are absent. Therefore, only the inner segments of the photoreceptors adjacent to choroidal capillaries display an oxidative metabolism. We demonstrate that in the retina of guinea pigs both neuroglobin and mitochondria are restricted to this layer. Our results clearly demonstrate an association of neuroglobin and mitochondria, thus supporting the hypothesis that neuroglobin is a respiratory protein that supplies oxygen to the respiratory chain. The visual function of the vertebrate retina relies on sufficient supply with oxygen. Neuroglobin is a respiratory protein thought to play an essential role in oxygen homeostasis of neuronal cells. For further understanding of its function, we compared the distribution of neuroglobin and mitochondria in both vascular and avascular mammalian retinae. In the vascular retinae of mouse and rat, oxygen is supplied by the outer choroidal, deep retinal, and inner capillaries. We show that in this type of retina, mitochondria are concentrated in the inner segments of photoreceptor cells, the outer and the inner plexiform layers, and the ganglion cell layer. These are the same regions in which oxygen consumption takes place and in which neuroglobin is present at high levels. In the avascular retina of guinea pig the deep retinal and inner capillaries are absent. Therefore, only the inner segments of the photoreceptors adjacent to choroidal capillaries display an oxidative metabolism. We demonstrate that in the retina of guinea pigs both neuroglobin and mitochondria are restricted to this layer. Our results clearly demonstrate an association of neuroglobin and mitochondria, thus supporting the hypothesis that neuroglobin is a respiratory protein that supplies oxygen to the respiratory chain. The vertebrate retina consumes huge amounts of metabolic energy, which is used for the maintenance of the dark current of ions (1Tsacopoulos M. Poitry-Yamate C.L. MacLeish P.R. Poitry S. Prog. Retin. Eye Res. 1998; 17: 429-442Crossref PubMed Scopus (84) Google Scholar) and to replenish GTP that is converted to cGMP during phototransduction (2Hsu S.C. Molday R.S. J. Biol. Chem. 1994; 269: 17954-17959Abstract Full Text PDF PubMed Google Scholar). The required ATP derives either form anaerobic glycolysis or from oxidative phosphorylation (3Anderson B. Saltzman H.A. Arch. Ophthalmol. 1964; 72: 792-795Crossref PubMed Scopus (103) Google Scholar, 4Ames A. Can. J. Pharmacol. 1992; 70: S158-S164Crossref Scopus (128) Google Scholar, 5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar). Because the latter process uses molecular oxygen (O2), it is not surprising that the retina is the highest oxygen-consuming tissue of the mammalian body (6Anderson B. Trans. Am. Ophthalmol. Soc. 1968; 66: 423-474PubMed Google Scholar). Lack of sufficient oxygen (hypoxia) has immediate effects on visual performance and is thought to be an important factor in a number of retinal diseases (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar, 7Osborne N.N. Casson R.J. Wood J.P.M. Chidlow G. Graham M. Melena J. Prog. Retin. Eye Res. 2004; 23: 91-147Crossref PubMed Scopus (826) Google Scholar). Oxygen is transported to the retina by blood capillaries (Fig. 1; cf. Ref. 5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar). The vascular retina of man, mouse, and most other mammals has a dual blood supply (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar, 7Osborne N.N. Casson R.J. Wood J.P.M. Chidlow G. Graham M. Melena J. Prog. Retin. Eye Res. 2004; 23: 91-147Crossref PubMed Scopus (826) Google Scholar) in which the outer retina is nourished by choroidal blood vessels that lie immediately behind the pigment epithelium. The inner retina is supplied by branches of the central retinal capillary: the deep capillary network located in the outer plexiform layer and the superficial capillaries adjacent to the ganglion cell layer. However, in species with an avascular retina, such as guinea pig or rabbit, oxygen is delivered solely by the choroidal vascular bed, whereas the deep retinal and superficial capillaries are essentially absent (8Chase J. Ophthalmology. 1982; 89: 1518-1525Abstract Full Text PDF PubMed Scopus (101) Google Scholar). Because oxygen diffuses just over a short distance, only the outer retina receives sufficient oxygen to sustain aerobic energy production. The inner retina of guinea pig is essentially anoxic, with oxygen partial pressures as low as 1 Torr (9Yu D.Y. Cringle S.J. Alder V.A. Su E.N. Yu P.K. Am. J. Physiol. 1996; 270: H965-H973PubMed Google Scholar). Respiratory proteins such as myoglobin (Mb) 1The abbreviations used are: Mb, myoglobin; Cygb, cytoglobin; Ngb, neuroglobin; PBS, phosphate-buffered saline; ROS, reactive oxygen species; RACE, rapid amplification of cDNA ends. enhance the supply of the respiratory chain with oxygen (10Wittenberg J.B. Wittenberg B.A. J. Exp. Biol. 2003; 206: 2011-2020Crossref PubMed Scopus (398) Google Scholar). Neuroglobin (Ngb) is a recently identified heme-protein that resembles the Mb and hemoglobin and shares a common evolutionary origin with some invertebrate nerve-globins (11Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (910) Google Scholar). Ngb is an intracellular protein preferentially expressed in the neurons of the central and peripheral nervous systems but also in some endocrine tissues (11Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (910) Google Scholar, 12Reuss S. Saaler-Reinhardt S. Weich B. Wystub S. Reuss M. Burmester T. Hankeln T. Neuroscience. 2002; 115: 645-656Crossref PubMed Scopus (167) Google Scholar, 13Wystub S. Laufs T. Schmidt M. Burmester T. Maas U. Saaler-Reinhardt S. Hankeln T. Reuss S. Neurosci. Lett. 2003; 346: 114-116Crossref PubMed Scopus (112) Google Scholar, 14Fuchs C. Heib V. Kiger L. Haberkamp M. Roesner A. Schmidt A. Hamdane D. Marden M.C. Hankeln T. Burmester T. J. Biol. Chem. 2004; 279: 24116-24122Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 15Laufs T. Wystub S. Reuss S. Burmester T. Saaler-Reinhardt S. Hankeln T. Neurosci. Lett. 2004; 362: 83-86Crossref PubMed Scopus (59) Google Scholar). Like other globins, Ngb binds O2 reversibly via an iron (Fe2+) ion of the heme group with an affinity (P50) of about 1 Torr, which is in the range of that of Mb (16Dewilde S. Kiger L. Burmester T. Hankeln T. Baudin-Creuza V. Aerts T. Marden M.C. Caubergs R. Moens L. J. Biol. Chem. 2001; 276: 38949-38955Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). The precise role of Ngb in the organism is currently still not well understood. Initially, it has been suggested that Ngb carries out an Mb-like function, thus ensuring oxygen homeostasis of neurons (11Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (910) Google Scholar). In fact, Ngb enhances the survival of cultured neuronal cells under hypoxia (17Sun Y. Jin K. Mao X.O. Zhu Y. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15306-15311Crossref PubMed Scopus (462) Google Scholar). However, the concentration of Ngb in the total brain is very low and in the range of 1 μm (11Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (910) Google Scholar), which appears to be hardly compatible with a respiratory role of this protein. Therefore, Ngb has also been considered e.g. as scavenger for peroxynitrite (18Herold S. Fago A. Weber R.E. Dewilde S. Moens L. J. Biol. Chem. 2004; 279: 22841-22847Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) or as sensor that transmits a hypoxia signal via interaction with other proteins (19Wakasugi K. Nakano T. Morishima I. J. Biol. Chem. 2003; 278: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Wakasugi K. Nakano T. Morishima I. Biochemistry. 2004; 43: 5119-5125Crossref PubMed Scopus (45) Google Scholar, 21Wakasugi K. Nakano T. Kitatsuji C. Morishima I. Biochem. Biophys. Res. Commun. 2004; 318: 453-460Crossref PubMed Scopus (65) Google Scholar). Given the variety of globin functions discovered in recent years, still other roles for Ngb are conceivable, such as degradation of reactive oxygen species (ROS), NO decomposition, or a function as terminal oxidase (22Pesce A. Bolognesi M. Ascenzi P. Bocedi A. Dewilde S. Moens L. Hankeln T. Burmester T. EMBO Rep. 2002; 3: 1146-1151Crossref PubMed Scopus (257) Google Scholar, 23Burmester T. Hankeln T. News Phys. Sci. 2004; 19: 110-113Crossref PubMed Scopus (123) Google Scholar, 24Hankeln T. Ebner B. Fuchs C. Gerlach F. Haberkamp M. Laufs T. Roesner A. Schmidt M. Weich B. Wystub S. Saaler-Reinhardt S. Reuss S. Bolognesi M. De Sanctis D. Marden M.C. Kiger L. Dewilde S. Moens L. Nevo E. Avivi A. Weber R.E. Fago A. Burmester T. J. Inorg. Biochem. 2005; 99: 110-119Crossref PubMed Scopus (259) Google Scholar). In a previous study, we found that Ngb concentration in the neuronal retina of mouse is about 50–100 μm (25Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) and thus about 100 times higher than in total brain extracts (11Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (910) Google Scholar). Because such concentration is in fact in the range of that of Mb in muscle cells, this observation can been taken as support for an oxygen supply function of Ngb. This hypothesis also predicts Ngb to be located in the vicinity of the mitochondria, which should be, in turn, associated with oxygen consumption rates. To further understand Ngb function, we decided to investigate the cellular and subcellular distribution of both mitochondria and Ngb in retinae that display different degrees of vascularization and thus oxygen supply. Animals—The procedures concerning animals complied with German legislation for the protection of animals and were approved by the county government office (Bezirksregierung Rheinhessen-Pfalz). Adult pigmented BALB/c mice (Mus musculus) and albino rats (Rattus norvegicus) were maintained under constant conditions, with a 12 h:12 h light:dark regimen at room temperature (21 ± 1 °C). Food and water were supplied ad libitum. Guinea pigs (Cavia porcellus) were sacrificed immediately after purchase at the middle of the light period. For perfusion-fixation, the animals were killed with an ether overdose and transcardially perfused with 100 ml of room temperature PBS (7.5 mm Na2HPO4, 2.5 mm NaH2PO4, 145 mm NaCl), supplemented with 15,000 IU heparin/liter. Perfusion-fixation was carried out at a constant rate of 10 ml/min with 200–300 ml of ice-cold 4% paraformaldehyde, 1.37% l-lysine, 0.21% sodium periodate in PBS. The eyes were removed, postfixed for 1 h in the same fixative, and stored at 4 °C in phosphate-buffered 30% sucrose. Cloning and Sequencing of Guinea Pig Ngb cDNA—Total RNA was extracted from guinea pig brain tissue by the guanidine hydrochloride method (26Chirgwin J.M. Przbyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar). Various degenerated oligonucleotide primers were designed according to the conserved segments of the aligned mammalian Ngb cDNA sequences. Ngb cDNA fragments were amplified by reverse transcription-PCR experiments employing the Qiagen OneStep kit according to the manufacturer's instructions. The PCR products were cloned into the pCR4-TOPO vector (Invitrogen). Sequences were obtained from both strands using a commercial sequencing service (GEN-terprise). The missing 3′-end of the cDNA was obtained using the RACE system of Invitrogen. The guinea pig Ngb cDNA sequence was deposited at the GenBank™/EMBL data base under the accession number AJ781213. Antibody Preparation—Two different antigenic peptides were designed according to the conserved mammalian Ngb amino acid sequences (cf. Fig. 2). The first synthetic peptide covers the Ngb acid positions 47–61, with a cysteine added at the N terminus for coupling (H2N-CRQFSSPEDCLSSPEF-CONH2), the second peptide includes amino acids 55–70 (H2N-CLSSPEFLDHIRKVML-CONH2). Peptide syntheses were carried out by Eurogentec or Seqlab. The peptides were coupled to KLH and used for immunization of rabbits, employing commercial services (Eurogentec or Seqlab). The specific anti-Ngb antibodies were purified from the serum employing the synthetic peptides coupled to SulfoLink columns (Pierce) according to the instructions of the manufacturer. After elution, the antibodies were stored at 4 °C in 50 mm Tris, 100 mm glycine, pH ∼7.4) supplemented with 1% bovine serum albumin and 0.05% NaN3. Immunohistochemistry—Indirect immunofluorescence studies on perfusion-fixed mammalian retinae were essentially performed as described previously (13Wystub S. Laufs T. Schmidt M. Burmester T. Maas U. Saaler-Reinhardt S. Hankeln T. Reuss S. Neurosci. Lett. 2003; 346: 114-116Crossref PubMed Scopus (112) Google Scholar, 25Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 27Schmidt M. Laufs T. Reuss S. Hankeln T. Burmester T. Neurosci. Lett. 2005; 374: 207-211Crossref PubMed Scopus (44) Google Scholar). Briefly, the eyes were frozen in melting isopentane and 14-μm longitudinal cryosections were obtained from the retina with a Microm HM500 O microtome. The sections were mounted on Superfrost-Plus® (Roth) slides, dried, and immediately used. Nonspecific binding sites were blocked with 10% bovine serum albumin in PBS. The retina sections were incubated 2 h at room temperature with the polyclonal anti-Ngb peptide antibodies (1:5 to 1:50), polyclonal anti-von Willebrand factor antibodies (1:1,000; Abcam, Cambridge, UK), or monoclonal mouse anti-cytochrome c antibodies (1:200; Dianova, Hamburg, Germany), each diluted in blocking solution. The sections were washed 3 × 8 min in PBS and incubated for 90 min at room temperature in the dark with the appropriate secondary antibody (goat anti-mouse coupled to Cy2 or Cy3, or goat anti-rabbit IgG coupled to Cy3; Dianova, Hamburg, Germany), each diluted 1:200 to 1:500 in the blocking solution. The sections were washed as above and embedded in Elvanol polyvinyl alcohol (Mowiol; Calbiochem). The Hoechst dye 33258 (0.3 μg/ml) was added to the Elvanol to stain the nuclei. The sections were evaluated by a Leitz DM RD or an Olympus BX51 microscope. Photographs were taken with a digital camera. The images were combined with the Adobe Photoshop 7 program, which was also used to adjust image contrast and brightness and to add labels. Signal profiles of Ngb and cytochrome c from double labeling experiments were obtained and quantified employing the Scion Image program (version Beta4.02). Cloning and Sequencing of Guinea Pig Ngb cDNA—A set of degenerated primers were deduced from the known mammalian Ngb coding regions and used to amplify fragments of the Ngb cDNA by reverse transcription-PCR from guinea pig total brain RNA. The 3′-end was obtained by RACE methods. However, 5′-RACE experiments failed, and the missing nucleotides of the 5′-coding region were determined by the help of degenerated primers that had been constructed according to conserved regions of rodent Ngb 5′-untranslated region. The complete Ngb coding region was eventually confirmed by reverse transcription-PCR experiments that use primers adjacent to the start and stop codons and subsequent sequencing. Like all other known mammalian Ngbs, the coding region of guinea pig cDNA covers 456 bp; 10 and 685 bp were recovered from the 5′- and 3′-untranslated regions, respectively. The deduced amino acid sequence of 151 residues displays about 90–95% amino acid identity to the other known mammalian Ngb proteins, with all key determinants required for reversible oxygen binding being conserved (Fig. 2). Blood Capillaries in Vascular and Avascular Mammalian Retinae—The mammalian retina is built by distinct layers that are well distinguishable by light microscopy (Fig. 3A). We first investigated the localization and distribution of blood vessels in 14 μm thick cryo-sections of mouse (Fig. 3B), rat (Fig. 3C), and guinea pig (Fig. 3D) retinae. Using a polyclonal antibody against the von Willebrand factor, we found in the retinae of all three species strong staining of the endothelial cells of the capillaries in the choroidal layer. In mouse and rat, additional spotted signals (arrows in Fig. 3, B and C) were observed that can be attributed to the superficial capillaries in the neurofiber layer and the deep capillaries in the outer plexiform layer. This positive anti-von Willebrand factor immunostaining of the inner retina was absent in the guinea pig retina, which showed strong positive reaction only in the region of the choroidal vessels. Ngb and Mitochondria in Vascular Retinae of Mouse and Rat—We raised two polyclonal antibodies against distinct peptides of the mammalian Ngb proteins (Fig. 2). The peptide sequences are conserved in mouse, rat, guinea pig, and other mammals. The antibodies were purified from the sera by the aid of the appropriate peptides. In immunohistochemical studies, the antibodies showed identical cytoplasmic staining on cryo-sections of mouse (Fig. 3, E and F) and rat retinae (Fig. 3H). In both species, positive anti-Ngb immunoreaction was found to be essentially restricted to the inner segments of the photoreceptors, the inner and outer plexiform layers, and the ganglion cells. Both nuclear layers showed only weak cytoplasmic staining. After preabsorption of the antibody with the appropriate peptide or with recombinantly expressed Ngb, the positive anti-Ngb staining disappeared (Figs. 3G). This demonstrated the specificity of the antibodies. Mitochondria were identified in tissue sections employing a commercial monoclonal antibody against cytochrome c. Mouse and rat retinae showed identical patterns with this antibody, with a clear cytoplasmic signal (Fig. 3, I and K). Within the neuronal retina, cytochrome c labeling showed a punctuate pattern, consistent with the location of the immune reaction being confined to the mitochondria. Strong immunoreaction was found mainly in the inner segments of the photoreceptors, the plexiform, and the ganglion cell layers. Minor staining was also observed in endothelial cells of choroidal capillaries, as well as in the nuclear layers. Double labeling experiments employing anti-cytochrome c and anti-Ngb antibodies showed that the cellular localizations of Ngb and cytochrome c in the inner segments of the photoreceptors, the plexiform layers, and the ganglion cells are essentially identical (Fig. 3, J and L). The intensities of Ngb and cytochrome c signals were quantified by Scion image, which confirms their largely identical distribution. Only in the inner segments the mitochondria appeared to be evenly distributed, whereas Ngb was more concentrated in the lower sections. Ngb and Mitochondria in the Avascular Retina of Guinea Pig—Both anti-Ngb antibodies were applied to cryo-sections of the guinea pig retina. In this species, positive anti-Ngb reaction was found to be essentially restricted to the inner segments of the photoreceptor cells (Fig. 3M). Strong anti-cytochrome c staining was observed in the inner segments, while a weak positive immunoreaction was visible in the outer segments and possibly also at some spots in the plexiform layers (Fig. 3N). Double staining experiments with anti-Ngb and anti-cytochrome c antibodies showed a largely overlapping, but slightly shifted, labeling pattern in the inner segments (Fig. 3O). While cytochrome c and thus the mitochondria are also present in the upper regions, Ngb labeling was more evenly distributed in the inner segments (Fig. 4B). Oxygen Consumption and Mitochondria in the Vertebrate Retina—Since many years it has been known that the vertebrate retina consumes large amounts of oxygen and that an adequate cellular oxygen environment is crucial for the appropriate function of retinal cells (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar, 6Anderson B. Trans. Am. Ophthalmol. Soc. 1968; 66: 423-474PubMed Google Scholar, 7Osborne N.N. Casson R.J. Wood J.P.M. Chidlow G. Graham M. Melena J. Prog. Retin. Eye Res. 2004; 23: 91-147Crossref PubMed Scopus (826) Google Scholar). Lack of oxygen has severe effects to the visual performance (28Wiedman M. Tabin G.C. Ophthalmology. 1999; 106: 1924-1926Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 29Karakucuk S. Mirza G.E. Ophthalmic Res. 2000; 32: 30-40Crossref PubMed Scopus (30) Google Scholar). The vertebrate retina is a highly specialized structure that is divided into morphologically and functionally distinct layers (Figs. 1 and 3A). This feature allows the easy correlation of measured oxygen levels with particular cellular and subcellular structures (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar). In the vascular retinae of mouse and rat, oxygen partial pressures have been found to be in the range of 10–30 Torr (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar, 30Yu D.Y. Cringle S.J. Comp. Biochem. Physiol. A. 2002; 132: 47-52Crossref PubMed Scopus (42) Google Scholar). It has been shown that oxygen consumption takes places mainly in the inner segments of the photoreceptor cells, the plexiform layers, and the ganglion cells (Fig. 4A). Here we have been able to associate differences in oxygen consumption rates conclusively with the distribution of mitochondria. In an aerobic metabolism, the respiratory chain in the mitochondria is the main site of oxygen consumption. Previous studies using electron microscopic techniques, histochemical labeling, or immunohistochemical methods agreed that mitochondria are concentrated in the inner segments but were not conclusive on their distribution in the inner retina (31Matschinsky F.M. Costa E. Giacobini E. Biochemistry of Simple Neuronal Models. 2. Raven Press, New York1970: 217-243Google Scholar, 32Uga S. Smelser G.K. Invest. Ophthal. Vis. Sci. 1973; 12: 434-448Google Scholar, 33Germer A. Biedermann B. Wolburg H. Schuck J. Grosche J. Kuhrt H. Reichelt W. Schousboe A. Paasche G. Mack A.F. Reichenbach A. J. Neurocytol. 1998; 27: 329-345Crossref PubMed Scopus (39) Google Scholar, 34Germer A. Schuck J. Wolburg H. Kuhrt H. Mack A.F. Reichenbach A. J. Neurocytol. 1998; 27: 347-359Crossref PubMed Scopus (20) Google Scholar). The present study using an anti-cytochrome c antibody confirms an accumulation of mitochondria in the inner segments in mouse and rat retinae and shows for the first time that mitochondria are also concentrated in the plexiform and ganglion cell layers. Mitochondria appear to be essentially absent from the nuclear layers and the outer segments. The results also strongly support the calculations by Yu and Cringle (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar, 30Yu D.Y. Cringle S.J. Comp. Biochem. Physiol. A. 2002; 132: 47-52Crossref PubMed Scopus (42) Google Scholar). These authors concluded from their measurements of intraretinal oxygen levels (Fig. 1) that the layers that we found in our study to contain most of the mitochondria were the main oxygen consumers of vascular mammalian retinae (Fig. 4A). Ngb in Vascular Mammalian Retinae Correlates with Mitochondria Localization and Oxygen Consumption—In striated and cardiac muscles, another highly active vertebrate tissue, Mb is employed for the storage of oxygen and facilitation of oxygen diffusion (10Wittenberg J.B. Wittenberg B.A. J. Exp. Biol. 2003; 206: 2011-2020Crossref PubMed Scopus (398) Google Scholar, 35Merx M.W. Flögel U. Stumpe T. Gödecke A. Decking U.K. Schrader J. FASEB J. 2002; 15: 1077-1079Crossref Scopus (91) Google Scholar). Recently, two additional globin types have been identified in vertebrates: Ngb and cytoglobin (Cygb) (11Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (910) Google Scholar, 36Burmester T. Ebner B. Weich B. Hankeln T. Mol. Biol. Evol. 2002; 19: 416-421Crossref PubMed Scopus (438) Google Scholar). These respiratory proteins are also present in the retinae of mouse (25Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), chicken (37Kugelstadt D. Haberkamp M. Hankeln T. Burmester T. Biochem. Biophys. Res. Commun. 2004; 325: 719-725Crossref PubMed Scopus (84) Google Scholar), and fish (14Fuchs C. Heib V. Kiger L. Haberkamp M. Roesner A. Schmidt A. Hamdane D. Marden M.C. Hankeln T. Burmester T. J. Biol. Chem. 2004; 279: 24116-24122Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), thus potentially contributing to the oxygen supply of this tissue. Because the retinal layers show distinct oxygen consumption rates (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar, 30Yu D.Y. Cringle S.J. Comp. Biochem. Physiol. A. 2002; 132: 47-52Crossref PubMed Scopus (42) Google Scholar, 38Cringle S.J. Yu D.Y. Comp. Biochem. Physiol. A. 2002; 132: 61-66Crossref PubMed Scopus (67) Google Scholar, 39Cringle S.J. Yu D.Y. Yu P.K. Su E.N. Invest. Ophthalmol. Vis. Sci. 2002; 43: 1922-1927PubMed Google Scholar), it is easy to compare the distribution of Ngb and Cygb to cellular and intracellular respiration rates. In mouse, Cygb is expressed only in a subset of neurons of the brain and of the ganglion cell and inner nuclear layers of the retina (27Schmidt M. Laufs T. Reuss S. Hankeln T. Burmester T. Neurosci. Lett. 2005; 374: 207-211Crossref PubMed Scopus (44) Google Scholar, 40Schmidt M. Gerlach F. Avivi A. Laufs T. Wystub S. Simpson J.C. Nevo E. Saaler-Reinhardt S. Reuss S. Hankeln T. Burmester T. J. Biol. Chem. 2004; 279: 8063-8069Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Minor amounts of Cygb protein are also present in the inner plexiform layer, while it is absent from the outer nuclear layer and the photoreceptor cells. Thus the localization of Cygb cannot be related to mitochondria or oxygen consumption and a respiratory function of this protein is unlikely (27Schmidt M. Laufs T. Reuss S. Hankeln T. Burmester T. Neurosci. Lett. 2005; 374: 207-211Crossref PubMed Scopus (44) Google Scholar). In contrast to Cygb, Ngb shows enhanced expression in the murine retina (25Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Its distribution in inner segments, inner and outer plexiform layers, and ganglion cells may easily be associated with the main sites of oxygen consumption. As we have shown here, Ngb distribution in mouse and rat retinae also correlates with mitochondria, which are essentially restricted to the same layers (Fig. 4). This provides an additional hint that the distribution of Ngb may in fact be correlated to oxygen consumption rates. It should be noted, however, that although the staining patterns of Ngb and mitochondria largely overlap, they are not identical. First, this observation provides evidence against a mitochondrial localization of Ngb. Second, higher cytochrome c levels were observed in regions that are close to capillaries and thus have higher oxygen levels. By contrast, Ngb is more concentrated in neighboring regions in which somewhat less mitochondria are present (Fig. 4) but where sufficient oxygen is still available for oxidative phosphorylation (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar). Here, the respiratory protein Ngb may increase the amount of oxygen available to the mitochondria more efficiently than in regions with higher oxygen levels (see below). Absence of Mitochondria and Ngb in the Avascular Inner Retina of Guinea Pig—By measuring intraretinal O2 levels, Cringle and Yu (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar, 9Yu D.Y. Cringle S.J. Alder V.A. Su E.N. Yu P.K. Am. J. Physiol. 1996; 270: H965-H973PubMed Google Scholar, 41Cringle S.J. Yu D.Y. Alder V. Su E.N. Yu P.K. Am. J. Physiol. 1996; 40: H1162-H1165Google Scholar, 42Cringle S.J. Yu D.Y. Alder V.A. Su E.N. Invest. Ophthalmol. Vis. Sci. 1998; 40: 2307-2313Google Scholar) concluded that the O2 requirement of the inner retina in the guinea pig is small. While the outer retina, which is mainly composed by photoreceptor cells, accounts for more than 90% of oxygen consumption, only about 5% of the total retinal oxygen usage can be attributed to the inner retina (41Cringle S.J. Yu D.Y. Alder V. Su E.N. Yu P.K. Am. J. Physiol. 1996; 40: H1162-H1165Google Scholar). The oxygen partial pressure within the inner retina is low (1 Torr) and below the range of the assumed minimal tissue oxygen tension for mitochondrial function (26Chirgwin J.M. Przbyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16652) Google Scholar, 43Epstein F.H. Kidney Int. 1997; 51: 381-385Abstract Full Text PDF PubMed Scopus (206) Google Scholar). Thus the metabolism must be sustained by anaerobic mechanisms such as fermentation. The absence of mitochondria in the inner retina of guinea pigs (Figs. 3N and 4B) demonstrates that the low oxygen consumption rates are not due to a limited availability of oxygen but is a physiological characteristic of this tissue. Because of the lack of mitochondria, even an excess of available oxygen (hyperoxia) does not increase oxygen consumption rates of the inner retina of guinea pigs (9Yu D.Y. Cringle S.J. Alder V.A. Su E.N. Yu P.K. Am. J. Physiol. 1996; 270: H965-H973PubMed Google Scholar). The lack of the mitochondria in the inner retina of guinea pigs also correlates with the absence of Ngb (Figs. 3O and 4B). While the Ngb protein is probably one of the best conserved mammalian proteins (Fig. 2), we could show here that its distribution differs among species. Our observations link for the first time Ngb to oxygen consumption, even at the subcellular level. Support for a Respiratory Function of Ngb—Intracellular globins such as the Mb are well known for their respiratory functions, supplying oxygen to the respiratory chain of the mitochondria (10Wittenberg J.B. Wittenberg B.A. J. Exp. Biol. 2003; 206: 2011-2020Crossref PubMed Scopus (398) Google Scholar, 35Merx M.W. Flögel U. Stumpe T. Gödecke A. Decking U.K. Schrader J. FASEB J. 2002; 15: 1077-1079Crossref Scopus (91) Google Scholar). However, the actual role of Ngb has remained elusive (22Pesce A. Bolognesi M. Ascenzi P. Bocedi A. Dewilde S. Moens L. Hankeln T. Burmester T. EMBO Rep. 2002; 3: 1146-1151Crossref PubMed Scopus (257) Google Scholar, 23Burmester T. Hankeln T. News Phys. Sci. 2004; 19: 110-113Crossref PubMed Scopus (123) Google Scholar, 24Hankeln T. Ebner B. Fuchs C. Gerlach F. Haberkamp M. Laufs T. Roesner A. Schmidt M. Weich B. Wystub S. Saaler-Reinhardt S. Reuss S. Bolognesi M. De Sanctis D. Marden M.C. Kiger L. Dewilde S. Moens L. Nevo E. Avivi A. Weber R.E. Fago A. Burmester T. J. Inorg. Biochem. 2005; 99: 110-119Crossref PubMed Scopus (259) Google Scholar). A function of Ngb in facilitating oxygen diffusion within neurons is supported (i) by its relationship to invertebrate nerve-globins (11Burmester T. Weich B. Reinhardt S. Hankeln T. Nature. 2000; 407: 520-523Crossref PubMed Scopus (910) Google Scholar), for which a role as Mb-like oxygen supply protein had been established (44Kraus D.W. Colacino J.M. Science. 1986; 232: 90-92Crossref PubMed Scopus (43) Google Scholar), (ii) by its ability to promote neuron survival under hypoxic/ischemic conditions (17Sun Y. Jin K. Mao X.O. Zhu Y. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15306-15311Crossref PubMed Scopus (462) Google Scholar), and (iii) by the enhanced Ngb concentration in the highly oxygen-consuming tissue of the retina (25Schmidt M. Giessl A. Laufs T. Hankeln T. Wolfrum U. Burmester T. J. Biol. Chem. 2003; 278: 1932-1935Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Others have considered Ngb as an oxidative stress sensor (19Wakasugi K. Nakano T. Morishima I. J. Biol. Chem. 2003; 278: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Wakasugi K. Nakano T. Morishima I. Biochemistry. 2004; 43: 5119-5125Crossref PubMed Scopus (45) Google Scholar, 21Wakasugi K. Nakano T. Kitatsuji C. Morishima I. Biochem. Biophys. Res. Commun. 2004; 318: 453-460Crossref PubMed Scopus (65) Google Scholar) or have suggested that Ngb is involved in the detoxification of NO or other reactive nitrogen species (18Herold S. Fago A. Weber R.E. Dewilde S. Moens L. J. Biol. Chem. 2004; 279: 22841-22847Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 45Mammen P.P.A. Shelton J.M. Goetsch S.C. Williams S.C. Richardson J.A. Garry M.G. Garry D.J. J. Histochem. Cytochem. 2002; 50: 1591-1598Crossref PubMed Scopus (128) Google Scholar). As already noted (23Burmester T. Hankeln T. News Phys. Sci. 2004; 19: 110-113Crossref PubMed Scopus (123) Google Scholar, 24Hankeln T. Ebner B. Fuchs C. Gerlach F. Haberkamp M. Laufs T. Roesner A. Schmidt M. Weich B. Wystub S. Saaler-Reinhardt S. Reuss S. Bolognesi M. De Sanctis D. Marden M.C. Kiger L. Dewilde S. Moens L. Nevo E. Avivi A. Weber R.E. Fago A. Burmester T. J. Inorg. Biochem. 2005; 99: 110-119Crossref PubMed Scopus (259) Google Scholar), those functions are less likely. In particular, no correlation between Ngb and known sites of NO synthase expression in brain or retina has been observed (12Reuss S. Saaler-Reinhardt S. Weich B. Wystub S. Reuss M. Burmester T. Hankeln T. Neuroscience. 2002; 115: 645-656Crossref PubMed Scopus (167) Google Scholar, 27Schmidt M. Laufs T. Reuss S. Hankeln T. Burmester T. Neurosci. Lett. 2005; 374: 207-211Crossref PubMed Scopus (44) Google Scholar). A role of Ngb in the detoxification of harmful ROS may not be formally excluded, in particular because mitochondria are known to be the main sites of ROS production (46Harper M.E. Bevilacqua L. Hagopian K. Weindruch R. Ramsey J.J. Acta Physiol. Scand. 2004; 182: 321-331Crossref PubMed Scopus (210) Google Scholar). However, taking into account that in the retina most ROS are induced by light in the outer segments of the photoreceptor cells (47Penn J.S. Naash M.I. Anderson R.E. Exp. Eye Res. 1987; 44: 779-788Crossref PubMed Scopus (135) Google Scholar, 48Organisciak D.T. Wang H.M. Xie A. Reeves D.S. Donoso L.A. Prog. Clin. Biol. Res. 1989; 314: 493-512PubMed Google Scholar) where Ngb is absent (Figs. 3 and 4), a ROS-related role of Ngb should be considered less likely. In summary, the present study supports a respiratory function of Ngb in retinal and possibly also brain neurons. In this scenario, Ngb has a similar function as Mb (10Wittenberg J.B. Wittenberg B.A. J. Exp. Biol. 2003; 206: 2011-2020Crossref PubMed Scopus (398) Google Scholar, 35Merx M.W. Flögel U. Stumpe T. Gödecke A. Decking U.K. Schrader J. FASEB J. 2002; 15: 1077-1079Crossref Scopus (91) Google Scholar), thus facilitating the diffusion of O2 and enhancing the flow of O2 from the capillaries to the mitochondria. It should be noted that, like in Mb, the oxygen affinity of Ngb is higher than that of Hb but lower than that of the cytochrome oxidase (49Gnaiger E. Adv. Exp. Med. Biol. 2003; 543: 39-55Crossref PubMed Scopus (104) Google Scholar). This positions Ngb between the hemoglobin in the capillaries and the cytochrome c oxidase of the respiratory chain. The oxygen partial pressures in the oxygenated, Ngb-containing regions of the retina are in the range of 10–30 Torr (5Yu D.Y. Cringle S.J. Prog. Retin. Eye Res. 2001; 20: 175-208Crossref PubMed Scopus (494) Google Scholar), corresponding to oxygen concentrations of 14 to 42 μm at 37 °C. Ngb concentration in the total murine retina was determined to be in the range of 50 to 100 μm (24Hankeln T. Ebner B. Fuchs C. Gerlach F. Haberkamp M. Laufs T. Roesner A. Schmidt M. Weich B. Wystub S. Saaler-Reinhardt S. Reuss S. Bolognesi M. De Sanctis D. Marden M.C. Kiger L. Dewilde S. Moens L. Nevo E. Avivi A. Weber R.E. Fago A. Burmester T. J. Inorg. Biochem. 2005; 99: 110-119Crossref PubMed Scopus (259) Google Scholar). Because Ngb is largely restricted to the oxygen-consuming areas, local cellular and subcellular concentrations may be much higher. Thus the concentration of Ngb probably largely exceeds the free oxygen concentrations, and thus Ngb may contribute significantly to the flow of oxygen to the mitochondria. This hypothesis agrees with the observed co-localization of Ngb and mitochondria and with its absence in the anaerobic inner retina of guinea pigs. However, alternative functions of Ngb that would support the aerobic ATP production in the mitochondria by a yet unknown mechanism are still conceivable and should be addressed in future studies. The excellent technical assistance of U. Disque-Kaiser and K. Lotz is gratefully acknowledged. We thank J. Harf for the schematic drawing of the retina. A. B., M. S., and T. B. thank J. Markl for excellent working facilities." @default.
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• W2143149723 title "Divergent Distribution in Vascular and Avascular Mammalian Retinae Links Neuroglobin to Cellular Respiration" @default.
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