Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia.

Exposure of cultured cells to changing gaseous environments is used as a model for understanding both the immediate and long-term effects of such exposures on lung cells in vivo. We conducted experiments with polystyrene tissue culture flasks and plates to determine the time course of changes in oxygen concentration occurring under in vitro conditions. Only a few minutes were required for the concentration of oxygen in the environmental chamber to reach equilibrium with that of the flushing gas. However, >3 h were required for the oxygen content in the medium in the tissue culture flasks and plates to achieve equilibrium. The low solubility of oxygen in aqueous solutions and the limited diffusion of oxygen through a boundary layer of gas above the medium are the major barriers to rapid oxygen transport into the culture medium. The delay in achieving the desired PO(2) within the culture medium limits the temporal precision of any assessment of the correlation of cellular events with the concentration of oxygen to which those cells are exposed.

Elevated expression of surfactant proteins in newborn rats during adaptation to hyperoxia.

The mechanisms whereby lung adaptation to hyperoxia occurs in the newborn period are incompletely understood. Pulmonary surfactant has been implicated in lung protection against hyperoxic injury, and elevated expression of certain surfactant proteins occurs in lungs of adult rats during adaptation to sublethal oxygen (85% O(2)). Here we report that newborn rats, which can adapt to even higher levels of hyperoxia (100% O(2)) than do adult rats, manifest changes in the lung surfactant proteins (SP), especially SP-A and SP-D. In newborn rats exposed to hyperoxia on Days 3 through 10 of life, lung messenger RNAs (mRNAs) for SP-A and SP-B gradually and progressively increased, relative to levels in age-matched, air-exposed newborns, over this 8-d period. By contrast, SP-C and SP-D mRNAs were maximally increased relative to values in simultaneously air-exposed control rats after 4 d of exposure. Lung mRNA for CC-10, a protein specific for Clara cells, was greater in hyperoxia-exposed rats than in air-exposed control rats on Day 4 of exposure, but not on other days. Lung mRNA for thyroid transcription factor (TTF)-1 was marginally increased on Days 1, 2, 4, and 6, and significantly increased on Day 8. Both SP-A and SP-D proteins were increased in lung lavage samples taken from hyperoxia-exposed newborns, relative to those taken from air-exposed controls, with the greatest increases occurring on Days 6 and 8 of exposure. However, the patterns of increase of the proteins were not identical to those of the respective mRNAs. In situ hybridization studies demonstrated increases in SP-D, and to a lesser extent in SP-A, in peripheral lung tissues from oxygen-exposed newborns. Taken together, these data indicate that specific surfactant proteins are upregulated at both the pretranslational and post-translational levels in distal lung epithelium during adaptation to hyperoxia in the newborn rat.

Glutamine protects mitochondrial structure and function in oxygen toxicity.

Glutamine is an important mitochondrial substrate implicated in the protection of cells from oxidant injury, but the mechanisms of its action are incompletely understood. Human pulmonary epithelial-like (A549) cells were exposed to 95% O2 for 4 days in the absence and presence of glutamine. Cell proliferation in normoxia was dependent on glutamine, and glutamine deprivation markedly accelerated cell death in hyperoxia. Glutamine significantly increased cellular ATP levels in normoxia and prevented the loss of ATP in hyperoxia seen in glutamine-deprived cells. Mitochondrial membrane potential as assessed by flow cytometry with chloromethyltetramethylrosamine was increased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-dependent increase in mitochondrial membrane potential was detected. Glutamine-supplemented, hyperoxia-exposed cells had a higher O2 consumption rate and GSH content. Electron and fluorescence microscopy revealed that, in hyperoxia, glutamine protected cellular structures, especially mitochondria, from damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase was partially protected by its indirect substrate, glutamine, indicating a mechanism of mitochondrial protection.

Hypoxia induces hexokinase II gene expression in human lung cell line A549.

During adaptation to hypoxic and hyperoxic conditions, the genes involved in glucose metabolism are upregulated. To probe involvement of the transcription factor hypoxia-induced factor-1 (HIF-1) in hexokinase (HK) II expression in human pulmonary cells, A549 cells and small-airway epithelial cells (SAECs) were exposed to stimuli such as hypoxia, deferoxamine (DFO), and metal ions. The largest increase in HK-II (20-fold for mRNA and 2.5-fold for enzymatic activity) was observed in A549 cells when exposed to DFO. All stimuli selectively increased the 5.5-kb rather than 4-kb transcript in A549 cells. Cycloheximide and actinomycin D inhibited these responses. In addition, cells were transfected with luciferase reporter constructs driven by the full-length HK-II 5'-regulatory region (4.0 kb) or various deletions of that region. A549 cells transfected with the 4.0-kb construct and exposed to hypoxia or DFO increased their luciferase activity 7- and 10-fold, respectively, indicating that HK-II induction is, at least in part, due to increased gene transcription. Sixty percent of the inducible activity of the 4.0-kb construct was shown to reside within the proximal 0.5 kb. Additionally, cotransfection with a stable HIF-1 mutant and the 4.0-kb promoter construct resulted in increased luciferase activity under normoxic conditions. These results strongly suggest that HK-II is selectively regulated in pulmonary cells by a HIF-1-dependent mechanism.

Changes in pulmonary expression of hexokinase and glucose transporter mRNAs in rats adapted to hyperoxia.

Impairment of lung aconitase activity, citric acid cycle, and mitochondrial respiration by hyperoxia necessitates the elevation of glycolysis for energy production and of pentose shunt activity for reducing equivalents. The molecular mechanisms that allow increased glucose utilization are unknown. Adult male and female rats were adapted to sublethal hyperoxia, equivalent to 83% oxygen at sea level, or air for 7 days. Lung RNA and protein increased in hyperoxia (197 and 57%, respectively), whereas total DNA was unchanged. In hyperoxia, lung total hexokinase (HK) activity increased threefold, and mRNAs for HK-II and -III were specifically upregulated. HK-I mRNA was unchanged. mRNAs for HK-II and -III gradually increased during the first 72 h in hyperoxia. HK-II mRNA was significantly elevated at 72 h, preceding changes in lung cell populations. Although virtually absent in air, HK-II activity was highly expressed in hyperoxia. Among lung glucose transporters, specific expression of mRNAs for GLUT-4 (insulin dependent) and sodium-glucose cotransporter-1 was decreased, whereas that for GLUT-1 was minimally changed. Adaptation to hyperoxia involves coordinated changes in gene expression for the proteins regulating pulmonary glucose transport.

Glucose modulates cell death due to normobaric hyperoxia by maintaining cellular ATP.

To determine whether glucose depletion is a principal determinant of hyperoxic cell death in vitro, human lung epithelial-like cells (A549) were exposed to hyperoxia (95% O2) in either 10, 30, or 50 ml of medium (Ham's F-12K). Glucose was depleted in the medium after 36, 60, or 96 h, respectively. Medium lactate dehydrogenase (LDH) activity increased only after glucose was depleted. To confirm that glucose depletion was critical to cell death, cells exposed to 95% O2 were supplemented with glucose at regular intervals to reestablish initial medium glucose concentrations. Other cells received no supplements. Without supplementation, glucose was depleted within 48 h, followed within 12 h by an almost complete loss of cell ATP and elevated medium LDH activity. Glucose-supplemented cells appeared normal microscopically and did not release LDH activity despite an extracellular pH of 6.5 due to fermentation. Additional experiments at sea-level pressure confirmed that glucose supplementation prevents extensive cell death in hyperoxia in cultured A549 cells.

Effects of dietary copper deficiency on relative food intake and growth efficiency in rats.

Weanling male rats were fed either copper-adequate (CuA) or copper-deficient (CuD) diets ad lib for 20 wk. A pair-fed group was also studied. Two relative food intake indices (RFIs) and a growth efficiency (GE) index were calculated. Body weights of the CuD group became reduced compared to the CuA group starting the sixth week of the study. Copper deficiency was found to affect both of the RFI indices and the GE index in a time-dependent manner. RFIs and GE were reduced early, but recovered towards the control values during the last half of the experimental period. At the end of 20 wk, organ copper content and serum ceruloplasmin was reduced in the CuD group. Dietary copper deficiency reduces growth rate through effects both on food intake and efficiency of food utilization for growth.

Isolated hearts from copper-deficient rats exhibit improved postischemic contractile performance.

Dietary copper deficiency affects a number of enzymes, the function of which may influence the outcome of myocardial ischemia-reperfusion injury. Male weanling rats were fed diets that were adequate (> 5 mg/kg) or deficient (< 1 mg/kg) in copper. After 4 wk, the rats' hearts were isolated and used to study the effects of ischemia-reperfusion on intraventricular developed pressure (DevP), positive and negative rates of intraventricular pressure change (+dp/dt and -dp/dt) and release of lactate dehydrogenase and creatine kinase from the heart. The ischemia-perfusion protocol included a 15-min equilibration period, 30 min of warm, total ischemia and reperfusion for 30 min. Preischemic hearts from copper-deficient rats produced lower DevP than hearts from copper-adequate rats at all levels of preload. However, postischemic recovery of DevP was significantly greater in the hearts of the copper-deficient group. Furthermore, the postischemic patterns of lactate dehydrogenase and creatine kinase release in the two groups were significantly different. These findings indicate that, although dietary copper deficiency adversely affects a number of enzymatic systems, the functional recovery of hearts subjected to ischemia-reperfusion injury is improved when the diet is restricted in copper.

Distributions of elements in the human retinal pigment epithelium.

Distributions of elements above the atomic number of sodium were mapped in the retinal pigment epithelia of eight human eyes. X-ray energy spectra and maps were collected from cryofixed, freeze-dried, and epoxy-embedded tissues using energy-dispersive x-ray microanalysis. All eyes had high concentrations of phosphorus in the nuclei of retinal pigment epithelial cells. Melanosomes were rich in sulfur, zinc, calcium, and iron. Lipofuscin and cytoplasm contained only phosphorus and sulfur in detectable amounts. Drusen, when present, contained phosphorus and calcium. Six eyes had a prominent aluminum peak recorded from melanosomes, nuclei, and Bruch's membrane. In one pair of 90-year-old eyes, small, electron-dense deposits surrounded many melanosomes and contained mercury and selenium. Retinal pigment epithelial melanosomes may bind and accumulate metals and other potentially toxic ions over time, preventing them from reaching the neural retina.

Conditioned medium-mediated photoreceptor differentiation in retina from embryonic rd chickens.

Retina cells from 8-day rd (retinal degenerate) chicken embryos were cultured in media supplemented with optic lobe conditioned medium (OLCM). The morphogenesis of rd photoreceptors is being described. Several differentiating photoreceptors depicted a membranous sac protruding from the apical end of the cell as revealed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Such structures were found mostly in 8-day-old cultures supplemented with OLCM and were absent in controls. They resembled rudimentary outer segments emanating from a cilium at the apical inner segments. TEM showed a few stacks of free floating disks within the membranous structure which was suggestive of a rudimentary outer segment development. The possible neurotrophic effect of OLCM on rd retina photoreceptor differentiation is being suggested.

Scanning electron microscopy of human drusen.

Drusen are small, yellowish deposits that form under the retinal pigment epithelium (RPE) with senescence or under certain pathological conditions. The present study examined these structures under the scanning electron microscope. Tissue came from four eyes of 66- and 75-year-old donors who demonstrated widespread drusen of the posterior fundus noted on postmortem examination. Specimens were prepared by either detaching the RPE from Bruch's membrane, or by cryofracturing the tissue for cross-sectional views. Drusen appeared to be composed of irregularly-shaped globular masses and of distinct spherical entities. These particles varied greatly in size, and were situated between the RPE's basement membrane and the outer collagenous zone of Bruch's membrane. Surface views showed drusen components to be embedded in the collagenous zone of Bruch's membrane. Pits corresponding to the sizes of the globular and spherical masses imply that some particles were lost during tissue processing. Fractured cross sections of the irregularly-shaped globular masses revealed a homogeneous, granular matrix with no distinct ultrastructural features, while some of the fractured spherical components demonstrated an internal core. Transmission electron microscopic analysis on the same specimens that were subjected to SEM corroborated these observations. Analytical x-ray microanalysis (Kevex, Foster City, CA) in the SEM revealed major peaks for calcium and phosphorous in the crystalline spherical components, and primarily potassium and chloride in the globular structures.

Scanning electron microscopic observations on differentiation and maintenance of photoreceptor cells in vitro.

Scanning electron microscopy was performed on cell cultures of embryonic and post-hatch chick retinas co-cultured with optic lobe neurons or in medium that had been pre-conditioned with optic lobe cells. The culture medium consisted of Eagles Basal Medium supplemented with glucose, fetal calf serum, glutamine and bicarbonate. Application of colchicine (5 micrograms/ml) to the cultures, encouraged the dissociation of retinal cell rosettes and optic lobe neuron aggregates, thereby allowing us to examine differentiation of isolated photoreceptor cells. Over time, developing photoreceptor cells gradually took on the morphological characteristics of rods and cones in the post-hatch chick: cells were polarized having a single neurite on one end of the cell and inner and outer segment-like structures on the other end. Developing cone cells elaborated an oil droplet and filopodial-like processes at the apical end of the inner segment. The latter may correspond to the calyceal processes which normally envelop the basal 1/3 of the outer segment. The sequence of events noted in vitro parallel those previously reported in vivo.

Scanning electron microscopic analysis of intraocular ossification in advanced retinal disease.

Chicken eyes from congenic blind (rd/rd) animals showing early, intermediate, and final stages of ossification, similar to the phthisis bulbi condition in man, were examined using scanning and transmission electron microscopy as well as light microscopy and X-ray microanalysis. Early stages of ossification were devoid of mineralized calcium apatite while intermediate and end stages of the disorder contained large amounts of calcium and phosphorus. This process resulted in metaplastic bone formation. An intact Bruch's membrane appeared to separate the choroid from the degenerated pigment epithelium and the developing bone suggesting that its possible origin was metaplasia of the retinal pigment epithelium and the degenerated sensory retina. The end-stage ossification resulted in "phthisic bone" formation which completely filled the vitreous cavity in a manner very similar to the human condition of phthisis bulbi.

Macular degeneration in a community of rhesus monkeys. Ultrastructural observations.

The eyes of 29 aged adult, (mean age, 20 years) rhesus monkeys were examined for the presence of age-related macular degeneration (AMD). This sample represented approximately 25% of the aged population in the seminatural colony at the Caribbean Primate Research Center (CPRC) of the University of Puerto Rico. Approximately 75% of the animals examined had drusen in the posterior pole. Ultrastructural analysis was used to determine whether the pathologic alteration of Bruch's membrane and drusen in the colony resembled those noted in aged or AMD-afflicted human retinas. There were abnormalities in all layers of Bruch's membrane. Deposits of heterogeneous material, comprised of membranous, granular, and cellular components, were seen in both the inner collagenous zone (ICZ) and the outer collagenous zone (OCZ). Accumulation of this drusenoid material in the ICZ produced a scalloping of the basal border of the retinal pigment epithelium (RPE). Dense bodies were seen in both Bruch's membrane and RPE cytoplasm near the basal infoldings. Cytoplasmic processes, as well as whole cells, were seen with regularity within the drusenoid material. In one case there was a cell with a basement membrane crossing the middle elastic layer of Bruch's membrane. These changes are consistent with those reported in human aging and AMD. Aged individuals in this colony appear to be predisposed to macular degenerative changes and may prove to be an invaluable animal model for studying AMD in humans.

Tunicamycin-induced dysgenesis of retinal rod outer segment membranes. I. A scanning electron microscopy study.

Incubation of Xenopus retinas with tunicamycin has been shown to block the glycosylation of opsin, the rod visual pigment apoglycoprotein, with concomitant accumulation of vesicular membrane material in the compartment between the rod inner and outer segments (i.e., the intersegmental space) (Fliesler et al, J Cell Biol 100:574-587, 1985). To further assess the morphology, topology, and cellular origin of this membranous material, Xenopus retinas were incubated in the presence or absence of tunicamycin and the photoreceptor cells were examined by scanning electron microscopy. The material which accumulated in the intersegmental space appeared to be a complex of membranous structures consisting of cisternae with numerous tubular projections, as well as closely associated individual vesicles of various sizes. This tubulo-vesicular material was exclusively associated with the basal surface of the rod outer segment. The connecting cilium, periciliary ridge complex, and the apical surface of the rod inner segment were devoid of such membrane material. Nascent (open) discs (i.e., evaginations of the plasma membrane at the base of the outer segment) often observed in control retinas were not present in tunicamycin-treated tissue. These results support the hypothesis that the membranous material which accumulates in the intersegmental space of rods in tunicamycin-treated retinas represents incompletely and aberrantly formed nascent disc membranes. The formation of this material is apparently a consequence of a deficiency in newly synthesized, asparagine-linked membrane glycoconjugates (e.g., the oligosaccharide chains of opsin) at the site of disc assembly.

Ultrastructural changes in the retinal pigment epithelium of congenitally blind chickens.

Pathological changes in the retinal pigment epithelium (RPE) in a strain of chickens having hereditary blindness and retinal degeneration were described at the ultrastructural level. Photoreceptors in the retinal degenerate (rd) chicken had previously been noted to degenerate within a week after hatching. Affected chicks have neural retinas that are morphologically comparable to normal animals prior to that time despite an obvious lack of vision. In the present study, no pathological changes were noted in rd RPE prior to the time of photoreceptor degeneration. However, while mitochondria in the normal chick's RPE underwent diurnal changes in morphology within a few days of hatching, pleomorphic or ring mitochondria were not seen with high frequency in the rd chick. After photoreceptors began degenerating, changes were seen in the rd RPE. By 2 weeks of age, we noted a reduction in the depth and number of basal infoldings, an increase in number and size of autophagic vacuoles and large whorls of membranous material within rd RPE cells. Membranous debris and what appeared to be broken off outer segments were seen in the subretinal space at that time. These phenomena became more prominent and prevalent with time. In 3-4 week old specimens, nearly intact outer segments were seen within RPE cytoplasm. At the same time very few intact outer segments were present on photoreceptors. After this time degenerative changes were seen in the RPE: a thinning of cells (apical to basal cell width), spreading out of cells (increased distance between intercellular junctional complexes), hypopigmentation of cells and presence of free cells in the sub-retinal space. Some RPE cells appeared in a rounded up configuration, bulging into the subretinal space and making junctional complexes with remaining photoreceptor inner segments or Mueller cell processes. Many RPE cells did appear to maintain their phagocytic abilities, as evidenced by presence of many microvilli and pinocytotic vacuoles in the apical cytoplasm.

Hereditary retinal degeneration in the Rhode Island Red chicken: ultrastructural analysis.

An electron microscopic analysis of photoreceptor degeneration in a congenitally blind strain of chickens is presented. The mutation was named rd, meaning 'retinal degeneration'. Although the chicks were behaviorally and electrophysiologically blind at the time of hatching, their retinas appeared morphologically comparable to normal chicks at this stage. Both groups had well-developed photoreceptor cells, although outer segments were typically disoriented or misaligned. In the normal, and to some degree in the rd, retina, outer segments became organized within the first week posthatching. In the rd retina at that time, however, more outer segments were disorganized and disoriented. Disc-like membranes were also seen in some inner segments. Many photoreceptors had distended inner segment tips containing a granular cytoplasm. Membraneous debris was present in the subretinal space. Over the next 2-3 weeks there was a reduction in number of inner segments, outer segments and photoreceptor nuclei of both rods and cones. Photoreceptor cell bodies in the outer nuclear layer were replaced by Mueller cell processes. By the end of the second month, a larger cone:rod ratio was apparent, and a large proportion of the remaining cones were double cones. Intact outer segments were rarely seen at that time. Few and sporadic cone cells, identified by a pale-staining oil droplet, were the predominant surviving photoreceptors by 6 months of age. At the later stages examined, the pigment epithelium (PE) appeared to be undergoing degenerative changes. A general thinning of cells and hypopigmentation of PE cells was apparent, although hyperpigmented, hypertrophied PE cells were also present which bulged into the subretinal space. Pigmented cells of unknown origin were also noted in the subretinal space at the later time points.