Lung oxygen consumption and mitochondria of alveolar epithelial and endothelial cells.

We examined oxygen consumption by lung slices and measured the volume density of mitochondria of granular pneumocytes, alveolar type I cells, and alveolar capillary endothelial cells in several species. We found that lung oxygen consumption (mu-1 02 times h-1 times mg DNA-1) varies inversely with the log of animal body weight and with the species alveolar diameter and directly with the species respiratory rate. The volume density of granular pneumocyte mitochondria show a direct linear correlation with the lung's oxygen consumption and the species respiratory rate, and an inverse linear correlation with the species alveolar diameter. The volume density of mitochondria in type I alveolar epithelial cells and capillary endothelial cells, considered together, did not differ in the two species studied (mouse and rat). We conclude that there are interspecies differences in oxygen consumption by lung cells and that granular pneumocytes contribute to these differences. We suggest that, at least part of these differences, are related to interspecies differences in surfactant secretory activity.     

Oxygen-induced changes in pulmonary phospholipids in the rat.

The composition and synthesis of alveolar and lung tissue phospholipids were investigated in normal and oxygen-poisoned rat lungs. Sixty-hour exposure to oxygen increased the total amount of phospholipids in the endobronchial extracts and lung tissue. Phosphatidyl glycerol was identified in both endobronchial extracts and lung tissue. The amount of unsaturated fatty acids in surfactant lecithin and phosphatidyl glycerol was slightly increased in oxygen-poisoned lungs whereas the composition of phospholipids in the endobronchial extracts was not affected by oxygen. After intraperitoneal administration of [32P]phosphate the specific activities of surfactant lecithin and phosphatidyl glycerol were clearly lower in oxygen-treated animals whereas the specific activities of lung tissue lecithin and phosphatidyl glycerol remained unaffected. The synthesis of lecithin from [14C]methionine through N-methyltransferase pathway was markedly depressed in lung slices but increased in liver tissue taken from oxygen-poisoned rats and incubated under oxygen indicating a difference between lung and liver methyltransferase enzymes. In conclusion, the present work suggests impaired synthesis and removal of alveolar phospholipids in oxygen-poisoned rats.     

The effect of DL-2-bromopalmitate on the utilization of palmitic acid by rat granular pneumocytes.

The effect of DL-2-bromopalmitate (BrPA), an analogue of palmitic acid (PA), on the utilization of this fatty acid by rat lungs was investigated by a combination of anatomic and biochemical methods. The experiments were performed in vitro on two types of preparations, isolated perfused lungs and lung slices. In the isolated lung preparation the substrate reached the lung via the capillaries, in lung slices via the alveolar epithelium. Electron microscope autoradiography showed that BrPA depressed uptake of PA by granular pneumocytes. Radioactivity recovered by tissue analysis and capture of CO2 established that PA oxidation and incorporation into phospholipids and triglycerides was depressed by BrPA. A close correlation was found between the reduction in radioactivity in phospholipids and the grain density over lamellar bodies. The study shows that BrPA reversibly interferes with the uptake and utilization of long chain fatty by granular pneumocytes. BrPA appears as a useful tool to study palmitate metabolism and surfactant production by the lung.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle

Cycloheximide (500 micrograms/ml) rapidly arrests cleavage, spindle assembly, and cycles of an M-phase-specific histone kinase in early Xenopus blastulae. 2 h after cycloheximide addition, most cells contained two microtubule asters radiating from perinuclear microtubule organizing centers (MTOCs). In contrast, blastomeres treated with cycloheximide for longer periods (3-6 h) contained numerous microtubule asters and MTOCs. Immunofluorescence with an anticentrosome serum and EM demonstrated that the MTOCs in cycloheximide-treated cells were typical centrosomes, containing centrioles and pericentriolar material. We conclude that centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. In addition, these observations suggest that Xenopus embryos contain sufficient material to assemble 1,000-2,000 centrosomes in the absence of normal protein synthesis.     

Effect of cycloheximide on protein biosynthesis in rat liver

1. The liver ribosomes of rats given cycloheximide by intraperitoneal injection incorporate less amino acid into protein than ribosomes from control rat liver when they are incubated in vitro with excess of Sephadex-treated cell sap. The effect is rapid, marked and persistent. 2. Cell sap from liver of cycloheximide-treated animals is inhibitory but the inhibition can be relieved almost entirely by treating the cell sap with Sephadex. No damage has been done to the cell-sap factors: it is suggested that the dissolved cycloheximide in the cell sap causes the inhibition. 3. Cycloheximide added in vitro inhibits amino acid incorporation into protein in the presence or absence of polyuridylic acid. The inhibition is lessened by addition of excess of cell sap but is not abolished. 4. The differences between these results and those obtained with mouse liver (Trakatellis, Montjar & Axelrod, 1965) might arise because of species differences in sensitivity to the drug.     

Evaluation of intra-amniotic surfactant administration for lung maturation in preterm sheep

We aimed to evaluate the effects of intra-amniotic surfactant administration on alveolar lecithin/sphingomyelin ratio, density of type II pneumocytes, and fetal lung function in preterm merino sheep. Pregnant ewes at 119 days gestation either received 200 mg intra-amniotic surfactant (n=4) or saline solution (n=4). After 24 h, the lambs were delivered by hysterotomy and mechanically ventilated. Lecithin/sphingomyelin ratios in alveolar fluid, inflating pressure–volume relationships, and type II pneumocyte counts in histological specimens were compared among the groups. All of the lambs completed the protocol. Mean lecithin/sphingomyelin ratio increased significantly in amniotic (p=0.03) and alveolar fluid (p=0.03) samples of surfactant-treated animals. Lung function in terms of pressure–volume curves did not differ between two groups. Type II pneumocyte density tended to be higher (p=0.057) after intra-amniotic surfactant administration. Single-dose treatment with intra-amniotic surfactant seems to improve amniotic and alveolar lecithin/sphingomyelin ratio questionably by increasing alveolar type II cells. Pressure–volume relationships from inflation of the lungs might be unaltered with intra-amniotic surfactant treatment.     

Control of cell protein catabolism in rat liver. Effects of starvation and administration of cycloheximide.

1. The loss of liver protein occurring in rats starved for 24 h was largely prevented by the administration of repeated doses of cycloheximide, an inhibitor of protein synthesis. Similar effects were produced on tubulin, a 'fixed' liver protein. 2. Starvation accelerated, whereas cycloheximide markedly lowered, the rate of protein radioactivity decay after labelling with [3H]valine or [14C]bicarbonate, indicating that changes in catabolic rates played an important role in the above regulations of liver protein mass. 3. The total activity of several lysosomal hydrolases showed little change in livers of starved rats, but a marked progressive decline developed after the administration of cycloheximide, particularly in the activities of cathepsins B, D and L as well as acid ribonuclease. There was no evidence that these changes might be due to endogenous inhibitors (at least for cathepsin B activity, which fell to less than 30% of the control values) or enzyme leakage into the bloodstream; rather, plasma beta-galactosidase and beta-N-acetylglucosaminidase activities fell progressively during the cycloheximide treatment. 4. Endogenous proteolytic rates, measured in vitro by incubating subcellular preparations from livers prelabelled in vivo with [3H]valine, were markedly decreased in cycloheximide-treated animals. 5. The osmotic fragility of hepatic lysosomes, appreciably enhanced in starved animals, after cycloheximide treatment was found to be even lower than in fed controls. 6. The present data are consistent with the view that in starved animals the loss of liver protein is mostly accounted for by increased breakdown, due, in part at least, to enhanced autophagocytosis. 7. Cycloheximide largely counteracted these effects of starvation, altering the liver from being 'poised' in a proteolytic direction to a protein-sparing condition. The present data suggest that, besides suppression of the autophagic processes, a decrease in the lysosomal proteolytic enzyme system may also play a role in this regulation, and they seem to provide further circumstantial evidence for the existence of co-ordinating mechanisms between protein synthesis and degradation.     

Mechanics of edematous lungs.

Using the parenchymal marker technique, we measured pressure (P)-volume (P-V) curves of regions with volumes of approximately 1 cm3 in the dependent caudal lobes of oleic acid-injured dog lungs, during a very slow inflation from P = 0 to P = 30 cmH2O. The regional P-V curves are strongly sigmoidal. Regional volume, as a fraction of volume at total lung capacity, remains constant at 0.4-0.5 for airway P values from 0 to approximately 20 cmH2O and then increases rapidly, but continuously, to 1 at P = approximately 25 cmH2O. A model of parenchymal mechanics was modified to include the effects of elevated surface tension and fluid in the alveolar spaces. P-V curves calculated from the model are similar to the measured P-V curves. At lower lung volumes, P increases rapidly with lung volume as the air-fluid interface penetrates the mouth of the alveolus. At a value of P = approximately 20 cmH2O, the air-fluid interface is inside the alveolus and the lung is compliant, like an air-filled lung with constant surface tension. We conclude that the properties of the P-V curve of edematous lungs, particularly the knee in the P-V curve, are the result of the mechanics of parenchyma with constant surface tension and partially fluid-filled alveoli, not the result of abrupt opening of airways or atelectatic parenchyma.     

Muscle growth and protein synthesis in the puparia of Calliphora vomitoria

The effects of cycloheximide on the development of the dorsal longitudinal flight muscle of 3- to 5-day-old puparia of Calliphora vomitoria have been investigated. One μg of cycloheximide injected into the puparia reduced the incorporation of ¹⁴C phenylalanine and lysine into protein to 5 and 8 per cent of their normal levels. The cycloheximide was found to have produced its maximum effect within 2 hr of its injection and increasing the concentration did not further depress the amount of amino acid incorporation. The sixth dorsal longitudinal muscles continued to increase in length after the injection of cycloheximide and the elongation of the muscle fibres was accompanied by an increase in protein content in normal and cycloheximide-treated animals. An injection of colchicine (which is believed to disrupt microtubules) immediately halted muscle growth. Electron microscopy of the muscle fibre revealed that fibres from cycloheximide-treated animals contained myofilaments, although there were some differences in myofilament structure between normal and treated animals. The formation of the muscle fibres in the absence of protein synthesis is discussed.     

Requirement of protein synthesis in the initiation of meiosis and other nuclear changes in conjugation of Blepharisma.

In conjugation of Blepharismajaponicum, cell contact between complementary mating types induces meiosis and other nuclear changes. How long the cells must be in contact in order to be induced to undergo these nuclear changes (activated) can be ascertained by surgically separating the united cells at different times after the onset of cell union and then examining the occurrence of the nuclear changes. Applying this technique to cycloheximide-treated cells, we investigated the role of protein synthesis in the activation. Cycloheximide was used at the concentration which was found to inhibit most incorporation of amino acid into protein in this ciliate. Newly formed conjugant pairs were incubated with and without cycloheximide, washed free of the inhibitor and surgically separated. Although untreated controls were activated in 1.8 h after cell-cell contact, no activation was observed in cycloheximide-treated cells after 5 h of contact. Removal of cycloheximide from the paired cells resulted in an activation delayed by the interval of exposure time to the inhibitor. If the pairs were first incubated in normal medium and then exposed to cycloheximide, operated, activated cells appeared and increased very slowly (activation rate, about $\text{1}\text{10}$ of the control). Protein synthesis is therefore required for the initiation of meiosis and other nuclear changes. We propose that heterotypic cell union induces and maintains the synthesis of a protein, whose accumulation to a certain threshold is required for activation.     

Effects of cycloheximide on protein degradation and gluconeogenesis in the perfused rat liver.

Cycloheximide at concentrations above 18 muM produced a 93% inhibition of total protein synthesis measured by valine incorporation in the perfused rat liver. Rates of protein degradation were estimated by perfusing livers prelabeled in vivo with L-[1-14C]valine with medium containing 15 mM L-valine. Thus labeled valine released from liver protein during perfusion was greatly diluted and reincorporation of label was minimized. Cycloheximide at 18 muM inhibited protein degradation by over 60%, after a delay of 15-20 min. Associated with these effects were dose-dependent increases in the rates of glucose and urea production. Glucose production increased 3 fold, from 0.54 +/- 0.07 in control to 1.85 +/- 0.24 mumol/min/100 g rat in cycloheximide-treated livers. Urea production increased from 0.24 +/- 0.02 to 0.62 +/- 0.06 mumol/min/100 g rat. No changes in liver glycogen or cyclic AMP content were seen. The data suggest that inhibition of protein synthesis provides an increased availability of intra-cellular amino acids and that many of these are rapidly degraded, yielding urea and glucose. This is supported by the fact that intracellular alanine levels were significantly increased following cycloheximide treatment. It is possible that the inhibition of protein degradation by cycloheximide is due to altered intra-cellular pools of amino acids or their metabolites.     

On defining total lung capacity in the mouse.

Maximal lung volume or total lung capacity in experimental animals is dependent on the pressure to which the lungs are inflated. Although 25-30 cm H2O are nominally used for such inflations, mouse pressure-volume (P-V) curves show little flattening on inflation to those pressures. In the present study, we examined P-V relations and mean alveolar chord length in three strains (C3H/HeJ, A/J, and C57BL/6J) at multiple inflation pressures. Mice were anesthetized, and their lungs were degassed in vivo by absorption of 100% O2. P-V curves were then recorded in situ with increasing peak inflation pressure in 10-cm H2O increments up to 90 cm H2O. Lungs were quickly frozen at specific pressures for morphometric analysis. The inflation limbs never showed the appearance of a plateau, with lung volume increasing 40-60% as inflation pressure was increased from 30 to 60 cm H2O. In contrast, parallel flat deflation limbs were always observed, regardless of the inflation pressure, indicating that the presence of a flat deflation curve cannot be used to justify measurement of total lung capacity in mice. Alveolar size increased monotonically with increasing pressure in all strains, and there was no evidence of irreversible lung damage from these inflations to high pressures. These results suggest that the mouse lung never reaches a maximal volume, even up to nonphysiological pressures >80 cm H2O.     

An apparatus for the measurement of lung volume and compliance in mice

Pressure-volume (P-V) curves and total lung capacity (TLC) were measured in excised lung of mice using a water manometer and a closed system in which the humidity and temperature were controlled. In pathogen-free mice there are no significant differences in elastic properties of these lungs in relation to their age. The measured TLC in those normal mice was approximately 2.9 ml. This relatively simple apparatus which allows one to make sensitive and accurate measurements of pulmonary function in mice and other small animals.     

The role of protein synthesis in regulation of oestradiol-17 beta in the pro-oestrous hamster

Injection (s.c.) of 2 mg cycloheximide at 14:00 h on the day of pro-oestrus prevented the normal rise in serum progesterone and significantly lowered progesterone levels at 15:00 h. Values then rose but only to approximately half of the control values between 16:00 h and 19:00 h. Oestradiol levels also decreased drastically by 15:00 h but were significantly higher in cycloheximide-treated animals until 19:00 h. FSH and LH concentrations were not affected when cycloheximide was given at 14:00 h but treatment at 10:00 h resulted in generally lower values. Animals treated with cycloheximide at 14:00 h failed to ovulate (N = 9), but simultaneous injection of 50 micrograms progesterone restored ovulation in 50% of the treated animals. In contrast, hamsters injected with cycloheximide at 10:00 h ovulated the next morning, suggesting that protein synthesis essential for ovulation is limited to the first 4-5 h after the release of LH.     

Ubiquitin depletion as a key mediator of toxicity by translational inhibitors

Cycloheximide acts at the large subunit of the ribosome to inhibit translation. Here we report that ubiquitin levels are critical for the survival of Saccharomyces cerevisiae cells in the presence of cycloheximide: ubiquitin overexpression confers resistance to cycloheximide, while a reduced ubiquitin level confers sensitivity. Consistent with these findings, ubiquitin is unstable in yeast (t(1/2) = 2 h) and is rapidly depleted upon cycloheximide treatment. Cycloheximide does not noticeably enhance ubiquitin turnover, but serves principally to block ubiquitin synthesis. Cycloheximide also induces UBI4, the polyubiquitin gene. The cycloheximide-resistant phenotype of ubiquitin overexpressors is also characteristic of partial-loss-of-function proteasome mutants. Ubiquitin is stabilized in these mutants, which may account for their cycloheximide resistance. Previous studies have reported that ubiquitin is destabilized in the absence of Ubp6, a proteasome-associated deubiquitinating enzyme, and that ubp6 mutants are hypersensitive to cycloheximide. Consistent with the model that cycloheximide-treated cells are ubiquitin deficient, the cycloheximide sensitivity of ubp6 mutants can be rescued either by ubiquitin overexpression or by mutations in proteasome subunit genes. These results also show that ubiquitin wasting in ubp6 mutants is proteasome mediated. Ubiquitin overexpression rescued cells from additional translational inhibitors such as anisomycin and hygromycin B, suggesting that ubiquitin depletion may constitute a widespread mechanism for the toxicity of translational inhibitors.     

Covalently closed circular DNAs of murine type C retrovirus: depressed formation in cells treated with cycloheximide early after infection.

Formation of viral closed circular supercoiled DNA duplexes and production of progeny virus were both inhibited in cultured mouse cells treated with cycloheximide in the first 4 h of type C retrovirus infection. With different doses of cycloheximide to cause different degrees of inhibition, the number of viral supercoiled DNA duplexes detected in the cells at 11 h showed an apparent correlation with the amount of progeny virus produced in the 12- to 22-h period of infection. A slight accumulation of the full-genome linear duplex and an open circular duplex of viral DNA intermediate was observed in the cycloheximide-treated cells. Cycloheximide given to the cells during the time of conversion of viral DNA from linear to supercoiled duplex forms (6 to 11 h after virus inoculation) did not inhibit the conversion. These kinetic data suggest that a cycloheximide-sensitive metabolic process, probably early viral protein synthesis, is required for retrovirus replication and supercoiled viral DNA formation in the cell.     

Interaction of cycloheximide and light on chlorophyll content and chloroplast ultrastructure inBrassica campestris

Cycloheximide retarded the loss of chlorophyll from detached komatsuna (Brassica campestris cv. Komatsuna) leaves during incubation in the dark but promoted its loss in light. Cycloheximide-induced chlorophyll bleaching in light was prevented by some active oxygen scavengers. Chloroplast envelopes of cycloheximide-treated leaves incubated in both the dark and light were destroyed within 48 h. The grana of cycloheximide-treated leaves incubated in light were dilated.