Relationships between hepatic melanogenesis and respiratory conditions in the newt, Triturus carnifex

The Kupffer cells (melanomacrophages) in the livers of lower vertebrates contain varying quantities of melanin according to the season. Specimens of Triturus carnifex raised for 2 months at 6 degrees C and then transferred to water at 22 degrees C show a rapid increase in the hepatic accumulation of the pigment. The Kupffer cells make up more than one fourth of the liver mass in chlorbutol-anesthetized animals isolated for 6-7 hr in hypoxic water at 18 degrees C (to bring the oxygen content in a 620-mL respiratory chamber from 1.1 ppm to 0.0). Thus, hepatic melanin is synthesized when the newt's oxygen supply is inadequate to meet its metabolic needs; melanogenesis, however, requires the presence of oxygen and does not occur in anesthetized specimens immersed in a totally anoxic fluid such as paraffin oil. The intraperitoneal injection prior to hypoxic treatment of 1 mg/g of body weight of kojic acid (inhibitor of the enzyme tyrosinase which catalyzes melanin synthesis) blocks melanogenesis and doubles oxygen consumption. The combination of hypoxia and tyrosinase inhibition causes permanent damage to essential functions of the nervous system, while hypoxic treatment alone has no irreversible consequences. The genic expression of tyrosinase in hypoxia appears to be a physiological response aimed at prolonging survival time in anaerobiosis by lowering the metabolic level; melanin would be an inert subproduct of this function.     

Thyroid and hypoxic stress in the newt Triturus carnifex

When specimens of the newt Triturus carnifex, under anaesthesia by submersion in a 0.2% chlorbutol solution for 25 min, are isolated in a respiratory chamber at 18 degrees C containing water with only 1.3 ppm of oxygen, they consume the oxygen completely in about 3 hr, but they can stay alive for many more hours and wake up with no apparent exterior consequences. Hypoxia induces rapid onset of hepatic steatosis and melanosis, as well as a controlled haemolytic process involving a pool of red blood cells of the same order of size as that held as a reserve in the spleen by animals in an aerial habitat. At the origin of the phenomena is an intense response by the hypophysis, histologically detectable 1 hr from the onset of treatment and confirmed 2 hr later by a highly significant increase in the plasma thyroidstimulating hormone (TSH) concentration compared with the controls (41.5 +/- 13.7 microU/L vs. 15.5 +/- 6.2; P < 0.005). The thyroid follicles react by reabsorbing their colloid, but instead of an increase in the plasma free T3 and T4 concentrations, fT3 falls significantly (1.5 +/- 0.3 pg/mL vs., the 2.4 +/- 0.7; P < 0.05), whereas fT4 remains stationary (4.0 +/- 0.5 pg/mL vs. 4.6 +/- 0.8; N.S.). After 6 hr, the plasmatic TSH concentration is still higher than in the controls (27.0 +/- 3.0 microU/L vs. 15.5 +/- 6.2; P < 0.05), whereas fT3 and fT4 remain stable (1.5 +/- 0.3 and 4.4 +/- 0.5 pg/mL, respectively). If T3 or T4 labelled with 125I is administered prior to hypoxia, after 6 hr of treatment the radioactivity is found to be limited exclusively to the liver and kidney; the thyroid, gall bladder and gut result negative, and this does not agree with hypotheses of hormone inactivation by deiodination, sulphation or glucuronidation. This apparently peculiar endocrine path has not been observed in previous studies on hypoxia in vertebrates, because the experiments were always designed to analyse plasma hormone levels after at least 24 hr of hypoxia or during chronic treatments, losing the most interesting phases of the endocrine response. The possibility that the hypoxic newt possesses alternative or complementary metabolic pathways to anaerobic glycolysis to sustain steatogenesis and melanogenesis and maintain the same cardiac activity as the controls is briefly discussed.     

N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia

Exposure to chronic hypoxia results in hypoxic pulmonary hypertension (HPH). In rats HPH develops during the first two weeks of exposure to hypoxia, then it stabilizes and does not increase in severity. We hypothesize that free radical injury to pulmonary vascular wall is an important mechanism in the early days of the hypoxic exposure. Thus antioxidant treatment just before and at the beginning of hypoxia should be more effective in reducing HPH than antioxidant therapy of developed pulmonary hypertension. We studied adult male rats exposed for 4 weeks to isobaric hypoxia (F(iO2) = 0.1) and treated with the antioxidant, N-acetylcysteine (NAC, 20 g/l in drinking water). NAC was given "early" (7 days before and the first 7 days of hypoxia) or "late" (last two weeks of hypoxic exposure). These experimental groups were compared with normoxic controls and untreated hypoxic rats (3-4 weeks hypoxia). All animals kept in hypoxia had significantly higher mean pulmonary arterial blood pressure (PAP) than normoxic animals. PAP was significantly lower in hypoxic animals with early (27.1 +/- 0.9 mmHg) than late NAC treatment (30.5 +/- 1.0 mmHg, P < 0.05; hypoxic without NAC 32.6 +/- 1.2 mmHg, normoxic controls 14.9 +/- 0.7 mmHg). Early but not late NAC treatment inhibited hypoxia-induced increase in right ventricle weight and muscularization of distal pulmonary arteries assessed by quantitative histology. We conclude that release of free oxygen radicals in early phases of exposure to hypoxia induces injury to pulmonary vessels that contributes to their structural remodeling and development of HPH.     

Zinc content of the gills of rainbow trout (S. gairdneri) after treatment with zinc solutions under normoxic and hypoxic conditions

Forty specimens of rainbow trout (54–127 g) were divided into 4 groups which were treated as follows: (a) normoxic clean water; (b) hypoxic clean water; (c) normoxic water with 10 ppm zinc for 10 h; (d) hypoxic water with 10 ppm zinc for 10 h. The zinc content was determined separately for each of the 4 gill arches on each side of the fish. Values for the zinc concentration were greater following the zinc treatments, but no significant difference between hypoxia and normoxia was observed. Differences in concentrations of zinc were found in different arches whether expressed per gram dry weight or per unit surface area of the secondary lamellae.     

Function of the hepatic melanogenesis in the newt, Triturus carnifex

Like the majority of lower vertebrates, the newt Triturus carnifex holds varying quantities of melanin and hemosiderin in the Kupffer cells of the liver. Following hypoxic treatment, the amount of these two pigments can increase to such an extent that they can occupy nearly a quarter of the surface of histological sections. A group of six specimens, anesthetised with chlorbutol, were subjected to hypoxic treatment by keeping them in a respiratory chamber containing degassed water under vacuum, with only 1.1 ppm of residual oxygen, until they had consumed the oxygen completely (4 hours, at a temperature of 18 degrees C). Using hematological and histochemical techniques and computerised image analysis, it has been shown that hypoxic animals not only increase the extent of the melanic areas of the liver from about 5-7% to almost 24% compared to control groups kept under two different respiratory conditions (6 anesthetised specimens exposed to the air and 6 submerged in normoxic water), they also went through a remarkable hemolytic process to justify a parallel increase in hemosiderin deposits. Melanin was extracted from the liver by keeping fragments of the organ for one hour at 37 degrees C in an oxidising solution (20 mL of benzyl alcohol, 10 mL of acetone, 5 mL of 10% hydrogen peroxide, and 4 drops of concentrated ammonia solution), then quickly rinsing them in 50% acetone and subsequently letting them stand for 6 hours in 10 mL of distilled water alkalised to pH 12 with a drop of ammonia solution. The extract was then left to sediment at pH 2.5 and the black precipitate washed and dried under vacuum. Elemental and spectrophotometric analyses revealed a significant presence of purines in the melanic pigment. This phenomenon can be explained by the animals' need under hypoxic crisis to rapidly neutralise purines resulting from lysis of the nucleated red blood cells by introducing them into an inert molecular complex. A partial model of structure is proposed here. Synthesis of the mixed polymer is possible through the well-known capacity of ferrous iron to activate tyrosinase (the enzyme responsible for melanogenesis) even in the absence of DOPA.     

高原低氧胁迫对小鼠肝脏功能及基因表达的影响

低氧作为青藏高原最为特殊的环境因素之一,对高原动物的适应进化产生了深刻的影响。持续的低氧暴露会损伤肝脏功能,引起动物机体代谢紊乱,但连续低氧处理对子代肝脏的影响仍缺乏相关研究。本研究将成年小鼠转移至高原低氧环境 (海拔3 220 m) 饲养并繁殖,以常氧条件下饲养小鼠为对照,统计低氧处理小鼠 (低氧第0代) 及其子代 (低氧第1 ~ 5代) 生长数据,发现长期低氧暴露导致小鼠肝脏比重增加,肝细胞肿胀,肝索间红细胞浸润,并且子一代小鼠肝小叶出现脂肪变性。血液生化指标显示,相比于对照组 (常氧第0代),低氧第0代和低氧第1代的谷丙转氨酶和谷草转氨酶水平显著上升 (P < 0.05);血清白蛋白、球蛋白、总胆红素和总胆固醇水平在低氧第0代中下降,低氧第1代中上升 (P < 0.05)。空腹注射葡萄糖和胰岛素后低氧组小鼠的葡萄糖耐受能力和胰岛素敏感性显著减弱 (P < 0.05)。常氧第0代、低氧第0代及低氧第1代肝脏RNA-seq分析发现,低氧第0代和低氧第1代共有的459个差异基因显著富集在MAPK、细胞凋亡、脂质代谢和内质网等信号通路。本研究发现低氧胁迫对子代小鼠肝脏具有重要影响,此结果对肝脏低氧生理和高原肝脏疾病的研究具有重要的借鉴意义。     

Strategies of hypoxia and anoxia tolerance in cardiomyocytes from the overwintering common frog, Rana temporaria

Using ventricular cardiomyocytes of the common frog, Rana temporaria, we investigated the metabolic strategies employed by the heart to tolerate 4 mo of hypoxic submergence (overwintering) as well as acute bouts of anoxia. In contrast to what is observed for the whole animal, there was no change in oxygen consumption in cardiomyocytes isolated from normoxic frogs compared with those isolated from 4-mo hypoxic animals. Furthermore, cells from both normoxic and hypoxic frogs were able to completely recover oxygen consumption following 30 min of acute anoxia. From estimates of ATP turnover, it appears that frog cardiomyocytes are capable of a profound, completely reversible metabolic depression, such that ATP turnover is reduced by >90% of control levels during anoxia but completely recovers with reoxygenation. Moreover, this phenomenon is also observed in frogs that have been subjected to 4 mo of extended hypoxia. We found a significant increase in the stress protein, hsp70, after 1 mo of hypoxic submergence, which may contribute to the heart's remarkable hypoxia and anoxia tolerance and may act to defend metabolism during the overwintering period.     

Hypoxia-Induced Generation of Nitric Oxide Free Radicals in Cerebral Cortex of Newborn Guinea Pigs

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca⁺⁺-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO₂ = 21%, n = 5) or hypoxic (FiO₂ = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO₄.7H₂O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 ± 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca²⁺- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.     

Chronic hypoxia increases MCA contractile response to U-46619 by reducing NO production and/or activity.

Chronic hypoxia alters contractile sensitivity of isolated arteries to alpha-adrenergic stimulation and other agonists. However, most studies have been performed in thoracic aortas or other large vessels making little contribution to vascular resistance in their respective circulations. To determine the effect of chronic hypoxia on the vasoconstrictor response in a small, resistance-sized vessel, we studied second and third generation middle cerebral arteries (MCA; approximately 75-microm internal diameter before mounting). MCA were isolated from normoxic (inspired oxygen = 125 Torr) and hypoxic (8 wk at 3,960 m; inspired oxygen = 90 Torr) guinea pigs, and their vasoconstrictor responses were determined to the thromboxane mimetic U-46619 by using dual-pipette video microscopy. Arteries from hypoxic animals had greater contractile sensitivity to U-46619 compared with those of the normoxic animals (-log EC50 = 7.86 +/- 0.11 vs. 7.62 +/- 0.06, respectively, P < 0.05). Addition of the nitric oxide (NO) inhibitor nitro-L-arginine (200 microM) to the vessel bath eliminated the differences in contractile sensitivity between the MCA from the normoxic and chronically hypoxic groups. Supplementation with L-arginine in the drinking water sufficient to raise plasma L-arginine levels 41% reduced MCA contractile sensitivity to U-46619 in the normoxic group (-log EC50 = 7.22 +/- 0.31, P < 0.05 compared with the nonsupplemented normoxic group) but not in the chronically hypoxic group. These results show that chronic hypoxia increases the sensitivity of the MCA to the vasoconstrictor U-46619, likely because of a reduction in NO production and/or activity.     

Effects of hypoxia on animal burrow construction and consequent effects on sediment redox profiles

We conducted a laboratory experiment to investigate the effects of mild hypoxia on the burrowing behavior of three marine species (the hard clam Mercenaria mercenaria, the polychaete worm Neanthes virens, and the amphipod Leptocheirus plumulosus) and consequent effects on sediment redox profiles. Animals were introduced into defaunated sediment and allowed to burrow for four months at mildly hypoxic (2 mg l^− 1) and normoxic (7 mg l^− 1) dissolved oxygen (DO) levels. Sediment redox profiles were measured 10 times during the course of the experiment. At the end of the experiment, the sediment was imaged using computer-aided tomography to quantify burrow volume and location. For the three species, burrow volume remained constant over DO treatments, but amphipod and clam burrows were shallower in hypoxic treatments compared with normoxic treatments. Redox profile discontinuity (RPD) depth was shallower in hypoxic treatments compared with normoxic treatments for experiments without animals, indicating that water column oxygen concentration alone influences diffusion of oxygen into the sediment. Worms, but neither clams nor amphipods, increased the RPD depth relative to no-animal controls in both hypoxic and normoxic treatments, but the effect was greater in normoxic conditions. These results suggest that although hypoxia can reduce burrowing depth, infauna can still increase the depth to which oxygen penetrates the sediment, but not to the same degree as they would under normoxic conditions.     

Influence of hypoxia on bacteremia in the Dungeness crab, Cancer magister

We examined the possibility that decreased environmental oxygen can elevate the levels of indigenous bacteria in the hemolymph of Cancer magister. Crabs were exposed to air-saturated and hypoxic (50% air-saturation) water for 3 days and levels of culturable bacteria in hemolymph were measured every 24 h as the total number of colony-forming units (CFU) per milliliter of hemolymph. Bacteremia increased after 24 h of exposure to hypoxia and persisted for 72 h, whereas crabs exposed to normoxia had no measurable change in number of culturable bacteria. The predominant persistent bacteria in the hemolymph was isolated and identified by DNA sequence-based methods as Psychrobacter cibarus. Crabs were injected with P. cibarus or with buffered saline as a control after 3 h of hypoxia. Levels of culturable bacteria were significantly higher in hypoxic crabs than in normoxic ones (about 2500 versus 1000 CFU ml(-1) 80 min post-injection, respectively), and circulating levels of oxygen were significantly reduced in infected animals compared to uninfected ones after 48 h in hypoxia and after 72 h in air-saturated water post-injection. These data demonstrate that P. cibarius is present in Dungeness crabs, that environmental hypoxia can dramatically elevate levels of persistent bacteria, and that hypoxia in the presence of hemolymph bacteria may ultimately reduce immune and respiratory ability.     

Effect of neonatal hypoxia on the development of hepatic lipase in the rat

Increases in plasma lipids occur during hypoxia in suckling but not in weaned rats and may result from altered hepatic enzyme activity. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Hypoxia led to an increase in hepatic lipid content in the suckling rat only. Hepatic lipase was decreased to approximately 45% of control in 7-day-old rats exposed to hypoxia but not in hypoxic 35-day-old rats. Hypoxic suckling rats also had a 50% reduction in lactate dehydrogenase activity, whereas transaminase activity and CYP1A and CYP3A protein content were not different between hypoxic and normoxic groups. Additional rats were studied 7 and 14 days after recovery from hypoxic exposure from birth to 7 days of age; hepatic lipase activity had recovered to 85% by 7 days and to 100% by 14 days in the rats previously exposed to hypoxia. Administration of dexamethasone to neonatal rats to simulate the hyperglucocorticoid state found in hypoxic 7-day-old rats led to a moderate decrease ( approximately 75% of control) in hepatic lipases. Developmentally, in the normoxic state, hepatic lipases increased rapidly after birth and reached levels more than twofold that of the newborn by 7 days of age. Hypoxia delays the maturation of hepatic lipases. We suggest that the decrease in hepatic lipase activity contributes to hyperlipemia in the hypoxic newborn rats.     

Short Communication

Anaerobic metabolism and oxygen carrying-capacity of white shrimp ( Penaeus vannamei ) exposed to short term (three days) and long term (two weeks) moderate hypoxia (2-2.6 mg/L) was investigated. Glucose and lactate levels in hemolymph increased under both hypoxic conditions, indicating an activation of anaerobic pathways during the two-weeks exposure period. In muscle, no differences of glucose and lactate levels were observed between the control group and the exposed groups. In animals exposed to hypoxia for two weeks, hemocyanin and copper in hemolymph were higher than in animals under normoxic conditions or exposed for three days. These results indicate that an increase in oxygen carrying-capacity in shrimp is evident only after a sustained condition of hypoxia. Copper levels in the hepatopancreas decreased in both hypoxic conditions, suggesting a mobilization of copper stores for hemocyanin synthesis. These results indicate that penaeid shrimp can tolerate moderate hypoxic conditions by physiological adaptations, such as anaerobic metabolism and increased oxygen carrying-capacity. These adaptations require an adequate dietary supply of proteins and copper for hemocyanin synthesis and of carbohydrates for anaerobic metabolism.     

Short Communication

Anaerobic metabolism and oxygen carrying-capacity of white shrimp (Penaeus vannamei) exposed to short term (three days) and long term (two weeks) moderate hypoxia (2-2.6 mg/L) was investigated. Glucose and lactate levels in hemolymph increased under both hypoxic conditions, indicating an activation of anaerobic pathways during the two-weeks exposure period. In muscle, no differences of glucose and lactate levels were observed between the control group and the exposed groups. In animals exposed to hypoxia for two weeks, hemocyanin and copper in hemolymph were higher than in animals under normoxic conditions or exposed for three days. These results indicate that an increase in oxygen carrying-capacity in shrimp is evident only after a sustained condition of hypoxia. Copper levels in the hepatopancreas decreased in both hypoxic conditions, suggesting a mobilization of copper stores for hemocyanin synthesis. These results indicate that penaeid shrimp can tolerate moderate hypoxic conditions by physiological adaptations, such as anaerobic metabolism and increased oxygen carrying-capacity. These adaptations require an adequate dietary supply of proteins and copper for hemocyanin synthesis and of carbohydrates for anaerobic metabolism.     

The Effect of Acute Hypoxia on Synaptosomes from Rat Brain

Abstract: Synaptosomes have been isolated from the brains of nonanesthetized and nembutal-anesthetized rats subjected to 30 min hypoxia induced by breathing 7% oxygen in nitrogen. The respiratory rate was depressed in synaptosomes from starved hypoxic animals but was not significantly different from the respective control values in preparations from fed hypoxic animals, anesthetized animals, and hypoxic nonanesthetized animals allowed to recover from the hypoxic episode by 60 min of normoxic conditions. Observations are also reported concerning the levels of various metabolites in the synaptosomes isolated from the brains of the same groups of animals. It is suggested that hypoxia results in damage to the synaptosomal and/or mitochondrial membrane, which modifies substrate oxidation in the mitochondria and decreases availability of reducing equivalents for the respiratory chain. Results obtained on afflicted and recovered animals indicate that synaptosomal preparations provide a useful model for the study of hypoxic damage.     

Chronic hypoxia alters the function of NOS nerves in cerebral arteries of near-term fetal and adult sheep.

In addition to adrenergic innervation, cerebral arteries also contain neuronal nitric oxide synthase (nNOS)-expressing nerves that augment adrenergic nerve function. We examined the impact of development and chronic high-altitude hypoxia (3,820 m) on nNOS nerve function in near-term fetal and adult sheep middle cerebral arteries (MCA). Electrical stimulation-evoked release of norepinephrine (NE) was measured with HPLC and electrochemical detection, whereas nitric oxide (NO) release was measured by chemiluminescence. An inhibitor of NO synthase, N(omega)-nitro-l-arginine methyl ester (l-NAME), significantly inhibited stimulation-evoked NE release in MCA from normoxic fetal and adult sheep with no effect in MCA from hypoxic animals. Addition of the NO donor S-nitroso-N-acetyl-dl-penicillamine fully reversed the effect of l-NAME in MCA from normoxic animals with no effect in MCA from hypoxic animals. Electrical stimulation caused a significant increase in NO release in MCA from normoxic animals, an effect that was blocked by the neurotoxin tetrodotoxin, whereas there was no increase in NO release in MCA from hypoxic animals. Relative abundance of nNOS as measured by Western blot analysis was similar in normoxic fetal and adult MCA. However, after hypoxic acclimitization, nNOS levels dramatically declined in both fetal and adult MCA. These data suggest that the function of nNOS nerves declines during chronic high-altitude hypoxia, a functional change that may be related to a decline in nNOS protein levels.     

Time-course of the polycythemic response in normoxic and hypoxic mice with high blood oxygen affinity induced by cyanate administration

The Hb-O₂ affinity and the erythropoietic response as a function of time were studied in mice treated with sodium cyanate for up to 2 months. Cyanate increased the Hb-O₂ affinity in normoxic mice more than in chronically hypoxic mice. The hemoglobin concentration rose as a function of time both in normoxic and hypoxic conditions but reached higher levels in hypoxia. After 42 days of study (21 days of hypoxia) hemoglobin reached maximum levels and thereafter showed a plateau in both cyanate and control animals. It is concluded that a chronic left-shifted oxygen dissociation curve does not avoid the development of hypoxic polycythemia in mice. Moreover, prolonged cyanate administration potentiates the crythropoietic response to chronic hypoxia. Since polycythemia is an index of tissue hypoxia, the results show that the high hemoglobin affinity did not prevent tissue hypoxia in low PO₂ conditions. Results showing beneficial effects of high hemoglobin oxygen affinity induced by cyanate based on acute hypoxic expositions should be cautiously interpreted with regard to their adaptive value in animals chronically exposed to natural or simulated hypoxia.Abbreviations Hb hemoglobin - NaOCN sodium cyanate - ODC oxygen dissociation curve - P ₅₀ PO₂ at which hemoglobin is half saturated with O₂     

Increased normoxic ventilation induced by repetitive hypoxia in conscious dogs.

To determine if a long-lasting increase in normoxic ventilatory drive is induced in conscious animals by repetitive hypoxia, we examined the normoxic [arterial O2 saturation (SaO2) > 93%] ventilatory response following successive episodes of 2-min eucapnic hypoxic challenges (SaO2 = 80%) in awake tracheotomized dogs. End-tidal CO2 was maintained at the resting level during and after repetitive hypoxia. The experimental protocol was performed twice in each of five dogs on separate days. To determine if changes in normoxic ventilation occurred between episodes of repetitive hypoxia, data were compared from six periods (epochs) for all experiments. The mean minute ventilation (VI) during three normoxic periods between episodes of intermittent hypoxia was 135, 154, and 169% of control (P < 0.05). VI during a 30-min recovery period was still higher at 183 and 172% of control (P < 0.05). Normoxic VI between hypoxic and recovery periods was significantly higher than the corresponding values in sham experiments. Our results indicate that a long-lasting increase in normoxic ventilation can be evoked in an awake unanesthetized dog by a short exposure to repetitive hypoxia.     

Development of hypoxia enhanced 111In-labeled Bombesin conjugates: design, synthesis, and in vitro evaluation in PC-3 human prostate cancer

The gastrin-releasing peptide receptor (BB2r) has shown great promise for tumor targeting due to the increase of the receptor expression in a variety of human cancers including prostate, breast, small-cell lung, and pancreatic cancer. From clinical investigations, prostate cancer has been shown to be among the most hypoxic of the cancers investigated. Many solid tumors contain regions of hypoxia due to poor organization and efficiency of the vasculature. However, hypoxia is typically not present in normal tissue. Nitroimidazoles, a thoroughly investigated class of hypoxia selective drugs, have been shown to be highly retained in hypoxic tissues. The purpose of this study is to determine if the incorporation of hypoxia trapping moieties into the structural paradigm of BB2r-targeted peptides will increase the retention time of the agents in prostate cancer tumors. The present work involves the design, syntheses, purification, and in vitro investigation of hypoxia enhanced (111)In-BB2r-targeted radioconjugates. A total of four BB2r-targeted conjugates (1-4) were synthesized and coupled with increasing numbers of 2-nitroimidazoles, a hypoxia trapping moiety. Conjugates were radiolabeled with (111)In and purified by HPLC prior to in vitro studies. Receptor saturation assays under both normoxic and hypoxic conditions showed that the BB2r receptor expression on the PC-3 human prostate cancer cell line was not significantly affected by oxygen levels. Competitive binding assays revealed that incorporation of 2-nitroimidazoles had a detrimental effect to BB2r binding when adequate spacer groups, between the hypoxia trapping agent and the pharmacophore, were not employed. All of the 2-nitroimidazole containing BB2r-targeted agents exhibited significantly higher longitudinal retention in PC-3 cells under hypoxic conditions compared to the analogous normoxic studies. Protein association analysis revealed a 3-fold increase in binding of a 2-nitroimidazole containing BB2r-targeted agent under hypoxic relative to normoxic conditions. The positive nature of these results indicate that further exploration into the potential of hypoxia selective trapping agents for BB2r-targeted agents, as well as other targeted compounds, is warranted.