Oxygen supply of the brain cortex in acute nitrite hypoxia in rodents with different ecological specialization

Microhemodynamics and oxygen tension (pO₂) in the brain cortex tissues as well as the heart rate were studied in rodents with different ecological specialization during hypoxia produced by subcutaneous injection of sodium nitrite (3 mg/100 g body mass). It was shown that the blood flow in animals with low (rats) and high (muskrats) resistance to hypoxia decreased by the 30th min of the nitrite action, with its subsequent restoration to 85% and 83% of the initial level by the 60th min. The interspecies difference consisted in an increase of the brain blood flow (by 24%) in muskrats and a decrease (by 33%) in rats 15 min after the injection. In rats, simultaneously with the blood-flow dynamics, a pO₂ increase was observed in some brain cortex microareas, while in others—a pO₂ decrease 15 min after the NaNO₂ injection: meanwhile, in muskrats, at this time period a significant pO₂ decrease was observed on the background of a blood flow increase. In both animal species, the pO₂ minimal value was reached by the 45th min, while restoration almost to the initial levels—by the 60th min of the nitrite action. Changes in the rats, synchronous and unidirectional with the heart rate frequency, of the brain blood-flow, as well as tachycardia developing throughout the whole experiment in rats allow suggesting that restoration of the oxygen regime in the brain cortex microareas is provided by activation of systemic mechanisms of regulation of circulation.     

Mechanism of hypoxic preconditionin in guinea pig papillary muscles

Brief ischemia or hypoxia has been found to protect the heart against susbsequent long-lasting ischemia and to improve contractile dysfunction as well to reduce cell necrosis and the incidence of lethal arrhythmias. This phenomenon, termed preconditioning (PC) has been demonstrated in different species. However, little is known about PC in guinea pigs. Moreover, electrophysiological changes underlying protection have not been studied so far in conjuntion with force recovery in a setting of PC. The aim of the study was to study PC in a guinea pig papillary muscle, using recovery of contractility after long hypoxic challenge as the main end-point of protection, and to investigate concominant electrophysiological alterations. In guinea pig papillary muscle preparations contracting isometrically (paced at 2 Hz), transmembrane action potentials (AP) and developed force (DF) were recorded by conventional microelectrode technique and a force tranducer. In addition, effective refractory periods (ERP) were determined. Hypoxia was induced by superfusion with 100% N₂ (pO₂ < 5 kPa) and pacing at 3,3 Hz. In the control group, long hypoxia lasted for 45 min and was followed by 30 min reoxygenation. In the PC group, muscles were subjected to 5 min hypoxia followed by 10 min recovery prior to sustained hypoxia/reoxygenation. Results: Long hypoxia induced a similar depression of DF in both, PC and control groups. However, a loss of contractile activity occured earlier in the PC group. AP duration and ERP decreased faster and were significantly shorter after PC. Upon reoxygenation, preconditioned muscles showed significantly better recovery of function (DF 86% of prehypoxic value vs. 36% in controls; p < 0,05). AP and ERP were completely restored in both, PC and control groups. Guinea pig papillary muscle can be preconditioned with a brief hypoxic challenge against contractile dysfunction upon long-lasting hypoxia/reoxygenation. Shortening of AP and loss of contractility occured more quickly during hypoxia and may participate in the protective effect of preconditioning. Possible mechanisms might involve facilitated opening of K_ATP-dependent channels.     

Quantitative imaging of pO2 in orthotopic murine gliomas: hypoxia correlates with resistance to radiation

Hypoxia is considered one of the microenvironmental factors associated with the malignant nature of glioblastoma. Thus, evaluating intratumoural distribution of hypoxia would be useful for therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI) is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl and monitoring its line broadening caused by oxygen. In this study, the feasibility of EPRI for assessment of oxygen distribution in the glioblastoma using orthotopic U87 and U251 xenograft model is examined. Heterogeneous distribution of pO₂ between 0 and 50?mmHg was observed throughout the tumours except for the normal brain tissue. U251 glioblastoma was more likely to exhibit hypoxia than U87 for comparable tumour size (median pO₂; 29.7 and 18.2?mmHg, p?=?.028, in U87 and U251, respectively). The area with pO₂ under 10?mmHg on the EPR oximetry (HF10) showed a good correlation with pimonidazole staining among tumours with evaluated size. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared with U87. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumour.     

Cardiovascular manifestations of acute nitrite intoxication in laboratory rats

The systemic and peripheral hemodynamics was studied in male white rats under conditions of acute nitrite hypoxia (subcutaneous administration of sodium nitrite at doses of 1, 3, and 5 mg/100 g body mass). By the electrocardiographic, rheographic, and other methods there were recorded the heart rate (HR), minute circulation volume (MCV), cardiac output (CO), skeletal muscle circulation (SMC), brain circulation (BC), and systemic arterial pressure (AP). Nitrite was shown to produce a fast, dose-dependent AP fall accompanied by a decrease of MCV due to development of bradycardia and a fall of CO. At the phase of the steady hypotension, MCV increased due to a significant rise of CO on the background of the continuing bradycardia. The systemic circulatory effects of NaNO₂ were found to be accompanied by a redistribution of peripheral circulation in the form of a dose-dependent increase of BC and a sharp fall of MCV. It was shown that 1–1.5 h after the nitrite injection the parameters of systemic and peripheral hemodynamics approached the initial levels. Possible triggering mechanisms of the initial stage of the rat cardiovascular adaptation to conditions of acute nitrite hypoxia are discussed.     

Effects of hypoxic factors on cardiac chronotropic reactions in the muskrat Ondatra zibethicus in free behavior

Effects of natural and artificial hypoxic factors on cardiac chronotropic reactions were studied in the muskrat Ondatra zibethicus naturally adapted to underwater hypoxia under conditions of free behavior. To record cardiac activity, original implanted ECG sensors designed in the laboratory were used. Under observations were muskrats in the states of rest, movement, swimming on the water surface, diving, underwater swimming, forced underwater immersion, and artificial apnea, in the low-pressure chamber during changes of pressure from 100 to 25 kPa (ascent to an altitude of 11 km) and in the atmosphere of hypoxic mixtures with 5–10% O₂ as well as under conditions of hemic nitrite hypoxia after injection of 3 mg/kg NaNO₂. Heart rate (HR) in muskrats is labile and can change within the limits from 15 to 360 beats/min. A characteristic feature of hypoxic action is development in muskrats of bradycardia that can appear either instantly—both as a conditional reflex and from the nose lobe receptors—or gradually at a decrease of pO₂ in inhaled air. Before diving and after coming to the surface a brief tachycardia can also be observed. The gradual development of tachycardia takes place in nitrite hypoxia. Development of bradycardia was eliminated at blockade of M-cholinoreceptors by atropine, and of tachycardia—at blockade of β-adrenoreceptors by propranolol. Blockade of α-adrenoreceptors by phentolamine did not affect cardiac chronotropic reactions, which indicates the absence of their connection with vasoconstriction. Analysis of the cardiac rhythm variability has revealed a large spectrum of slow cardiointerval fluctuations connected with animal functional states. Regulation of cardiac chronotropic reactions in muskrats under effect of hypoxic factors operates along both sympathetic and parasympathetic pathways of the autonomic nervous system, the leading role in these processes being played by vagus influences. Original Russian Text. V. I. Shereshkov, T. E. Shumilova, D. A. Kuzmin, I. N. Yanvareva, and A. D. Nozdrachev, 2006, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2006, Vol. 42, No. 4, pp. 371–377.     

Individual characteristics of external respiration during intermittent normobaric hypoxia

Correlation and regression relationships between the indices of the body responsiveness to hypoxic impacts and initial individual values of indices of the respiratory system and heart activity were studied in a group of subjects during three repeated cycles of breathing alternately a hypoxic gas mixture (11 vol % O₂) for 5 min and normal air for 5 min. A steady negative correlation between the most important regulatory indicator, the increase in the CO₂ content of the lungs, and its initial level in individual subjects was found. This may determine the known “normalizing” curative and prophylactic effects of intermittent normobaric hypoxia on the gas transport system of the body. A correlation between the individual increase in the CO₂ content of the lungs in response to hypoxia and changes in the heart rate and initial inhalation rate and depth, rather than oxygen consumption by the body, was found.     

Effects of oxygenation conditions on the respiratory activity generated by medullo-spinal preparations of rats

On isolated medullo-spinal preparations (IMSP) of newborn rats that generate rhythmic respiratory activity, we observed specific features of the reactions to a decrease in the O₂ tension in the superfusing medium (saturation with an isocapnic anoxic gas mixture) manifested in a standard configuration of the preparation, after separation of the rostral part of the medulla and after pH modifications. Using microelectrode amperometric measurements of pO₂ above the ventral medullary surface and of pO₂ profiles into the medullary tissues, we demonstrated the dependence of oxygen supply of IMSP on the rate of superfusion and the existence of hypoxic and anoxic zones in its tissues. The mechanisms of sensitivity of the IMSP-generated respiratory rhythm to hypoxia are discussed.     

Hypobaria and hypoxia affects phytochemical production, gas exchange, and growth of lettuce

Hypobaria (low total atmospheric pressure) is essential in sustainable, energy-efficient plant production systems for long-term space exploration and human habitation on the Moon and Mars. There are also critical engineering, safety, and materials handling advantages of growing plants under hypobaria, including reduced atmospheric leakage from extraterrestrial base environments. The potential for producing crops under hypobaria and manipulating hypoxia (low oxygen stress) to increase health-promoting bioactive compounds is not well characterized. Here we showed that hypobaric-grown lettuce plants (25 kPa ≈ 25% of normal pressure) exposed to hypoxia (6 kPa pO₂ ≈ 29% of normal pO₂) during the final 3 d of the production cycle had enhanced antioxidant activity, increased synthesis of anthocyananins, phenolics, and carotenoids without reduction of photosynthesis or plant biomass. Net photosynthetic rate (P _N) was not affected by total pressure. However, 10 d of hypoxia reduced P _N, dark respiration rate (R _D), P _N/R _D ratio, and plant biomass. Growing plants under hypobaria and manipulating hypoxia during crop production to enhance health-promoting bioactive compounds is important for the health and well-being of astronauts exposed to space radiation and other stresses during long-term habitation.     

Tissue oxygen partial pressure in organs of chickens in the second half of embryogenesis and first days after hatching

The aim of this study is to measure the oxygen partial pressure (pO₂) in developing chicken tissues, namely, in the cerebral hemispheres, liver, m. pectoralis, and m. gastrocnemius, and to estimate the correlation of pO₂ with the earlier measured values (laser Doppler flowmetry) of volume blood flow (BF) in these organs. We have studied 10-, 15-, and 19-day-old embryos and 4-day-old chickens anesthetized with urethane. The pO₂ has been measured in the surface layers of organs with a membrane amperometric Clark-type O₂ electrode (cathode diameter of approximately 50 μm) placed in the center of the sensor unit (outer diameter of 3.4 mm). Noticeable distinctions between both the tissue pO₂ values in different organs and the dynamics of their changes during the observation time have been recorded. The following differences are the most important: (1) the lowest pO₂} {cm(and BF) is observed in the brain and, especially, in the liver of 10-day-old embryos; (2) in the subsequent period of embryogenesis, the pO₂ in the brain increases 1.9-fold (BF also increases), falls 1.7-fold in m. pectoralis, and displays minor changes in the liver and m. gastrocnemius on the background of constant BF value in the liver and both muscles; and (3) after hatching, pO₂ in the liver and m. pectoralis increases severalfold (BF increases too) but does not change in a statistically significant manner in the brain and m. gastrocnemius despite an increase in BF (more pronouncedly in the muscle). Two possible mechanisms underlying the changes in the tissue pO₂ in developing chicken organs have been proposed: one is determined by the specific features of intracardiac blood flows and the other is associated with the oxyhemoglobin dissociation pattern in the blood capillary circulation in the organs, determined by the specific features in its oxidative metabolism.     

Steady and nonsteady state gas exchange characteristics of soybean nodules in relation to the oxygen diffusion barrier

An open gas exchange system was used to monitor the nonsteady state and steady state changes in nitrogenase activity (H₂ evolution in N₂:O₂ and Ar:O₂) and respiration (CO₂ evolution) in attached, excised, and sliced nodules of soybean (Glycine max L. Merr.) exposed to external pO₂ of 5 to 100%. In attached nodules, increases in external pO₂ in steps of 10 or 20% resulted in sharp declines in the rates of H₂ and CO₂ evolution. Recovery of these rates to values equal to or greater than their initial rates occurred within 10 to 60 minutes of exposure to the higher pO₂. Recovery was more rapid at higher initial pO₂ and in Ar:O₂ compared to N₂:O₂. Sequential 10% increments in pO₂ to 100% O₂ resulted in rates of H₂ evolution which were 1.4 to 1.7 times the steady state rate at 20% O₂ in Ar. This was attributed to a relief at high pO₂ from the 40% decline in nitrogenase activity that was induced by Ar at a pO₂ of 20%. Changes in nodule respiration rate could not account for the nodules' ability to adjust to high external pO₂, supporting the hypothesis that soybean nodules have a variable barrier to O₂ diffusion which responds slowly (within minutes) to changes in pO₂. Nodule excision and slicing resulted in 45 and 78% declines, respectively, in total specific nitrogenase activity at 20% O₂. In contrast with the result obtained with intact nodules, subsequent 10% increases in pO₂ in Ar:O₂ did not result in transient declines in H₂ evolution rates, but in the rapid attainment of new steady state rates. Also, distinct optima in nitrogenase activity were observed at about 60% O₂. These results were consistent with an increase in the diffusive resistance of the nodule cortex following nodule excision or nodule slicing. This work also shows the importance of using intact plants and continuous measurements of gas exchange in studies of O₂ diffusion and nitrogenase activity in legume nodules.     

Effect of Oxygen and Malate on NO(3) Inhibition of Nitrogenase in Soybean Nodules

Soybean (Glycine max cv Hodgson) nitrogenase activity (C₂H₂ reduction) in the presence or absence of nitrate was studied at various external O₂ tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O₂ tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO₂, although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO₂ increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O₂. Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis.     

Hypoxia stimulates osteoclast formation from human peripheral blood

Active pathological bone destruction in humans often occurs in locations where oxygen tension (pO₂) is likely to be low, for example, at the sites of tumours, inflammation, infections and fractures, or the poorly vascularized yellow fatty marrow of the elderly. We examined the effect of pO₂ on formation of osteoclasts, the cells responsible for bone resorption, in 14‐day cultures of normal human peripheral blood mononuclear cells (hPBMCs) on ivory discs. Hypoxia (1–2% O₂) caused threefold increases in the number of osteoclasts formed, compared with 20% O₂. Hypoxia also caused a twofold increase in the number of nuclei per osteoclast, leading to stimulations of resorption pit formation of up to 10‐fold. Exposure to hypoxia led to stabilization of the hypoxia‐inducible factors, HIF1α and HIF2α, and upregulation of vascular endothelial growth factor and interleukin‐6 expression by hPBMCs. These findings help explain why extravasation of mononuclear precursors into relatively O₂‐deficient bone microenvironments could result in osteoclast formation and suggest a new mechanism for the bone loss associated with the pathophysiological conditions where hypoxia commonly occurs. Copyright © 2010 John Wiley & Sons, Ltd.     

A Comparative pO2 Probe and [18F]-Fluoro-Azomycinarabino-Furanoside ([18F]FAZA) PET Study Reveals Anesthesia-Induced Impairment of Oxygenation and Perfusion in Tumor and Muscle

Tumor hypoxia can be identified by [¹⁸F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [¹⁸F]FAZA uptake and partial oxygen pressure (pO₂) in carcinoma and muscle tissue of air- and oxygen-breathing mice.

Methods

CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O₂). We performed 10 min static PET scans 1 h, 2 h and 3 h after [¹⁸F]FAZA injection and calculated the [¹⁸F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO₂ probe in the tumor as well as in the gastrocnemius muscle to measure the pO₂ and perfusion.

Results

Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [¹⁸F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O₂: 4.4 vs. IF-O₂: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [¹⁸F]FAZA uptake in the carcinomas. Invasive pO₂ probe measurements yielded enhanced intra-tumoral pO₂ values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O₂ 9.73 mmHg, IF-O₂: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003).

Conclusion

[¹⁸F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [¹⁸F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO₂-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.     

Noninvasive Monitoring of Small Intestinal Oxygen in a Rat Model of Chronic Mesenteric Ischemia

We noninvasively monitored the partial pressure of oxygen (pO₂) in rat’s small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30 % of blood flow for experimental rats. Noninvasive measurement of pO₂ was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO₂ for control rats remained stable over the 7-day period. The pO₂ on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO₂ post banding with a progressive decline to a final pO₂ of 20.9 ± 4.5 mmHg (mean ± SE; p = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe? was used to validate the low tissue pO₂. Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO₂ values had a higher intensity of staining. Thus, monitoring SI pO₂ using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO₂ in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease.     

Regulation of peripheral and systemic blood circulation in rats in conditions of acute nitrite hypoxia

Regulation of the systemic and peripheral hemodynamics in the conditions of acute nitrite hypoxia (doses of NaNO₂ 10, 30, and 50 mg/kg of the body mass) were studied on white male rats. It was shown that NaNO₂ causes a quick dose-dependent decrease in the blood pressure with an intensification of the parasympathetic tonus and development of bradycardia. The hemodynamics was restored as the oxygen capacity of the blood decreased with an increase in the sympathetic tonus and development of tachycardia. The role of intracardial metasympathetic structures and the renin-angiotensin system in cardiovascular adaptation to hypoxia was established. Adaptation to nitrite hypoxia is accomplished by a coordinated interaction of neurogenic and humoral factors. A combination of pharmacological agents, which include separate links of regulator systems of the organism, leads to failure of the adaptation process.     

Survival and function of mesenchymal stem cells (MSCs) depend on glucose to overcome exposure to long‐term,severe and continuous hypoxia

Use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for various diseases but has generated marginally successful results. A consistent finding of most studies is massive death of transplanted cells. The present study examined the respective roles of glucose and continuous severe hypoxia on MSC viability and function with respect to bone tissue engineering. We hereby demonstrate for the first time that MSCs survive exposure to long‐term (12 days), severe (pO₂ < 1.5 mmHg) hypoxia, provided glucose is available. To this end, an in vitro model that mimics the hypoxic environment and cell‐driven metabolic changes encountered by grafted sheep cells was established. In this model, the hallmarks of hypoxia (low pO₂, hypoxia inducible factor‐1α expression and anaerobic metabolism) were present. When conditions switched from hypoxic (low pO₂) to ischemic (low pO₂ and glucose depletion), MSCs exhibited shrinking, decreased cell viability and ATP content due to complete exhaustion of glucose at day 6; these results provided evidence that ischemia led to the observed massive cell death. Moreover, MSCs exposed to severe, continuous hypoxia, but without any glucose shortage, remained viable and maintained both their in vitro proliferative ability after simulation with blood reperfusion at day 12 and their in vivo osteogenic ability. These findings challenge the traditional view according to which severe hypoxia per se is responsible for the massive MSC death observed upon transplantation of these cells and provide evidence that MSCs are able to withstand exposure to severe, continuous hypoxia provided that a glucose supply is available.     

Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

In order to investigate the effects of root hypoxia (1–2% oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio and nitrite content were significantly increased. Contrary to that in leaves, glutamine synthetase activity was significantly enhanced in roots. The activities of nitrate and nitrite reductase were enhanced in roots as well as leaves. The higher increase in the NH₄⁺ content and in the protease activities in roots and leaves of hypoxically treated plants coincide with a greater decrease in soluble protein contents. Taken together, these results suggest that root hypoxia leaded to higher protein degradation. The hypoxia-induced increase in the aminating glutamate dehydrogenase activity may be considered as an alternative N assimilation pathway involved in detoxifying the NH₄⁺, accumulated under hypoxic conditions. With respect to hypoxic stress, the distinct sensitivity of the enzymes involved in N assimilation is discussed.Key words: tomato, hypoxia, nitrogen, glutamine synthetase, protease, glutamate dehydrogenase     

Nodule structure and nitrogenase activity ofCoriaria arborea in response to varying pO2

When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO₂ they show a broad optimum between 20 and 40 kPa O₂, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO₂ nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO₂. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO₂ and decrease in resistance at below ambient pO₂. Plants grown with root systems at pO₂ from 5–40 kPa O₂ did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO₂ than at low pO₂. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele.     

Respiration and oxygen transport in soybean nodules

Summary The respiration rate of individual soybean (Glycine max Merr.) nodules was measured as a function of pO₂ and temperature. At 23°, as the pO₂ was increased from 0.1 to 0.9 atm, there was a linear increase in respiration rate. At 13°, similar results were obtained, except that there was an abrupt saturation of respiration at approximately 0.5 atm pO₂. When measurements were made on the same nodule, the rate of increase in respiration with pO₂ was the same at 13° and 23°. Additional results were that 5% CO in the gas phase had no effect on respiration, except for a small decrease in the pO₂ at which respiration became saturated. Also, nodules still attached to the soybean root displayed the same respiratory behavior as detached nodules. A model for oxygen transport in the nodule is presented which explains these results quantitatively. The essence of the model is that the respiration rate of the central tissue of the nodule is almost entirely determined by the rate of oxygen diffusion to the respiratory enzymes. Evidence is given that the nodule cortex is the site of almost all of the resistance to oxygen diffusion within the nodule.     

Respiration and nitrogenase activity in nodules ofCasuarina cunninghamiana and cultures ofFrankia sp. HFP020203: Effects of temperature and partial pressure of O2

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant.Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction.Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO₂ (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O₂ (vesicles absent) or 20 kPa O₂ (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO₂ at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO₂, but the Q₁₀ was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O₂-saturated at low pO₂ values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.