Hours Of Severe Hypoxia Research Articles

A large aerobic scope and complex regulatory abilities confer hypoxia tolerance in larval toadfish, Opsanus beta, across a wide thermal range

Few studies have been performed on early-life stage toadfish, and none have addressed their tolerance to temperature and hypoxia despite large seasonal temperature fluctuations and daily hypoxia in their natural environment. The first directed captive breeding of Opsanus beta allowed the examination of larval oxygen demands and hypoxia tolerance across the range of their environmental temperatures (23–33 °C). Larval toadfish exhibited a surprisingly large aerobic scope across the tested temperature range. In response to progressive hypoxia, larval toadfish showed early metabolic depression and a low regulation index (RI), while juveniles had higher regulatory abilities but, unexpectedly, a lower aerobic scope. Larval and juvenile toadfish survived hours of severe hypoxia, but larval fish had a higher excessive post-hypoxia oxygen consumption, yet their metabolic rate returned to RMR in the same timeframe as the juveniles, likely due to their higher aerobic scope. We defined hypoxia tolerance using a physiological trait, p50, the oxygen tension in which oxygen uptake is reduced to 50 % of the metabolic rate at rest and determined it at all tested temperatures. Comparing these p50 values to environmental conditions in Florida Bay using hourly temperature and oxygen measurements from January 2014–October 2021 revealed that larval toadfish rarely experience < p50 conditions (11 % of events). Further, the median duration of these events was 3 h. The metabolic performance of larval toadfish combined with temperature and oxygen observations from their natural environment reveals the fascinating strategy in which larval toadfish survive diel hypoxia across seasons.

In This Issue

HomeCirculation ResearchVol. 129, No. 10In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published28 Oct 2021https://doi.org/10.1161/RES.0000000000000516Circulation Research. 2021;129:891is related toMacrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NGChaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion InjuryEfficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG (p 895)Ma et al perform base editing in mouse embryos to prevent hypertrophic cardiomyopathy.Download figureDownload PowerPointInherited hypertrophic cardiomyopathy (HCM), characterized by thickening and dysfunction of the heart muscle, is the most common genetic heart disorder. Mutations causing HCM often affect a single gene, such as the myosin heavy chain (MHC) gene, and as such have the potential to be corrected by gene editing style approaches. Not all editing techniques are the same, however, with some potentially introducing unwanted insertions or deletions of nucleotides (indels) at the target site. Ma and colleagues wondered whether base editing—in which a single nucleotide basepair is corrected and which typically doesn’t lead to indels—might be a safer approach. The team created a base-editor system to convert an HCM-causing A-T basepair into the wildtype G-C at a single point in the mouse Mhc6 gene. Transfection of the base-editing system into single-cell zygotes resulted in a correction rate of approximately 60-70 percent and, after transfer of the corrected embryos into surrogate females, prevented HCM in the postnatal animals. The team also performed base editing on mouse embryos in utero, achieving a 25 percent gene correction rate. The approach did not cause detectable indels or off-target editing, the team showed, suggesting it might have potential for development as a clinical tool.Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4 (p 909)Heart-repair via MST1 inhibition may be hindered by inflammatory effects, say Liu et al, who suggest a pharmacological work-around.Download figureDownload PowerPointAfter a myocardial infarction, the injured heart muscle is largely unable to regenerate and instead forms a dysfunctional scar that can ultimately lead to heart failure. Cardiomyocyte-specific inhibition of the kinase MST1—shown to boost repair and regeneration in other organs—can prevent infarction-induced death of these cells and preserve heart function, suggesting it may have clinical utility. However, MST1 also has anti-inflammatory properties in macrophages. Its inhibition in those cells may thus delay inflammation resolution after infarction and impair proper healing. Liu and colleagues have now examined mice lacking MST1 in macrophages and found that, sure enough, after myocardial infarction the inflammatory mediator leukotriene B4 was upregulated in macrophages and the animals’ heart function was reduced compared to that of wildtype controls. Importantly, blocking the action of leukotriene B4 in mice reduced infarction injuries in the hearts of MST1-lacking animals and enhanced repair in the injured hearts of wildtype animals given an MST1 inhibitor. The results suggest that, if MST1 inhibition is used as a future post-infarction regenerative therapy, then leukotriene B4 blockade may prevent its inflammatory side-effects.Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion Injury (p 930)Maintaining nitric oxide synthase function after reperfusion injury protects the heart from damage, say Subramani et al.Download figureDownload PowerPointRestoring blood flow to ischemic heart muscle after a myocardial infarction is critical for salvaging muscle function, but can itself cause damage—so-called reperfusion injury. The generation of reactive oxygen species (ROS) and loss of nitric oxide (NO) both contribute to such injury, and the situation is exacerbated by the NO-producing enzyme, endothelial NO synthase (eNOS), producing damaging superoxide anions instead of NO. This switch in eNOS function is caused by glutathionylation of the enzyme (SG-eNOS), but how long the malfunction lasts and how it is rectified is unclear. Subramani and colleagues show that in human endothelial cells exposed to several hours of hypoxia followed by reoxygenation, the level of SG-eNOS steadily increases until, at 16 hours, it decreases sharply. By blocking several different cellular degradation pathways, the team discovered that this decrease in SG-eNOS was due to chaperone-mediated autophagy, with chaperone protein HSC70 being responsible for SG-eNOS destruction. Importantly, the team went on to show that pharmacological deglutathionylation of eNOS in mice promoted NO production and reduced reperfusion injury, suggesting this approach may be of clinical benefit after a myocardial infarction. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMacrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4Mingming Liu, et al. Circulation Research. 2021;129:909-926Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NGShuhong Ma, et al. Circulation Research. 2021;129:895-908Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion InjuryJaganathan Subramani, et al. Circulation Research. 2021;129:930-945 October 29, 2021Vol 129, Issue 10Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000516 Originally publishedOctober 28, 2021 PDF download Advertisement

Hypoxia Tolerance Declines with Age in the Absence of Methionine Sulfoxide Reductase (MSR) in Drosophila melanogaster.

Unlike the mammalian brain, Drosophila melanogaster can tolerate several hours of hypoxia without any tissue injury by entering a protective coma known as spreading depression. However, when oxygen is reintroduced, there is an increased production of reactive oxygen species (ROS) that causes oxidative damage. Methionine sulfoxide reductase (MSR) acts to restore functionality to oxidized methionine residues. In the present study, we have characterized in vivo effects of MSR deficiency on hypoxia tolerance throughout the lifespan of Drosophila. Flies subjected to sudden hypoxia that lacked MSR activity exhibited a longer recovery time and a reduced ability to survive hypoxic/re-oxygenation stress as they approached senescence. However, when hypoxia was induced slowly, MSR deficient flies recovered significantly quicker throughout their entire adult lifespan. In addition, the wildtype and MSR deficient flies had nearly 100% survival rates throughout their lifespan. Neuroprotective signaling mediated by decreased apoptotic pathway activation, as well as gene reprogramming and metabolic downregulation are possible reasons for why MSR deficient flies have faster recovery time and a higher survival rate upon slow induction of spreading depression. Our data are the first to suggest important roles of MSR and longevity pathways in hypoxia tolerance exhibited by Drosophila.

IL-8 as mediator in the microenvironment-leukaemia network in acute myeloid leukaemia.

The bone marrow microenvironment is physiologically hypoxic with areas being as low as 1% O2, e.g. the stem cell niche. Acute myeloid leukaemia (AML) blasts misuse these bone marrow niches for protection by the local microenvironment, but also might create their own microenvironment. Here we identify IL-8 as a hypoxia-regulated cytokine in both AML cell lines and primary AML samples that is induced within 48 hours of severe hypoxia (1% O2). IL-8 lacked effects on AML cells but induced migration in mesenchymal stromal cells (MSC), an integral part of the bone marrow. Accordingly, MSC were significantly increased in AML bone marrow as compared to healthy bone marrow. Interestingly, mononuclear cells obtained from healthy bone marrow displayed both significantly lower endogenous and hypoxia-induced production of IL-8. IL-8 mRNA expression in AML blasts from 533 patients differed between genetic subgroups with significantly lower expression of IL-8 in acute promyelocytic leukaemia (APL), while in non APL-AML patients with FLT ITD had the highest IL-8 expression. In this subgroup, high IL-8 expression was also prognostically unfavourable. In conclusion, hypoxia as encountered in the bone marrow specifically increases IL-8 expression of AML, which in turn impacts niche formation. High IL-8 expression might be correlated with poor prognosis in certain AML subsets.

Contrasting effects of ascorbate and iron on the pulmonary vascular response to hypoxia in humans.

Hypoxia causes an increase in pulmonary artery pressure. Gene expression controlled by the hypoxia‐inducible factor (HIF) family of transcription factors plays an important role in the underlying pulmonary vascular responses. The hydroxylase enzymes that regulate HIF are highly sensitive to varying iron availability, and iron status modifies the pulmonary vascular response to hypoxia, possibly through its effects on HIF. Ascorbate (vitamin C) affects HIF hydroxylation in a similar manner to iron and may therefore have similar pulmonary effects. This study investigated the possible contribution of ascorbate availability to hypoxic pulmonary vasoconstriction in humans. Seven healthy volunteers undertook a randomized, controlled, double‐blind, crossover protocol which studied the effects of high‐dose intravenous ascorbic acid (total 6 g) on the pulmonary vascular response to 5 h of sustained hypoxia. Systolic pulmonary artery pressure (SPAP) was assessed during hypoxia by Doppler echocardiography. Results were compared with corresponding data from a similar study investigating the effect of intravenous iron, in which SPAP was measured in seven healthy volunteers during 8 h of sustained hypoxia. Consistent with other studies, iron supplementation profoundly inhibited hypoxic pulmonary vasoconstriction (P < 0.001). In contrast, supraphysiological supplementation of ascorbate did not affect the increase in pulmonary artery pressure induced by several hours of hypoxia (P = 0.61). We conclude that ascorbate does not interact with hypoxia and the pulmonary circulation in the same manner as iron. Whether the effects of iron are HIF‐mediated remains unknown, and the extent to which ascorbate contributes to HIF hydroxylation in vivo is also unclear.

Temporal and Spatial Dynamics of Diel-Cycling Hypoxia in Estuarine Tributaries

The distribution and intensity of hypoxia (low dissolved oxygen) in estuaries is increasing worldwide due to cultural eutrophication. This study quantifies the strength of associations between the duration of diel-cycling severe hypoxia (≤2 mg O2 l−1) in bottom water (∼15 cm above bottom) of a shallow (<2 m) coastal lagoon estuary (Delaware, USA) and abiotic environmental variables (water temperature, insolation, tide, streamflow, and wind) and predicts the duration of severe hypoxia given different combinations of these variables. The intensity and spatial extent and dynamics of diel-cycling severe hypoxia events were defined. Vertical variability in dissolved oxygen (DO) concentration during the daytime was also determined. During the summers of 2001–2005, bottom DO data were collected for periods of weeks to months at multiple sites using automated sondes. Multiple linear regression (MLR) and regression tree analysis (RTA) were used to determine the relative importance of the environmental variables in predicting the number of hours of severe hypoxia per day. Key findings of the study were that severe hypoxia events of minutes to hours in duration occurred frequently in all four tributaries sampled, primarily between 0200 and 1000 hours. Severe hypoxia duration and diel-cycling amplitudes of DO concentration increased in the up-tributary direction. Hierarchically, the duration of severe hypoxia was influenced mostly by the mean daily water temperature, then by preceding days’ total insolation, percentage of morning hours (02:00 to 10:00 a.m.) ebb tide, and daily streamflow. Collectively, the variables examined by the MLR and the RTA approaches accounted for 62% and 65% of the variability in the duration of severe hypoxia, respectively. RTA demonstrated that daily mean water temperature above 26.3°C and previous day’s total insolation below 13.6 kW m−2 were associated with the longest lasting severe hypoxic events (9.56 h). The environmental variables and combinations of conditions that modulate or augment diel-cycling hypoxia presented in this paper enhance understanding of this widespread and growing phenomenon and provide additional insight regarding the extent to which it can impact food webs in very shallow estuarine waters that often serve as nursery habitat.

PD184352 Releases the Regular Hypoxic Reversible DNA Replication Arrest in T24 Cells

The oxygen dependent regulation of DNA replication is an essential property of proliferating mammalian cells. In human T24 bladder cancer cells, several hours of hypoxia leads to reversible DNA replication arrest and re-entry of oxygen induces a burst of replication initiation. This short communication provides strong evidence that PD184352 initiates DNA replication in living hypoxic cells without elevating the oxygen level. PD184352 releases the regular hypoxic replicon arrest, however, at a low intensity compared to the effect of reoxygenation. Moreover, PD184352 shows no effect on normoxically incubated as well as reoxygenated T24 cells.

Cdk2 activity is dispensable for triggering replicon initiation after transient hypoxia in T24 cells

We examined whether the fast release of replicon initiation after sudden O2 recovery of hypoxically incubated mammalian cells depends on kinase activity of Cdk2. We used a system based on starved/refed T24 cells elaborated previously for such investigations [van Betteraey-Nikoleit M, Eisele KH, Stabenow D and Probst H (2003) Eur J Biochem270, 3880-3890]. Cells subjected to hypoxia concurrently with refeeding accumulate the G1 DNA content within 5-6 h. In this state they are ready to perform, within 1-2 min after O2 recovery, a burst of replicon initiations that marks the start of a synchronous S-phase. We found that Cdk2 binds to the chromatin fraction within 4-6 h after refeeding with fresh medium, irrespective of whether the cells were incubated normoxically or hypoxically. However, inhibition of Cdk2 by olomoucine, roscovitine or the Cdk2/cyclin inhibitory peptide II had no influence on the synchronous burst of replicon initiations. Cdc6 and pRb, possible targets of Cdk2 phosphorylation, behaved differentially. Inhibition did not affect phosphorylation of Cdc6 after reoxygenation, whilst chromatin bound pRb remained hypophosphorylated beyond the initiation burst. Thus, neither Cdk2 activity, though present at the end of the hypoxic period, nor pRb phosphorylation are necessary for releasing the burst of replicon initiations upon oxygen recovery. Consequentially, Cdk2 dependent phosphorylation(s) cannot be a critical trigger of replicon initiation in response to reoxygenation after several hours of hypoxia, at least in the T24 cells studied.

Adenosinergic and cholinergic control mechanisms during hypoxia in the epaulette shark (Hemiscyllium ocellatum), with emphasis on branchial circulation

Coral reef platforms may become hypoxic at night during low tide. One animal in that habitat, the epaulette shark (Hemiscyllium ocellatum), survives hours of severe hypoxia and at least one hour of anoxia. Here, we examine the branchial effects of severe hypoxia (<0.3 mg oxygen l(-1) for 20 min in anaesthetized epaulette shark), by measuring ventral and dorsal aortic blood pressure (P(VA) and P(DA)), heart rate (fh), and observing gill microcirculation using epi-illumination microscopy. Hypoxia induced a flow of blood in two parallel blood vessels, termed longitudinal vessels, in the outer borders of the free tip of the gill filament. Hypoxia also induced significant falls in fh, P(VA) and P(DA), and a biphasic change in ventilation frequency (increase followed by decrease). Adenosine injection (1 micromol kg(-1)) also initiated blood flow in the longitudinal vessels, in addition to significant drops in P(VA), P(DA) and fh, and a biphasic response in ventilation frequency (decrease followed by increase) indicating that adenosine influences ventilation. Aminophylline (10 mg kg(-1)), an A(1) and A(2) adenosine receptor antagonist, blocked the effects of adenosine injection, and also significantly reduced blood flow in the longitudinal vessels during hypoxia. In the second part of the study, we examined the cholinergic influence on the cardiovascular circulation during severe hypoxia (<0.3 mg l(-1)) using antagonists against muscarinic (atropine 2 mg kg(-1)) and nicotinic (tubocurarine 5 mg kg(-1)) receptors. Injection of acetylcholine (ACh; 1 micromol kg(-1)) into the ventral aorta caused a marked fall in fh, a large increase in P(VA), but small changes in P(DA) (suggesting increased R(gill)). Atropine was able to inhibit the branchial vascular responses to ACh but not the hypoxic bradycardia, suggesting the presence of muscarinic receptors on the heart and gill vasculature, and that the hypoxia induced bradycardia is of non-cholinergic origin. The results suggest that adenosine mediates increases in the arterio-venous circulation in the gill during hypoxia. This may serve to increase blood supply to heart and gill tissue.

Oxygen-dependent Regulation of in VivoReplication of Simian Virus 40 DNA Is Modulated by Glucose

Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200-400 ppm O(2) or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.

Coregulation of Glucose Uptake and Vascular Endothelial Growth Factor (VEGF) in Two Small-Cell Lung Cancer (SCLC) Sublines In Vivo and In Vitro

We examined the relationship between 18BF- labeled 2-fluro-2-deoxy -D-glucose (FDG) uptake, and expression of glucose transporters (GLUTs) in two human small-cell lung cancer (SCLC) lines CPH 54A and CPH 54B. Changes in the expression of GLUTs and vascular endothelial growth factor (VEGF) during 12-, 18-, and 24 hours of severe hypoxia in vivo (xenograffs) and in vitro (cell cultures) were recorded for both tumor lines. The two SCLC lines are subpopulations of the same patient tumor. In spite of their common genomic origin they represent consistently different metabolic and microenvironmental phenotypes as well as treatment sensitivities. There were higher levels of Glut-1 protein in 54B and a correspondingly higher FDG uptake in this tumor line (P<.001). During hypoxia a significant upregulation of in VEGF mRNA, GLUT-1 mRNA, and Glut-1 and -3 protein occurred with a distinctly different time course in the two cell lines. A similar co-upregulation of GLUT and VEGF was seen in hypoxic tumors of both lines. There were no significant changes of HIF-1α mRNA during hypoxia in either of the cell lines. A more detailed understanding of such correlations between glucose metabolism, angiogenesis, and microenvironmental phenotype of tumors, by positron emission tomography (PET) and molecular techniques might further sophisticate our interpretation of glycolytic predominance in tumors as seen by 18BFFDG PET.

Alterations in structure of elastic laminae of rat pulmonary arteries in hypoxic hypertension.

The effect of hypoxic hypertension on the remodeling process of the elastic laminae of the rat hilar pulmonary arteries (PAs) was studied by electron microscopy. Rats were exposed to hypoxia (10% O2) for periods of 0.5, 2,6,12,48,96,144, and 240 h. Changes in the structure of the PA elastic laminae were examined and analyzed with respect to changes in the PA wall tensile stress. The PA blood pressure increased rapidly within the first several hours of hypoxia and reached a stable level within 2 days, whereas the PA wall tensile stress increased initially due to elevated blood pressure and then decreased after 48 h due to vessel wall thickening and returned to the control level after 4 days. In association with these changes, the elastic laminae, which appeared homogeneous in normal control rats, changed into structures composed of randomly oriented filaments and edematous contents with an increase in the volume during the early period of hypoxia and regained their homogeneous appearance and normal volume after 4 days. The changes in the elastic laminae were correlated with changes in the tensile stress. These changes were associated with a transient decrease in the stiffness of the PAs. In hypoxic rats given nifedipine, no change was found in the blood pressure, the tensile stress, or the structure of the elastic laminae of the PAs despite continuous exposure to hypoxia. These results suggested that altered tensile stress in the PA wall played a critical role in the initiation and regulation of structural changes in the elastic laminae and that these changes might contribute to alterations in the mechanical properties of the PA in hypoxic hypertension.

Impaired cerebral autoregulation in the newborn lamb during recovery from severe, prolonged hypoxia, combined with carotid artery and jugular vein ligation

To study the effect of severe prolonged hypoxia combined with ligation of the carotid artery and jugular vein (simulating pre-extracorporeal membrane oxygenation [ECMO] events) on cerebral autoregulation in the newborn lamb. Animal studies, using the newborn lamb, with comparison of two randomized treatment groups. Newborn lambs of mixed breed, 1 to 7 days of age, were used for the study. Two groups of animals were studied: a normoxic control group (n = 7) and a hypoxic group (n = 8). Work was conducted in the research laboratories of the Department of Anesthesiology, Critical Care Medicine at The Johns Hopkins Medical Institutions, Baltimore, MD. Animals were anesthetized (pentobarbital), intubated, and mechanically ventilated. We examined the effect of prolonged severe hypoxia combined with carotid artery and jugular vein ligation on cerebral autoregulation during recovery from this insult. Control animals were maintained in a normoxic state (3 hrs) without carotid artery or jugular vein ligation. Hypoxic animals with carotid artery and jugular vein ligation were exposed to a 2-hr period of hypoxia (arterial oxygen saturation 44 +/- 14%; PaO2 30 +/- 3 torr [4 +/- 0.4 kPa]) followed by a 1-hr normoxic recovery period. Cerebral autoregulation was evaluated at the end of the 1-hr recovery period in hypoxic animals, and after 3 hrs of normoxia in control animals. Cerebral perfusion pressure was decreased by increasing intracranial pressure, with infusion of artificial cerebrospinal fluid into an intracranial pressure catheter in the lateral ventricle of the brain. Studies were taken at four ranges of cerebral perfusion pressure: > 55 mm Hg; 55 to 40 mm Hg; 39 to 26 mm Hg; and < or = 25 mm Hg. Cerebral blood flow was measured using the radiolabeled microsphere technique. Cerebral oxygen consumption, fractional oxygen extraction, and oxygen transport values were calculated at each study period. Two hours of severe hypoxia increased cerebral blood flow by 110%, whereas cerebral oxygen consumption was unchanged. In hypoxic animals, cerebral autoregulation was altered, with both cerebral blood flow and cerebral oxygen consumption decreasing at a cerebral perfusion pressure of 39 to 26 mm Hg compared with unchanged cerebral blood flow or cerebral oxygen consumption at a cerebral perfusion pressure of < or = 25 mm Hg in control animals. At the point of loss of autoregulation, significant right-to-left hemispheric cerebral blood flow changes occurred in hypoxic animals. In hypoxic animals, cerebellar cerebral blood flow changes were similar to those changes in the total cerebrum, while brain stem and caudate decreased cerebral blood flow only at a cerebral perfusion pressure of < or = 25 mm Hg. These findings indicate that cerebral autoregulation is disrupted during the recovery phase from an insult caused by prolonged, severe hypoxia with carotid artery and jugular vein ligation. This insult results in significant differences in right and left hemispheric cerebral blood flow rates when cerebral autoregulation is lost. If these results can be extrapolated to the human state, they may help to explain the role of pre-ECMO hypoxia combined with vessel ligation as a risk factor in cerebral injury in ECMO patients.

Benign-to-malignant B16 melanoma progression induced in two stages in vitro by exposure to hypoxia.

Benign tumors apparently become malignant by generating a succession of variants with ever-greater growth potential and autonomy. Such stepwise progression has not been achieved in vitro under conditions likely to occur in developing tumors. Tumors initiated by clone G3.5 of the mouse B16 melanoma regularly generate stable variants that are more malignant. We investigated the possibility that hypoxia might promote stepwise progression along a benign-to-malignant pathway in monolayer cultures of G3.5 cells. Confluent monolayers of metastatic clone G3.5 and the nonmetastatic clone G3.15 were subjected to severe hypoxia (< 50 ppm O2) for up to 72 hours, or to moderate hypoxia (300-1200 ppm O2) for up to 12 days, and were then maintained subconfluent or at confluence for several weeks to permit emergence of progression variants. The relative malignancy of variants was assayed in vivo after subcutaneous injection into mice, by measuring tumor growth rate and counting lung metastases, and after intravenous injection, by counting lung colonies. In vitro assessment of the variants involved growth as monolayers with or without serum, growth in soft agar, and measurement of invasiveness. G3.5 cells were converted to a more malignant variant (G3.5*) by 12-48 hours of severe hypoxia, or longer periods of moderate hypoxia, when followed by maintenance at confluence for 3-5 weeks. Conversions occurred in discrete foci of morphologically-discernible cells (optimum focus formation about one in 1-2 x 10(5) cells) that rapidly expanded to dominate the cultures. The G3.5* phenotype was comparable to the conversion phenotype generated in tumors and included acquisition of growth autonomy in serum-free medium. G3.15 cells were converted to a G3.5-like phenotype by one round of exposure to hypoxia and confluence, and then to the G3.5* phenotype during a second round, at a low frequency (one focus in 5 x 10(6) cells). This behavior was consistent with a failure of all but the largest G3.15 tumors to generate G3.5* conversion cells. Progression from a relatively benign phenotype, G3.15, to a highly malignant phenotype, G3.5*, can be produced in monolayer culture in two stable stages by sequential rounds of exposure to hypoxia and confluence. The resulting conversions corresponded to phenotypes generated within tumors. Both conversions resulted in populations with enhanced growth capabilities, which could establish dominance within tumors. The stepwise conversion of B16 melanoma clones provides a unique model for the in vitro investigation of mechanisms underlying acquisition of malignancy during tumor development.

Distribution of erythropoietin producing cells in rat kidneys during hypoxic hypoxia

We have used in situ hybridization to determine the localization and distribution of cells expressing the erythropoietin (EPO) gene in kidneys of rats exposed to reduced oxygen tensions to characterize the control of renal EPO formation during hypoxic hypoxia. Animals were subjected to severe hypoxia (7.5% O2) for 4, 8 and 32 hours to assess changes related to the duration of hypoxic exposure, and additionally to 9% and 11.5% O2 for eight hours to define changes related to the degree of hypoxia. The number of cells containing EPO mRNA were counted on tissue sections and compared to tissue concentrations of EPO mRNA and to the serum hormone concentrations. In situ hybridization revealed expression of the EPO gene exclusively in peritubular cells that were predominantly located in the cortical labyrinth under all conditions tested. After four hours of severe hypoxia (7.5% O2) approximately 170-fold more cells were found to contain EPO mRNA than under normoxic conditions. The number of EPO producing cells did not change significantly between four and eight hours exposure to 7.5% O2, but the amount of EPO mRNA per kidney increased approximately threefold. Further continuation of hypoxia resulted in down-regulation of renal EPO mRNA levels, which was mainly due to a reduction in the number of cells containing EPO mRNA. Comparison of graded degrees of hypoxia applied for eight hours showed an inverse exponential relationship between oxygen tension and the number of EPO producing cells. This recruitment of cells expressing the EPO gene occurred along a gradient extending from the corticomedullary border to the subcapsular tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy

(31)P-, (13)C-, and (15)N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and gamma-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gln, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from Glc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.

Endogenous opioids and ventilatory adaptation to prolonged hypoxia in goats

To investigate whether endogenous opioid peptides mediate time-dependent changes in ventilatory control during prolonged hypoxia, we studied four adult goats at rest during 14 days at simulated high altitude in a hypobaric chamber (P B∼450 Torr). Arterial PCO 2 fell during the first several hours of hypoxia, remained stable over the next 7 days, and then rose slightly (but without statistical significance) by day 14. Ventilatory responsiveness to CO 2 increased during the first week of hypoxia. By day 14, while still greater than control, the ventilatory response to CO 2 was less than that observed on day 7. Immunoactive β-endorphin levels in plasma and CSF did not change during the 14-day period. Administration of naloxone on day 14 did not restore the ventilatory response to CO 2 to the level observed during the first week of acclimatization. We conclude that in adult goats, time-dependent changes in ventilatory response to CO 2 during acclimatization to prolonged hypoxia are not primarily attributable to alterations in endogenous opioid peptide activity.

Influence of the arachidonic acid cascade on the in vitro hepatic response to hypoxia

Studies were undertaken to determine the effect of arachidonic acid, the precursor of bisenoic prostanoic acid derivatives, on the response of the isolated, perfused rabbit liver to hypoxia. Two and one half hours of severe hypoxia resulted in significant increases in hepatic vascular perfusion pressure, tissue wet weight, and the rates of cellular loss of lactic dehydrogenase, malic dehydrogenase, and acid phosphatase into the perfusing medium. Hypoxia also increased the rate of hepatic PGF 2α production by 25% after 2 1 2 hours (p<0.05, hypoxia vs sham). The addition of arachidonic acid (0.1 μg/g/min for 150 minutes) to the perfusion medium of hypoxic livers significantly attenuated the changes in perfusion pressure, tissue wet weight, and loss of cellular enzymes. Arachidonic acid administration increased the rate of PGF 2α production by 100% (p<0.05, sham vs hypoxia + arachidonic acid) within 30 min after hypoxia and maintained this rate for the duration of the study. These results demonstrate that hypoxia mediated prostaglandin F 2α synthesis in the rabbit liver can occur in the absence of neural and blood borne components and that significant activation of the arachidonic acid cascade via the administration of exogenous arachidonic acid has a salutary effect on hepatic hemodynamics and cellular integrity during hypoxia.