scholarly journals Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay

2006 ◽  
Vol 401 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Dominic Ehrismann ◽  
Emily Flashman ◽  
David N. Genn ◽  
Nicolas Mathioudakis ◽  
Kirsty S. Hewitson ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Recombinant Protein ◽  
Synthetic Peptides ◽  
Hypoxia Inducible Factor ◽  
Oxygen Sensors ◽  
Hydroxylase Activity ◽  
Protein Substrates ◽  
Physiological Conditions ◽  
The Relationship

The activity and levels of the metazoan HIF (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2OG (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIF hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent Km (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent Km (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1α. The present results, albeit obtained under non-physiological conditions, imply that the apparent Km (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.

scholarly journals Use of Water-Soluble Oxygen Sensors to Study the Oxygen Consumption during Photosensitization with Hypericin in Vitro and in Vivo

2016 ◽  
Vol 110 (3) ◽  
pp. 507a-508a
Author(s):  
Veronika Huntosova ◽  
Dominik Belej ◽  
Emmanuel Gerelli ◽  
Pavol Miskovsky ◽  
Georges Wagnieres
Keyword(s):  
Oxygen Consumption ◽  
Oxygen Sensors ◽  
Water Soluble ◽  
Soluble Oxygen

scholarly journals Hypoxia-inducible factor-1α is the therapeutic target of the SGLT2 inhibitor for diabetic nephropathy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ryoichi Bessho ◽  
Yumi Takiyama ◽  
Takao Takiyama ◽  
Hiroya Kitsunai ◽  
Yasutaka Takeda ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Oxygen Consumption ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Sglt2 Inhibitor ◽  
Inhibitory Effect ◽  
Sglt2 Inhibitors ◽  
Proximal Tubular Epithelial Cells ◽  
Renal Proximal Tubular

Abstract Previous studies have demonstrated intrarenal hypoxia in patients with diabetes. Hypoxia-inducible factor (HIF)-1 plays an important role in hypoxia-induced tubulointerstitial fibrosis. Recent clinical trials have confirmed the renoprotective action of SGLT2 inhibitors in diabetic nephropathy. We explored the effects of an SGLT2 inhibitor, luseogliflozin on HIF-1α expression in human renal proximal tubular epithelial cells (HRPTECs). Luseogliflozin significantly inhibited hypoxia-induced HIF-1α protein expression in HRPTECs. In addition, luseogliflozin inhibited hypoxia-induced the expression of the HIF-1α target genes PAI-1, VEGF, GLUT1, HK2 and PKM. Although luseogliflozin increased phosphorylated-AMP-activated protein kinase α (p-AMPKα) levels, the AMPK activator AICAR did not changed hypoxia-induced HIF-1α expression. Luseogliflozin suppressed the oxygen consumption rate in HRPTECs, and subsequently decreased hypoxia-sensitive dye, pimonidazole staining under hypoxia, suggesting that luseogliflozin promoted the degradation of HIF-1α protein by redistribution of intracellular oxygen. To confirm the inhibitory effect of luseogliflozin on hypoxia-induced HIF-1α protein in vivo, we treated male diabetic db/db mice with luseogliflozin for 8 to 16 weeks. Luseogliflozin attenuated cortical tubular HIF-1α expression, tubular injury and interstitial fibronectin in db/db mice. Together, luseogliflozin inhibits hypoxia-induced HIF-1α accumulation by suppressing mitochondrial oxygen consumption. The SGLT2 inhibitors may protect diabetic kidneys by therapeutically targeting HIF-1α protein.

scholarly journals A novel intra-ventricular assist device enhances cardiac performance in normal and acutely failing isolated porcine hearts

2021 ◽  
pp. 039139882110039
Author(s):  
Daniël IM van Dort ◽  
Jos Thannhauser ◽  
Wim J Morshuis ◽  
Guillaume SC Geuzebroek ◽  
Dirk J Duncker
Keyword(s):  
Oxygen Consumption ◽  
Myocardial Oxygen Consumption ◽  
Ex Vivo ◽  
Acute Hypoxia ◽  
Mechanical Efficiency ◽  
Cardiac Performance ◽  
Pump Failure ◽  
Physiological Conditions ◽  
The Impact

Background: We recently demonstrated that a novel intra-ventricular membrane pump (IVMP) was able to increase the pump function of isolated beating porcine hearts. In follow-up, we now investigated the impact of the IVMP on myocardial oxygen consumption and total mechanical efficiency (TME) and assessed the effect of IVMP-support in acutely failing hearts. Methods: In 10 ex vivo beating porcine hearts, we studied hemodynamic parameters, as well as arterial and coronary venous oxygen content. We assessed cardiac power (CP), myocardial oxygen consumption (MVO2), and TME (CP divided by MVO2) under baseline conditions and during IVMP-support. Additionally, five isolated hearts were subjected to global hypoxia to investigate the effects of IVMP-support on CP under conditions of acute heart failure. Results: Under physiological conditions, baseline CP was 0.36 ± 0.10 W, which increased to 0.65 ± 0.16 W during IVMP-support (increase of 85% ± 24, p < 0.001). This was accompanied by an increase in MVO2 from 18.6 ± 6.2 ml/min at baseline, to 22.3 ± 5.0 ml/min during IVMP-support (+26 ± 31%, p = 0.005). As a result, TME (%) increased from 5.9 ± 1.2 to 8.8 ± 1.8 (50 ± 22% increase, p < 0.001). Acute hypoxia-induced cardiac pump failure reduced CP by 35 ± 6%, which was fully restored to baseline levels during IVMP-support in all hearts. Conclusion: IVMP-support improved mechanical efficiency under physiological conditions, as the marked increase in cardiac performance only resulted in a modest increase in oxygen consumption. Moreover, the IVMP rapidly restored cardiac performance under conditions of acute pump failure. These observations warrant further study, to evaluate the effects of IVMP-support in in vivo animal models of acute cardiac pump failure.

scholarly journals Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2

2020 ◽  
Vol 6 (27) ◽  
pp. eaaz8534
Author(s):  
Anindya Dey ◽  
Shubhangi Prabhudesai ◽  
Yushan Zhang ◽  
Geeta Rao ◽  
Karthikeyan Thirugnanam ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Hypoxia Inducible Factor ◽  
Point Mutations ◽  
Prolyl Hydroxylase ◽  
Zinc Finger Motif ◽  
Hydroxylase Activity ◽  
Zebrafish Model ◽  
Enzymatic Action

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine β-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

scholarly journals The relationship between initial creatine phosphate breakdown and recovery oxygen consumption for a single isometric tetanus of the frog sartorius muscle at 20 degrees C.

1979 ◽  
Vol 73 (2) ◽  
pp. 159-174 ◽  
Author(s):  
M Mahler
Keyword(s):  
Oxygen Consumption ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Creatine Phosphate ◽  
Sartorius Muscle ◽  
Exothermic Reactions ◽  
Rate Of Oxygen Consumption ◽  
The Relationship ◽  
Frog Sartorius

A previous paper (Mahler, M. 1978 J. Gen. Physiol. 71:559--580) describes the time-course of the suprabasal rate of oxygen consumption (delta QO2) in the sartorius muscle of R. pipiens after isometric tetani of 0.1--1.0 s at 20 degrees C. To test whether these were the responses to impulse changes in the rate of ATP hydrolysis, we compared the total suprabasal oxygen consumption during recovery (delta[O2]) with the amount of ATP hydrolyzed during a contraction, measured indirectly as the decrease in creatine phosphate (delta[CP]O). If suprabasal ATP hydrolysis during recovery is negligible in comparison with that during contraction, delta[CP]0/delta[O2] should approximate the P:O2 ratio for oxidative metabolism, which has an expected value of 6.1--6.5. We found: formula; see text. We conclude that in this muscle at 20 degrees C: (a) after a tetanus of 0.2--1.0 s, delta QO2(t) can be considered the response to an impulse increase in the rate of ATP hydrolysis; (b) the reversal during recovery of unidentified exothermic reactions occurring during the contraction (Woledge, R. C. 1971. Prog. Biophys. Mol. Biol. 22:39--74) can be coupled to an ATP hydrolysis that is at most a small fraction of delta[CP]0; (c) the pooled mean for delta[CP]0/delta[O2], 6.58 +/- 0.55, sets an experimental lower bound for the P:O2 ratio in vivo.

Renal cAMP and 1,25(OH)2D3 synthesis in estrogen-treated chick embryos and hens

1985 ◽  
Vol 249 (6) ◽  
pp. E626-E633 ◽  
Author(s):  
A. Martz ◽  
L. R. Forte ◽  
S. G. Langeluttig
Keyword(s):  
Adenylate Cyclase ◽  
Sexual Maturity ◽  
Egg Laying ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Chick Embryos ◽  
Hydroxylase Activity ◽  
Avian Kidney ◽  
The Relationship

Onset of sexual maturity in female chickens or administration of estrogen to mature males or to juveniles of either sex results in increased parathyroid hormone (PTH)-dependent adenylate cyclase activity and increased 25-hydroxyvitamin D3-1-hydroxylase activity in kidney. The relationship between estrogen-mediated alterations of these two enzyme systems was investigated in embryonic and mature, egg-laying chickens treated in vivo with 17 beta-estradiol (E2). Basal and PTH- and forskolin-stimulated adenylate cyclase activity in kidney plasma membrane preparations was not affected by E2 treatment of 19-day-old chick embryos or of 41-wk-old egg-laying females. High, possibly maximal, levels of catalytic activity in control embryos and hens may have precluded further stimulation by E2. In contrast, E2 significantly enhanced 25-hydroxyvitamin D3-1-hydroxylase activity of embryonic kidney up to 10-fold (P less than 0.005). In mature females, E2 caused cessation of egg laying accompanied by a significant reduction (P less than 0.005) of 25-hydroxyvitamin D3-1-hydroxylase activity. These results indicate that the PTH-dependent adenylate cyclase and the 25-hydroxyvitamin D3-1-hydroxylase systems of avian kidney can be regulated independently and suggest that factors in addition to estrogen are involved in their regulation.

Mitochondrial coupling in vivo in mouse skeletal muscle

2004 ◽  
Vol 286 (2) ◽  
pp. C457-C463 ◽  
Author(s):  
David J. Marcinek ◽  
Kenneth A. Schenkman ◽  
Wayne A. Ciesielski ◽  
Kevin E. Conley
Keyword(s):  
Skeletal Muscle ◽  
Oxygen Consumption ◽  
Quantitative Measurement ◽  
Atp Synthesis ◽  
Mouse Skeletal Muscle ◽  
Physiological Conditions ◽  
Consumption Ratio ◽  
Novel Approach ◽  
Wide Range

The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen fluxes, P/O) plays a central role in cellular bioenergetics. Reduced P/O values are associated with mitochondrial pathologies that can lead to reduced capacity for ATP synthesis and tissue degeneration. Previous work found a wide range of values for P/O in normal mitochondria. To measure mitochondrial coupling under physiological conditions, we have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATPase and oxygen consumption rates during ischemia with 31P magnetic resonance and optical spectroscopy, respectively. This novel approach allows the independent quantitative measurement of ATPase and oxygen flux rates in intact tissue. The quantitative measurement of oxygen consumption is made possible by our ability to independently measure the saturations of hemoglobin (Hb) and myoglobin (Mb) from optical spectra. Our results indicate that the P/O in skeletal muscle of the mouse hindlimb measured in vivo is 2.16 ± 0.24. The theoretical P/O for resting muscle is 2.33. Systemic treatment with 2,4-dinitrophenol to partially uncouple mitochondria does not affect the ATPase rate in the mouse hindlimb but nearly doubles the rate of oxygen consumption, reducing in vivo P/O to 1.37 ± 0.22. These results indicate that only a small fraction of the oxygen consumption in resting mouse skeletal muscle is nonphosphorylating under physiological conditions, suggesting that mitochondria are more tightly coupled than previously thought.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Do Extra-Platelet Sources Contribute to the Plasma Level of Thrombospondin?

1988 ◽  
Vol 59 (02) ◽  
pp. 273-276 ◽  
Author(s):  
J Dawes ◽  
D A Pratt ◽  
M S Dewar ◽  
F E Preston
Keyword(s):  
Platelet Count ◽  
Background Concentration ◽  
Serum Concentrations ◽  
Bone Marrow Depression ◽  
Serial Sampling ◽  
Control Serum ◽  
Platelet Release ◽  
Highly Correlated ◽  
The Relationship

SummaryThrombospondin, a trimeric glycoprotein contained in the platelet α-granules, has been proposed as a marker of in vivo platelet activation. However, it is also synthesised by a range of other cells. The extraplatelet contribution to plasma levels of thrombospondin was therefore estimated by investigating the relationship between plasma thrombospondin levels and platelet count in samples from profoundly thrombocytopenic patients with marrow hypoplasia, using the platelet-specific α-granule protein β-thromboglobulin as control. Serum concentrations of both proteins were highly correlated with platelet count, but while plasma β-thromboglobulin levels and platelet count also correlated, there was no relationship between the number of platelets and thrombospondin concentrations in plasma. Serial sampling of patients recovering from bone marrow depression indicated that the plasma thrombospondin contributed by platelets is superimposed on a background concentration of at least 50 ng/ml probably derived from a non-platelet source, and plasma thrombospondin levels do not simply reflect platelet release.

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