Search

Carotid body (CB) type I cells sense the blood Po 2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1 ) mitochondrial electron transport chain metabolism, 2 ) heme oxygenase 2 (HO-2)-generating CO, and/or 3 ) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp+NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN - , diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O 2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47 phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47 phox tandem to alter type I cell potassium channels and therewith CND.

Carotid body (CB) type I cell hypoxia-sensing function is assumed to be based on potassium channel inhibition. Subsequent membrane depolarization initiates an intracellular calcium increase followed by transmitter release for excitation of synapses with linked nerve endings. Several reports, however, contradict this generally accepted concept by showing that type I cell oxygen-sensing properties vary significantly depending on the method of their isolation. We report therefore for the first time noninvasive mapping of the oxygen-sensing properties of type I cells within the intact adult mouse CB ex vivo by using multifocal Nipkow disk-based imaging of oxygen-, calcium- and potential-sensitive cellular dyes. Characteristic type I cell clusters were identified in the compact tissue by immunohistochemistry because of their large cell nuclei combined with positive tyrosine hydroxylase staining. The cellular calcium concentrations in these cell clusters either increased or decreased in response to reduced tissue oxygen concentrations. Under control conditions, cellular potential oscillations were uniform at ∼0.02 Hz. Under hypoxia-induced membrane depolarization, these oscillations ceased. Simultaneous increases and decreases in potential of these cell clusters resulted from spontaneous burstlike activities lasting ∼1.5 s. type I cells, identified during the experiments by cluster formation in combination with large cell nuclei, seem to respond to hypoxia with heterogeneous kinetics.

The cellular oxygen sensing system of the body ensures appropriate adaptation of cellular functions toward hypoxia by regulating gene expression and ion channel activity. Two-photon laser microscopy is an ideal tool to study and prove the relevance of the molecular mechanisms within oxygen sensing pathways on the cellular and complex tissue or organ level. Images of hypoxia inducible factor 1 (HIF-1) subunit nuclear mobility and protein-protein interaction in living cells, of hypoxia-induced changes in membrane potential and intracellular calcium of live ex vivo carotid bodies as well as of rat kidney proximal tubulus function in vivo, will be shown.

Current concepts of cellular oxygen-sensing include an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial electron transport chain (ETC), heme oxygenase-2 (HO-2), and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) and HIF asparagyl hydroxylase FIH-1 (factor-inhibiting HIF). Different oxygen sensitivities, cell-specific distribution and subcellular localization of specific oxygen-sensing cascades involving reactive oxygen species (ROS) as second messengers may help to tailor various adaptive responses according to differences in tissue oxygen availability. Herein, we propose an integrated model for these various oxygen-sensing mechanisms that very efficiently regulate HIF-alpha activity and plasma membrane potassium-channel (PMPC) conductivity.

Structural and functional integrity of organ function profoundly depends on a regular oxygen and glucose supply. Any disturbance of this supply becomes life threatening and may result in severe loss of organ function. Particular reductions in oxygen availability (hypoxia) caused by respiratory or blood circulation irregularities cannot be tolerated for longer periods due to an insufficient energy supply by anaerobic glycolysis. Complex cellular oxygen sensing systems have evolved to tightly regulate oxygen homeostasis. In response to variations in oxygen partial pressure (PO2), these systems induce adaptive and protective mechanisms to avoid or at least minimize tissue damage. These various responses might be based on a range of oxygen sensing signal cascades including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low PO2 affinity cytochrome c oxidase (aa3) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl-hydroxylase and HIF asparaginyl hydroxylase called factor-inhibiting HIF (FIH-1). Thus, specific oxygen sensing cascades involving reactive oxygen species as second messengers may by means of their different oxygen sensitivities, cell-specific and subcellular localization help to tailor various adaptive responses according to differences in tissue oxygen availability.

Structural and functional integrity of brain function profoundly depends on a regular oxygen and glucose supply. Any disturbance of this supply becomes life threatening and may result in severe loss of brain function. In particular, reductions in oxygen availability (hypoxia) caused by systemic or local blood circulation irregularities cannot be tolerated for longer periods due to an insufficient energy supply to the brain by anaerobic glycolysis. Hypoxia has been implicated in central nervous system pathology in a number of disorders including stroke, head trauma, neoplasia and neurodegenerative disease. Complex cellular oxygen sensing systems have evolved for tight regulation of oxygen homeostasis in the brain. In response to variations in oxygen partial pressure (P(O(2))) these induce adaptive mechanisms to avoid or at least minimize brain damage. A significant advance in our understanding of the hypoxia response stems from the discovery of the hypoxia inducible factors (HIF), which act as key regulators of hypoxia-induced gene expression. Depending on the duration and severity of the oxygen deprivation, cellular oxygen-sensor responses activate a variety of short- and long-term energy saving and cellular protection mechanisms. Hypoxic adaptation encompasses an immediate depolarization block by changing potassium, sodium and chloride ion fluxes across the cellular membrane, a general inhibition of protein synthesis, and HIF-mediated upregulation of gene expression of enzymes or growth factors inducing angiogenesis, anaerobic glycolysis, cell survival or neural stem cell growth. However, sustained and prolonged activation of the HIF pathway may lead to a transition from neuroprotective to cell death responses. This is reflected by the dual features of the HIF system that include both anti- and proapoptotic components. These various responses might be based on a range of oxygen-sensing signal cascades, including an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial chain such as a specialized mitochondrial, low P(O(2)) affinity cytochrome c oxidase (aa(3)) and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF prolyl-hydroxylase (PHD) and HIF asparaginyl hydroxylase, known as factor-inhibiting HIF (FIH-1). Thus specific oxygen-sensing cascades, by means of their different oxygen sensitivities, cell-specific and subcellular localization, may help to tailor various adaptive responses according to differences in tissue oxygen availability.

Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 alpha accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.

Purpose: Investigation of the oxygenation status of organotypic multicellular spheroids (OMS) and their response to irradiation., Materials and Methods: Tumour specimens of glioblastoma multiforme patients (n = 16) were initiated as OMS. Following 20 Gy gamma-irradiation, the cell migratory capacity was evaluated. Spheroid oxygenation was determined by micro-electrode pO2 measurements and pimonidazole immunostaining. Spheroids prepared from established human glioma cell lines were used as a reference., Results: Irradiation inhibited spheroid outgrowth by 12 to 88% relative to the non-irradiated controls. A large interpatient variation was noticed. Oxygen measurements revealed a gradual decrease in pO2 level from the periphery to the core of the spheroids, but the pO2 values remained within an oxygenated range. However, in the cell line spheroids an intermediate layer of hypoxia surrounding the central core was observed., Conclusion: Cell line spheroids with a hypoxic cell fraction and well-oxygenated OMS both show high resistance to irradiation, indicating that hypoxia may not be the biological factor determining the radioresistance of glioma spheroids in vitro.

Light-absorption spectra and afferent chemoreceptor discharge were simultaneously recorded on superfused rat carotid bodies (CBs) under the influence of cytochrome a3-CuB ligands (O2, CN-, CO) in order to identify the primary mitochondrial cytochrome c oxidase (CCO) oxygen sensor. Spectra could be described on the basis of weighted light-absorption spectra of cytochrome b558 of the NAD(P)H oxidase and mitochondrial cytochromes b and c, CCO, cytochrome a3, and an unusual cytochrome a peaking at 592 nm. Discharge signals were deconvoluted into phasic and tonic activity for comparing different CB responses. The spectral weight of cytochrome a592 decreased significantly starting at high PO2 (100 mm Hg) and low sodium cyanide (CN-, 10 mM) accompanied by increasing phasic peak discharge. Combined CO-hypoxia or CO-CN- application inhibited photolysis of CO-stimulated chemoreceptor discharge, revealing photometrically cytochrome a592 as central in oxygen sensing. Control spectra in tissue from sympathetic and nodose ganglia did not show any cytochrome a592 contribution. According to these results, cytochrome a592 is assumed as a unique component of CB CCO, revealing in contrast to other cytochromes an apparent low PO2 and high CN- affinity, probably due to a shortcut of electron flow within CCO between CuA and cytochrome a3-CuB.