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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.

Reactive oxygen species (ROS) generated by the NADPH oxidase have been proposed to play an important role in the carotid body (CB) oxygen sensing process (Cross et al. 1990). Up to now it remains unclear whether hypoxia causes an increase or decrease of CB ROS levels. We transfected CBs with the ROS sensitive HSP-FRET construct and subsequently measured the intracellular redox state by means of Förster resonance energy transfer (FRET) microscopy. In a previous study we found both increasing and decreasing ROS levels under hypoxic conditions. The transition from decreasing to increasing ROS levels coincided with the change of the caging system from ambient environment caging (AEC) to individually ventilated caging (IVC) (Bernardini A, Brockmeier U, Metzen E, Berchner-Pfannschmidt U, Harde E, Acker-Palmer A, Papkovsky D, Acker H, Fandrey J, Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET based study. Am J Physiol Cell Physiol. doi: 10.1152/ajpcell.00370.2013 , 2014). In this work we analyze hypoxia induced ROS reaction of animals from an IVC system that had been exposed to AEC conditions for 5 days. The results further support the hypothesis of an important impact of the caging system on CB ROS reaction.

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.

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.

Carotid body physiology meets cytochrome c oxidase crystallography Commentary to Ortega-Sáenz P, López-Barneo J. Physiology of the Carotid Body: From Molecules to Disease. Changing gas composition of the super-fusion medium from 20% O SB 2 sb , 3% CO SB 2 sb , 77% N SB 2 sb (normoxia) to 97% N SB 2 sb , 3% CO SB 2 sb (full hypoxia) is followed by a characteristic hypoxia-induced increased chemoreceptor sinus nerve activity (registration in black) serving as control. 1 CcO crystallographic changes of about 3 Ångström are displayed by CB chemoreceptor discharge. a Chemoreceptor discharge under normoxic and hypoxic CO application (red registration line) The effect of CO is shown as a percentage of peak chemoreceptor activity induced by 4-min hypoxia. [Extracted from the article]