Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET-based study

Abstract

Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels (n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47(phox) and p67(phox). In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells (n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O2-sensitive dye. We conclude that attenuated p47(phox) subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.

Measurement of ROS Levels and Membrane Potential Dynamics in the Intact Carotid Body Ex Vivo

“…7.2a shows a remarkable heterogeneity in the hypoxic ROS response of type I cells. 3 CBs responded with an increase in ROS levels as recently shown for animals from IVC systems (Bernardini et al 2014 ). 1 CB did not show any notable change in ROS levels and 2 CBs The reversal of chronic hypoxic into normoxic animals (as far as the CB ROS response is concerned) seems to have been partially successful and might require therefore more adaptation time for a full transition.…”

“…As recently shown by our group through FRET measurements using the HSP-FRET construct CB type I cells can show both an increase and a decrease as response to hypoxic challenges (Bernardini et al 2014 ). Figure 7.1 shows an example of a successful transfection of HSP-FRET into CB type I cells (Fig.…”

“…Preparations of CBs were made as previously described (Bernardini et al 2014 ). Briefl y, animals were euthanized by isofl urane inhalation and the bifurcation of the common carotid artery dissected immediately thereafter and kept in icecold phosphate buffered saline until fi ne preparation was done, where connective and fat tissues were removed.…”

“…Recent work from our group (Bernardini et al 2014 ) showed that both ROS results (increase and decrease) can be observed in mouse CB preparations. We suspected differences in the used caging systems to be the cause for the observed heterogeneity.…”

“…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.…”