1. In this, and the accompanying paper (Duchen & Biscoe, 1992), we test the hypothesis that the oxygen sensitivity of mitochondrial electron transport forms a basis for transduction in the carotid body, the primary peripheral arterial oxygen sensor. We here describe for isolated type I cells the changes in autofluorescence of mitochondrial NAD(P)H that accompany changes in P(o2). 2. NAD(P)H autofluorescence (excitation, 340-360 nm; emission peak, 450 nm) increased with anoxia, reflecting a rise in the NAD(P)H/NAD(P) ratio. Graded increases in autofluorescence were seen in response to graded decreases in P(o2), suggesting that mitochondrial function is progressively altered below a P(o2) of about 60 mmHg. 3. A mitochondrial origin for the NAD(P)H autofluorescence was suggested by the mutual exclusion of the responses to anoxia and cyanide. 4. Oxidized flavoproteins fluoresce when excited at 450 nm with an emission peak at 550 nm. The small signals obtained under these conditions increased with uncoupler and showed a graded decrease with falling P(o2) reflecting a rise in the FADH/FAD ratio. 5. Hypoxia raises [Ca2+]i. The hypoxia-induced changes in mitochondrial function were not secondary to this rise. A brief K+-induced depolarization leads to a transient increase in [Ca2+]i. At the same time there is a rapid decrease in NAD(P)H autofluorescence followed by an increase that far outlasts the rise in [Ca2+]i. This delayed increase in autofluorescence was smaller than was the increase with anoxia, even though K+-induced depolarization raised [Ca2+]i more than does anoxia. In Ca2+-free solutions the depolarization-induced changes were abolished, while those associated with hypoxia were maintained. 6. The changes of autofluorescence with K+-induced depolarization appear to reflect (i) oxidation of NAD(P)H by stimulation of respiration following mitochondrial Ca2+ uptake and (ii) reduction of NAD(P) by the Ca2+-dependent activation of mitochondrial dehydrogenases. This activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in P(o2). 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the P(o2) fell below about 5 mmHg. 8. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (DELTA-F(FCCP)). Blockade of electron transport by anoxia or CN- decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (DELTA-F(CN)). The fractional change in autofluorescence with FCCP, DELTA-F(FCCP)/(DELTA-F(FCCP + F(CN)), is thus a measure of resting O2 consumption. In type I cells, this gave a mean of 0.27 (S.D. +/- 0.08, n = 18). This contrasts with similarly dissociated mouse dorsal root ganglion (DRG) neurons, for which the mean ratio was 0.53 (S.D. +/- 0.14, n = 14), with DRG cells in culture (mean of 0.64, S.D. +/- 0.16, n = 16) or with freshly dissociated chromaffin cells (mean 0.45 +/- 0.05, n = 10). This suggests that the type I cells have an unusually high resting O2 consumption, near their maximal capacity. 9. These data suggest that (i) mitochondrial function alters over the physiologically relevant range of P(o2) values and (ii) that the high oxygen consumption of the tissue reflects that of the type I cell. They are consistent with the hypothesis that specialized mitochondrial electron transport confers on these cells their unusual oxygen sensitivity.
