The mechanism of oxygen sensing in arterial chemoreceptors is unknown but has often been linked to mitochondrial function. This proposal is supported by a wealth of data demonstrating that arterial chemoreceptors are powerfully excited by all inhibitors of oxidative phosphorylation (Heymans 1931; Shen & Hauss, 1939; Anichkov & Belenkii, 1963; Mulligan 1981; Mulligan & Lahiri, 1982; Wilson 1994). In the type-1 cell these metabolic poisons elicit a classic pattern of sensory neuronal excitation comprising modulation of ion channels to generate a receptor potential which then stimulates electrical activity, calcium influx and neurosecretion (Obeso 1989; Buckler & Vaughan-Jones, 1998; Ortega Saenz 2003; Williams & Buckler, 2004; Wyatt & Buckler, 2004; Varas 2007). The existence of a metabolic signalling pathway in these cells therefore seems well established; the contentious issue is whether this pathway is the same as that used for acute oxygen sensing. The standard criticism of the metabolic hypothesis is that cytochrome oxidase has such a high affinity for oxygen that physiological hypoxia should have no effect upon mitochondrial energy metabolism. This assertion has rarely been directly tested, but three key studies have reported mitochondrial function in the carotid body to have an extraordinarily high sensitivity to hypoxia (Mills & J?bsis, 1970, 1972; Nair 1986; Duchen & Biscoe, 1992converter, with gain 10 nA V-1, connected to the PMT. An offset was applied such that 0 (volts) corresponds to Licofelone IC50 the level of background signal in the absence of cells. Figure 3 Effects of graded hypoxia on NADH autofluorescence in SCG neuron Measurement of mitochondrial membrane potential using rhodamine 123 Mitochondrial membrane potential (m) was monitored using rhodamine 123 (Rh123) in the dequench mode. Rh123 is a fluorescent membrane-permeant cation which passively distributes across membranes according to the membrane potential. When cells are incubated in a solution containing Rh123 it is therefore taken up into the cell and then concentrated within the mitochondria, which have a very negative membrane potential. Under suitable loading conditions Rh123 uptake into mitochondria is associated with partial quenching of its fluorescence (Emaus 1986). Once loaded, changes in m cause redistribution of Rh123 between mitochondria and cytosol with consequent changes in the degree of fluorescence quenching; for example, mitochondrial depolarisation causes Rh123 efflux from the mitochondrial matrix into the cytosol resulting in decreased quenching and thus an increase in overall Rh123 fluorescence (Emaus 1986; Chen, 1988). Although we cannot directly calibrate Rh123 fluorescence changes in terms of m (see Discussion) we have applied some correction for dye bleaching/leakage. Baseline Rh123 fluorescence (measured under specified conditions) and the maximum fluorescence observed in the presence of 1 m carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, a mitochondrial uncoupler) were determined before and after experiments with hypoxia. Linear interpolation was then used to estimate Mouse monoclonal to C-Kit baseline and maximum fluorescence during the intervening recording so that data obtained at any time point could be normalised to a 0% (baseline) to 100% (FCCP) scale. The baseline condition varied depending on the experiment (see Results and figure legends). Raw data traces (Figs 4and 6converter (gain 10 nA V-1). Figure 4 Effects of hypoxia on mitochondrial membrane potential in type-1 cells Figure 5 Effects of hypoxia on electron transport Figure 6 Effects of hypoxia on cytochrome oxidase activity Solutions Standard bicarbonate-buffered Tyrode solutions contained (in mm): NaCl, 117; KCl, 4.5; CaCl2, 2.5; MgCl2, 1; NaHCO3, 23; glucose, 11. In Ca2+-free solutions CaCl2 was omitted and 100 m EGTA added. Normoxic solutions were equilibrated with 5% CO2 and 95% air, hypoxic solutions were equilibrated with 5% CO2 and 10, 5, 2.5, 1 and 0.5% oxygen (balance nitrogen). Anoxic solutions were produced by equilibration with 5% CO2/95% N2 followed by the addition of 100C200 m Na2S2O4 (in experiments involving nickel, Licofelone IC50 Na2S2O4 was added immediately before use). The of the control solution was assumed to be 150 mm Hg and that of the anoxic solution to be 0 mm Hg. The of hypoxic solutions was determined in the recording chamber using a Licofelone IC50 50 m Licofelone IC50 fibre optic oxygen sensor (PreSens, Regensburg, Germany) placed close to the bottom of the recording chamber and calibrated using control and anoxic solutions test for simple comparisons (in Excel, Microsoft), (ii) repeated-measures one-way analysis of variance (RM-ANOVA) with testing against control by the Holm Sidak method (Sigmaplot 12, Sysstat Software Inc, Germany), (iii) RM-ANOVA on ranks with testing against control by the Dunnet method (Sigmaplot 12). Significance was assumed at < 0.05. response curves shown in Figs 2C6 are three-parameter hyperbolas of the form is the at which a half maximal effect would.