Acute Oxygen-Sensing via Mitochondria-Generated Temperature Transients in Rat Carotid Body Type I Cells

The Carotid Bodies (CB) are peripheral chemoreceptors that detect changes in arterial oxygenation and, via afferent inputs to the brainstem, correct the pattern of breathing to restore blood gas homeostasis. Herein, preliminary evidence is presented supporting a novel oxygen-sensing hypothesis which suggests CB Type I cell “hypoxic signaling” may in part be mediated by mitochondria-generated thermal transients in TASK-channel-containing microdomains. Distances were measured between antibody-labeled mitochondria and TASK-potassium channels in primary rat CB Type I cells. Sub-micron distance measurements (TASK-1: 0.33 ± 0.04 µm, n = 47 vs TASK-3: 0.32 ± 0.03 µm, n = 54) provided evidence for CB Type I cell oxygen-sensing microdomains. A temperature-sensitive dye (ERthermAC) indicated that inhibition of mitochondrial activity in isolated cells caused a rapid and reversible inhibition of mitochondrial thermogenesis and thus temperature in these microdomains. Whole-cell perforated-patch current-clamp electrophysiological recordings demonstrated sensitivity of resting membrane potential (Vm) to temperature: lowering bath temperature from 37°C to 24°C induced consistent and reversible depolarizations (Vm at 37°C: -48.4 ± 4.11 mV vs 24°C: -31.0 ± 5.69 mV; n = 5; p \u3c 0.01). These data suggest that hypoxic inhibition of mitochondrial thermogenesis may play an important role in oxygen chemotransduction in the CB. A reduction in temperature within cellular microdomains will inhibit plasma membrane ion channels, influence the balance of cellular phosphorylation–dephosphorylation, and may extend the half-life of reactive oxygen species. The characterization of a thermosensory chemotransduction mechanism, that may also be used by other oxygen-sensitive cell types and may impact multiple other chemotransduction mechanisms is critical if we are to fully understand how the CBs, and potentially other oxygen-sensitive cells, respond to hypoxia

Measuring the Effects of High-Fat Diet on Breathing and Oxygen-Sensitivity of the Carotid Body Type I Cell

The carotid bodies (CB), the primary peripheral chemoreceptors, respond to changes in blood gases with neurotransmitter release, thereby increasing carotid sinus nerve firing frequency and ultimately correcting the pattern of breathing. It has previously been demonstrated that acute application of the adipokine leptin caused perturbations of intracellular calcium and membrane ion movement in isolated CB Type I cells (Pye et al, 2015) and augmented the response of the intact CB to hypoxia (Pye et al, 2016). This study\u27s aim was to examine, in-vivo, if elevated leptin modulated CB function and breathing. Rats were fed high-fat chow or control chow for 16-weeks. High-fat fed (HFF) animals gained significantly more weight compared to control fed (CF) animals (n=18; p\u3c.001; 512.56 g +/- 14.70 g vs. 444.11 g +/- 7.09 g). HFF animals also had significantly higher serum leptin levels compared to CF (n=18; p\u3c.0001; 3.05 ng/mL +/- 0.24 ng/mL vs. 1.29 ng/mL +/- 0.12 ng/mL). Whole-body plethysmography was used to test the acute hypoxic ventilatory response (HVR) in unrestrained, conscious animals. HFF animals had an attenuated 2nd-phase of the HVR when compared to CF (n=18; p\u3c.05; 710.1 +/- 41.9 mL kg-1 min-1 vs. 855.4 +/- 44.05 mL kg-1 min-1). CB Type I cells were isolated and intracellular calcium measured; no significant differences in the cellular hypoxic responses between groups were observed. These data show differences in the 2nd-phase of the HVR caused by high fat feeding are unlikely to be caused by an action of leptin on the Type I cells. However the possibility remains that leptin may have in-vivo postsynaptic effects on the carotid sinus nerve; this remains to be investigated

Measuring the Effects of High-Fat Diet on Breathing and Oxygen-Sensitivity of the Carotid Body Type I Cell

The carotid bodies (CB), the primary peripheral chemoreceptors, respond to changes in blood gases with neurotransmitter release, thereby increasing carotid sinus nerve firing frequency and ultimately correcting the pattern of breathing. It has previously been demonstrated that acute application of the adipokine leptin caused perturbations of intracellular calcium and membrane ion movement in isolated CB Type I cells (Pye et al, 2015) and augmented the response of the intact CB to hypoxia (Pye et al, 2016). This study\u27s aim was to examine, in-vivo, if elevated leptin modulated CB function and breathing. Rats were fed high-fat chow or control chow for 16-weeks. High-fat fed (HFF) animals gained significantly more weight compared to control fed (CF) animals (n=18; p\u3c.001; 512.56 g +/- 14.70 g vs. 444.11 g +/- 7.09 g). HFF animals also had significantly higher serum leptin levels compared to CF (n=18; p\u3c.0001; 3.05 ng/mL +/- 0.24 ng/mL vs. 1.29 ng/mL +/- 0.12 ng/mL). Whole-body plethysmography was used to test the acute hypoxic ventilatory response (HVR) in unrestrained, conscious animals. HFF animals had an attenuated 2nd-phase of the HVR when compared to CF (n=18; p\u3c.05; 710.1 +/- 41.9 mL kg-1 min-1 vs. 855.4 +/- 44.05 mL kg-1 min-1). CB Type I cells were isolated and intracellular calcium measured; no significant differences in the cellular hypoxic responses between groups were observed. These data show differences in the 2nd-phase of the HVR caused by high fat feeding are unlikely to be caused by an action of leptin on the Type I cells. However the possibility remains that leptin may have in-vivo postsynaptic effects on the carotid sinus nerve; this remains to be investigated

Acute Oxygen Sensing by the Carotid Body: A Rattlebag of Molecular Mechanisms

The molecular underpinnings of the oxygen sensitivity of the carotid body Type I cells are becoming better defined as research begins to identify potential interactions between previously separate theories. Nevertheless, the field of oxygen chemoreception still presents the general observer with a bewildering array of potential signalling pathways by which a fall in oxygen levels might initiate Type I cell activation. The purpose of this brief review is to address five of the current oxygen sensing hypotheses: the lactate–Olfr 78 hypothesis of oxygen chemotransduction; the role mitochondrial ATP and metabolism may have in chemotransduction; the AMP‐activated protein kinase hypothesis and its current role in oxygen sensing by the carotid body; reactive oxygen species as key transducers in the oxygen sensing cascade; and the mechanisms by which H2S, reactive oxygen species and haem oxygenase may integrate to provide a rapid oxygen sensing transduction system. Over the previous 15 years several lines of research into acute hypoxic chemotransduction mechanisms have focused on the integration of mitochondrial and membrane signalling. This review places an emphasis on the subplasmalemmal–mitochondrial microenvironment in Type I cells and how theories of acute oxygen sensing are increasingly dependent on functional interaction within this microenvironment

Acute Oxygen Sensing by the Carotid Body: A Rattlebag of Molecular Mechanisms

The molecular underpinnings of the oxygen sensitivity of the carotid body Type I cells are becoming better defined as research begins to identify potential interactions between previously separate theories. Nevertheless, the field of oxygen chemoreception still presents the general observer with a bewildering array of potential signalling pathways by which a fall in oxygen levels might initiate Type I cell activation. The purpose of this brief review is to address five of the current oxygen sensing hypotheses: the lactate–Olfr 78 hypothesis of oxygen chemotransduction; the role mitochondrial ATP and metabolism may have in chemotransduction; the AMP‐activated protein kinase hypothesis and its current role in oxygen sensing by the carotid body; reactive oxygen species as key transducers in the oxygen sensing cascade; and the mechanisms by which H2S, reactive oxygen species and haem oxygenase may integrate to provide a rapid oxygen sensing transduction system. Over the previous 15 years several lines of research into acute hypoxic chemotransduction mechanisms have focused on the integration of mitochondrial and membrane signalling. This review places an emphasis on the subplasmalemmal–mitochondrial microenvironment in Type I cells and how theories of acute oxygen sensing are increasingly dependent on functional interaction within this microenvironment

Ethanol and Opioids Do Not Act Synergistically To Depress Excitation in Carotid Body Type I Cells

The combination of opioids and ethanol can synergistically depress breathing and the acute ventilatory response to hypoxia. Multiple studies have shown that the underlying mechanisms for this may involve calcium channel inhibition in central neurons. But we have previously identified opioid receptors in the carotid bodies and shown that their activation inhibits calcium influx into the chemosensitive cells. Given that the carotid bodies contribute to the drive to breathe and underpin the acute hypoxic ventilatory response, we hypothesized that ethanol and opioids may act synergistically in these peripheral sensory organs to further inhibit calcium influx and therefore inhibit ventilation. Methods Carotid bodies were removed from 56 Sprague–Dawley rats (1021 days old) and then enzymatically dissociated to allow calcium imaging of isolated chemosensitive type I cells. Cells were stimulated with high K+ in the presence and absence of the µ-opioid agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) (10 µM), a maximal sublethal concentration of ethanol (3 g L-1, 65.1 mM) or a combination of both. Results DAMGO alone significantly inhibited Ca2+ influx but this effect was not potentiated by the high concentration of ethanol. Conclusion These results indicate for the first time that while opioids may suppress breathing via an action at the level of the carotid bodies, ethanol is unlikely to potentiate inhibition via this pathway. Thus, the synergistic effects of ethanol and opioids on ventilatory parameters are likely mediated by central rather than peripheral actions

High Fat Feeding in Rats Alters Respiratory Parameters by a Mechanism That Is Unlikely to Be Mediated by Carotid Body Type I Cells

The carotid bodies (CB) respond to changes in blood gases with neurotransmitter release, thereby increasing carotid sinus nerve firing frequency and ultimately correcting the pattern of breathing. It has previously been demonstrated that acute application of the adipokine leptin augments the hypoxic sensory response of the intact in-vitro CB (Pye RL, Roy A, Wilson RJ, Wyatt CN. FASEB J 30(1 Supplement):983.1, 2016) and isolated CB type I cell (Pye RL, Dunn EJ, Ricker EM, Jurcsisn JG, Barr BL, Wyatt CN. Arterial chemoreceptors in physiology and pathophysiology. Advances in experimental medicine and biology. Springer, Cham, 2015). This study’s aim was to examine, in-vivo, if elevated leptin modulated CB function and breathing. Rats were fed high fat or control chow for 16-weeks. High fat fed (HFF) animals gained significantly more weight compared to control fed (CF) animals and had significantly higher serum leptin levels compared to CF. Utilizing whole-body plethysmography, HFF animals demonstrated significantly depressed breathing compared to CF at rest and during hypoxia. However, amplitudes in the change in breathing from rest to hypoxia were not significantly different between groups. CB type I cells were isolated and intracellular calcium levels recorded. Averaged and peak cellular hypoxic responses were not significantly different. Despite a small but significant rise in leptin, differences in breathing caused by high fat feeding are unlikely caused by an effect of leptin on CB type I cells. However, the possibility remains that leptin may have in-vivo postsynaptic effects on the carotid sinus nerve; this remains to be investigated