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.

Selective mu and kappa Opioid Agonists Inhibit Voltage-Gated Ca2+ Entry in Isolated Neonatal Rat Carotid Body Type I Cells.

It is known that opioids inhibit the hypoxic ventilatory response in part via an action at the carotid body, but little is known about the cellular mechanisms that underpin this. This study's objectives were to examine which opioid receptors are located on the oxygen-sensing carotid body type I cells from the rat and determine the mechanism by which opioids might inhibit cellular excitability.Immunocytochemistry revealed the presence of µ and κ opioid receptors on type I cells. The µ-selective agonist DAMGO (10 µM) and the κ-selective agonist U50-488 (10 µM) inhibited high K(+) induced rises in intracellular Ca(2+) compared with controls. After 3 h incubation (37 °C) with pertussis toxin (150 ng ml(-1)), DAMGO (10 µM) and U50-488 (10 µM) had no significant effect on the Ca(2+) response to high K(+).These results indicate that opioids acting at µ and κ receptors inhibit voltage-gated Ca(2+) influx in rat carotid body type I cells via G(i)-coupled mechanisms. This mechanism may contribute to opioid's inhibitory actions in the carotid body.

The CamKKß Inhibitor STO609 Causes Artefacts in Calcium Imaging and Selectively Inhibits BKCa in Mouse Carotid Body Type I Cells.

It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells. This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKß, in the acute response to hypoxia in isolated mouse type I cells.Initial data indicated several previously unreported artefacts associated with using the CamKKß inhibitor STO609 and Ca(2+) imaging techniques. Most importantly Fura-2 and X-Rhod1 imaging revealed that STO609 quenched emission fluorescence even in the absence of intracellular Ca(2+) ([Ca(2+)](I)). Furthermore, STO609 (100 µM) rapidly inhibited outward macroscopic currents and this inhibition was abolished in the presence of the selective BK(Ca) inhibitor paxilline.Taken together these data suggest that ST0609 should be used with caution during Ca(2+) imaging studies as it can directly interact with Ca(2+) binding dyes. The rapid inhibitory effect of STO609 on BK(Ca) was unexpected as the majority of studies using this compound required an incubation of approximately 10 min to inhibit the kinase. Furthermore, as AMPK activation inhibits BK(Ca), inhibiting AMPK's upstream kinases would, if anything, be predicted to have the opposite effect on BK(Ca). Future work will determine if the inhibition of BK(Ca) is via CamKKß or via an off target action of STO609 on the channel itself.

Acutely Administered Leptin Increases [Ca2+] i and BK Ca Currents But Does Not Alter Chemosensory Behavior in Rat Carotid Body Type I Cells.

Obesity related pathologies are the health care crisis of our generation. The fat cell derived adipokine leptin has been shown to be a central stimulant of respiration. Very high levels of leptin, however, are associated with the depressed ventilatory phenotype observed in obesity hypoventilation syndrome. Leptin receptors have been identified on carotid body type I cells but how their activation might influence the physiology of these cells is not known.The acute application of leptin evoked calcium signaling responses in isolated type I cells. Cells increased their Fura 2 ratio by 0.074 ± 0.010 ratio units (n = 39, P < 0.001). Leptin also increased the peak membrane currents in 6 of 9 cells increasing the peak macroscopic currents at +10 mV by 61 ± 14 % (p < 0.02). Leptin administered in the presence of the selective BK(Ca) channel inhibitor Paxilline (0.5 µM) failed to increase membrane currents (n = 5). Interestingly, leptin did not significantly alter the resting membrane potential of isolated type I cells (n = 9) and anoxic/acidic depolarizations were unaffected by leptin (n = 7, n = 6).These data suggest that leptin receptors are functional in type I cells but that their acute activation does not alter chemosensory properties. Future studies will use chronic models of leptin dysregulation.

Evidence for functional, inhibitory, histamine H3 receptors in rat carotid body Type I cells.

The Type I cells are the sensory elements of the carotid bodies and play a critical role in defining the ventilatory response to hypoxia and hypercapnia. Type I cells release multiple neurotransmitters during a chemosensory stimulus resulting in increased firing of the carotid sinus nerve and modification of the breathing pattern. While much is known about the actions of individual neurotransmitters in this system, very little is known about how multiple neurotransmitters may integrate to shape the output of the carotid body. Recent data has indicated that the neurotransmitter histamine does not excite isolated Type I cells despite being released during hypoxia and its receptors being present on the Type I cells. Here the hypothesis that histamine might modulate an excitatory neurotransmitter such as acetylcholine was tested. Using calcium imaging techniques it was found that histamine attenuated calcium signaling events initiated by the muscarinic acetylcholine receptor agonist acetyl-beta-methylcholine via an H3 receptor mediated mechanism. In summary, these results suggest that when acetylcholine and histamine are co-released from Type I cells in response to chemostimuli, histamine may attenuate or modulate the excitatory presynaptic actions of acetylcholine.

Ion channel regulation by AMPK: the route of hypoxia-response coupling in thecarotid body and pulmonary artery.

Vital homeostatic mechanisms monitor O2 supply and adjust respiratory and circulatory function to meet demand. The pulmonary arteries and carotid bodies are key systems in this respect. Hypoxic pulmonary vasoconstriction (HPV) aids ventilation-perfusion matching in the lung by diverting blood flow from areas with an O2 deficit to those rich in O2, while a fall in arterial pO2 increases sensory afferent discharge from the carotid body to elicit corrective changes in breathing patterns. We discuss here the new concept that hypoxia, by inhibiting oxidative phosphorylation, activates AMP-activated protein kinase (AMPK) leading to consequent phosphorylation of target proteins, such as ion channels, which initiate pulmonary artery constriction and carotid body activation. Consistent with this view, AMPK knockout mice exhibit an impaired ventilatory response to hypoxia. Thus, AMPK may be sufficient and necessary for hypoxia-response coupling and may regulate O2 and thereby energy (ATP) supply at the whole body as well as the cellular level.