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 < 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.

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.

Preventing acute asthmatic symptoms by targeting a neuronal mechanism involving carotid body lysophosphatidic acid receptors.

Asthma accounts for 380,000 deaths a year. Carotid body denervation has been shown to have a profound effect on airway hyper-responsiveness in animal models but a mechanistic explanation is lacking. Here we demonstrate, using a rat model of asthma (OVA-sensitized), that carotid body activation during airborne allergic provocation is caused by systemic release of lysophosphatidic acid (LPA). Carotid body activation by LPA involves TRPV1 and LPA-specific receptors, and induces parasympathetic (vagal) activity. We demonstrate that this activation is sufficient to cause acute bronchoconstriction. Moreover, we show that prophylactic administration of TRPV1 (AMG9810) and LPA (BrP-LPA) receptor antagonists prevents bradykinin-induced asthmatic bronchoconstriction and, if administered following allergen exposure, reduces the associated respiratory distress. Our discovery provides mechanistic insight into the critical roles of carotid body LPA receptors in allergen-induced respiratory distress and suggests alternate treatment options for asthma.

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 H2 S, 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.

AMPK Knockdown in Placental Labyrinthine Progenitor Cells Results in Restriction of Critical Energy Resources and Terminal Differentiation Failure.

Placental abnormalities can cause Pregnancy-Associated Disorders, including preeclampsia, intrauterine growth restriction, and placental insufficiency, resulting in complications for both the mother and fetus. Trophoblast cells within the labyrinthine layer of the placenta facilitate the exchange of nutrients, gases, and waste between mother and fetus; therefore, the development of this cell layer is critical for fetal development. As trophoblast cells differentiate, it is assumed their metabolism changes with their energy requirements. We hypothesize that proper regulation of trophoblast metabolism is a key component of normal placental development; therefore, we examined the role of AMP-activated kinase (AMPK, PRKAA1/2), a sensor of cellular energy status. Our previous studies have shown that AMPK knockdown alters both trophoblast differentiation and nutrient transport. In this study, AMPKα1/2 shRNA was used to investigate the metabolic effects of AMPK knockdown on SM10 placental labyrinthine progenitor cells before and after differentiation. Extracellular flux analysis confirmed that AMPK knockdown was sufficient to reduce trophoblast glycolysis, mitochondrial respiration, and ATP coupling efficiency. A reduction in AMPK in differentiated trophoblasts also resulted in increased mitochondrial volume. These data indicate that a reduction in AMPK disrupts cellular metabolism in both progenitors and differentiated placental trophoblasts. This disruption correlates to abortive trophoblast differentiation that may contribute to the development of Pregnancy-Associated Disorders.

Physiological, Behavioral, and Histological Responses of Male C57BL/6N Mice to Different CO2 Chamber Replacement Rates.

Rodent euthanasia with CO2 by using gradual displacement of 10% to 30% of the chamber volume per minute is considered acceptable by the AVMA Panel on Euthanasia. However, whether a 50% to 100% chamber replacement rate (CRR) of CO2 is more painful or distressful than 10% to 30% CRR is unclear. Therefore, we examined physiological and behavioral parameters, corticosterone and ACTH levels, and lung histology of mice euthanized at CRR of 15%, 30%, 50%, or 100%. Adult male C57BL/6N mice were euthanized at different CO2 CRR as physiological parameters were recorded telemetrically. Video recordings were reviewed to determine when the mouse first became ataxic, when it was fully recumbent (characterized by the mouse's nose resting on the cage floor), and when breathing stopped. Overall, CO2 euthanasia increased cardiovascular parameters and activity. Specific significant differences that were associated with 50% to 100% compared with 15% to 30% CO2 CRR included an increase in systolic blood pressure per second from initiation of CO2 until ataxia, a decrease in total diastolic blood pressure until ataxia, and a decrease in total heart rate until ataxia, immobility, and death. All physiological responses occurred more rapidly with higher CRR. Activity levels, behavioral responses, plasma adrenocorticotropic hormone and corticosterone levels, and lung pathology were not different between groups. We found no physiological, behavioral, or histologic evidence that 15% or 30% CO2 CRR is less painful or distressful than is 50% or 100% CO2 CRR. We conclude that 50% to 100% CO2 CRR is acceptable for euthanizing adult male C57BL/6N mice.

Adrenaline release evokes hyperpnoea and an increase in ventilatory CO2 sensitivity during hypoglycaemia: a role for the carotid body.

KEY POINTS: Hypoglycaemia is counteracted by release of hormones and an increase in ventilation and CO2 sensitivity to restore blood glucose levels and prevent a fall in blood pH. The full counter-regulatory response and an appropriate increase in ventilation is dependent on carotid body stimulation. We show that the hypoglycaemia-induced increase in ventilation and CO2 sensitivity is abolished by preventing adrenaline release or blocking its receptors. Physiological levels of adrenaline mimicked the effect of hypoglycaemia on ventilation and CO2 sensitivity. These results suggest that adrenaline, rather than low glucose, is an adequate stimulus for the carotid body-mediated changes in ventilation and CO2 sensitivity during hypoglycaemia to prevent a serious acidosis in poorly controlled diabetes. ABSTRACT: Hypoglycaemia in vivo induces a counter-regulatory response that involves the release of hormones to restore blood glucose levels. Concomitantly, hypoglycaemia evokes a carotid body-mediated hyperpnoea that maintains arterial CO2 levels and prevents respiratory acidosis in the face of increased metabolism. It is unclear whether the carotid body is directly stimulated by low glucose or by a counter-regulatory hormone such as adrenaline. Minute ventilation was recorded during infusion of insulin-induced hypoglycaemia (8-17 mIU kg(-1)  min(-1) ) in Alfaxan-anaesthetised male Wistar rats. Hypoglycaemia significantly augmented minute ventilation (123 ± 4 to 143 ± 7 ml min(-1) ) and CO2 sensitivity (3.3 ± 0.3 to 4.4 ± 0.4 ml min(-1)  mmHg(-1) ). These effects were abolished by either ß-adrenoreceptor blockade with propranolol or adrenalectomy. In this hypermetabolic, hypoglycaemic state, propranolol stimulated a rise in P aC O2, suggestive of a ventilation-metabolism mismatch. Infusion of adrenaline (1 µg kg(-1)  min(-1) ) increased minute ventilation (145 ± 4 to 173 ± 5 ml min(-1) ) without altering P aC O2 or pH and enhanced ventilatory CO2 sensitivity (3.4 ± 0.4 to 5.1 ± 0.8 ml min(-1)  mmHg(-1) ). These effects were attenuated by either resection of the carotid sinus nerve or propranolol. Physiological concentrations of adrenaline increased the CO2 sensitivity of freshly dissociated carotid body type I cells in vitro. These findings suggest that adrenaline release can account for the ventilatory hyperpnoea observed during hypoglycaemia by an augmented carotid body and whole body ventilatory CO2 sensitivity.

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.

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.

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.

AMPK knockdown in placental trophoblast cells results in altered morphology and function.

The placenta is a transient organ that develops upon the initiation of pregnancy and is essential for embryonic development and fetal survival. The rodent placenta consists of distinct lineages and includes cell types that are analogous to those that make up the human placenta. Trophoblast cells within the labyrinth layer, which lies closest to the fetus, fuse and come in contact with maternal blood, thus facilitating nutrient and waste exchange between the mother and the baby. Abnormalities of the placenta may occur as a result of cellular stress and have been associated with pregnancy-associated disorders: such as preeclampsia, intrauterine growth restriction, and placental insufficiency. Cellular stress has also been shown to alter proliferation and differentiation rates of trophoblast cells. This stress response is important for cell survival and ensures continued placental functionality. AMP-activated protein kinase is an important sensor of cellular metabolism and stress. To study the role of AMPK in the trophoblast cells, we used RNA interference to simultaneously knockdown levels of both the AMPK alpha isoforms, AMPKα1 and AMPKα2. SM10 trophoblast progenitor cells were transduced with AMPKα1/2 shRNA and stable clones were established to analyze the effects of AMPK knockdown on important cellular functions. Our results indicate that a reduction in AMPK levels causes alterations in cell morphology, growth rate, and nutrient transport, thus identifying an important role for AMPK in the regulation of placental trophoblast differentiation.

Ion channel regulation by the LKB1-AMPK signalling pathway: the key to carotid body activation by hypoxia and metabolic homeostasis at the whole body level.

Our recent investigations provide further support for the proposal that, consequent to inhibition of mitochondrial oxidative phosphorylation, activation of AMP-activated protein kinase (AMPK) mediates carotid body excitation by hypoxia. Consistent with the effects of hypoxia, intracellular dialysis from a patch pipette of an active (thiophosphorylated) recombinant AMPK heterotrimer (α2ß2γ1) or application of the AMPK activators AICAR and A769662: (1) Inhibited BK(Ca) currents and TASK K(+) currents in rat carotid body type I cells; (2) Inhibited whole-cell currents carried by KCa1.1 and TASK3, but not TASK1 channels expressed in HEK293 cells; (3) Triggered carotid body activation. Furthermore, preliminary studies using mice with conditional knockout in type I cells of the primary upstream kinase that activates AMPK in response to metabolic stresses, LKB1, appear to confirm our working hypothesis. Studies on mice with knockout of the catalytic α1 subunit and α2 subunits of AMPK, respectively, have proved equally consistent. Accumulating evidence therefore suggests that the LKB1-AMPK signalling pathway is necessary for hypoxia-response coupling by the carotid body, and serves to regulate oxygen and therefore energy supply at the whole body level.

Knockdown of AMP-activated protein kinase alpha 1 and alpha 2 catalytic subunits.

AMP-activated protein kinase (AMPK) is a master metabolic regulator that responds to the AMP: ATP ratio and promotes ATP production when the cell is low on energy. There are two isoforms of the catalytic alpha subunit, AMPKα1 and AMPKα2. Here, we describe the production of a small interfering RNA (siRNA) and a short hairpin RNA (shRNA) targeting both catalytic isoforms of AMPK in human, mouse, and rat. Multiple loop sequences were tested to generate the most effective shRNA. The shRNA causes significant knockdown of both isoforms of AMPKα in mouse and human cells. The shRNA effectively knocked down AMPKα1 and AMPKα2 protein levels, compared to a five basepair mismatch-control shRNA in mouse fibroblast NIH3T3 cells and significantly knocked down AMPKα1 (63%) and AMPKα2 (72%) levels compared to control in human embryonic kidney cells, HEK293s. The shRNA also causes a significant reduction in AMPK activity, measured as phosphorylation of acetyl-CoA carboxylase (ACC), a direct phosphorylation target. While the protein levels of total ACC remained the same between the AMPKα1and α2 shRNA and control shRNA-treated cells, there was a 41% reduction in phospho-ACC protein levels. The generation of this AMPKα1and α2 shRNA can be used to stably knock down protein levels and activity of both catalytic isoforms of AMPK in different species to assess function.

Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase.

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

Inhibition of adenylate cyclase attenuates muscarinic Ca²(+) signaling by a PKA-independent mechanism in rat carotid body Type I cells.

Carotid body (CB) Type I cells respond to hypoxia by releasing excitatory and inhibitory neurotransmitters. This mechanism leads to increased firing of the carotid sinus nerve (CSN) which alters breathing to maintain blood gases within the physiological range. Acetylcholine targets both muscarinic and nicotinic receptors in the rat CB, acting postsynaptically on CSN and presynaptically on Type I cells. Muscarinic Ca²(+) signaling is inhibited by the activation of G(i)-coupled receptors including histamine H3 receptors. Here inhibition of adenylate cyclase with SQ22536 mimicked H3 receptor activation. Using Ca²(+) imaging techniques it was observed that inhibition of muscarinic Ca²(+) signaling was independent of protein kinase A (PKA) as PKA inhibitors H89 and KT5720 were without effect on the muscarinic Ca²(+) response. By contrast the Epac (exchange protein activated by cAMP) inhibitor brefeldin A inhibited muscarinic Ca²(+) signaling whereas the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiated Ca²(+) signaling. Thus in Type I cells inhibition of adenylate cyclase inhibited muscarinic Ca²(+) signaling via a PKA-independent pathway that may rely upon modulation of Epac.

Mechanisms for acute oxygen sensing in the carotid body.

Hypoxic chemotransduction in the carotid body requires release of excitatory transmitters from type I cells that activate afferent sensory neurones. Transmitter release is dependent on voltage-gated Ca2+ entry which is evoked by membrane depolarization. This excitatory response to hypoxia is initiated by inhibition of specific O2 sensitive K+ channels, of which several types have been reported. Here, we discuss mechanisms which have been put forward to account for hypoxic inhibition of type I cell K+ channels. Whilst evidence indicates that one O2 sensitive K+ channel, BKCa, may be regulated by gasotransmitters (CO and H2S) in an O2-dependent manner, other studies now indicate that activation of AMP-activated protein kinase (AMPK) accounts for inhibition of both BKCa and 'leak' O2 sensitive K+ channels, and perhaps also other O2 sensitive K+ channels reported in different species. We propose that type I cell AMPK activation occurs as a result of inhibition of mitochondrial oxidative phosphorylation, and does not require increased production of reactive oxygen species. Thus, AMPK activation provides the basis for unifying the 'membrane' and 'mitochondrial' hypotheses, previously regarded as disparate, to account for hypoxic chemotransduction.

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.

Presynaptic regulation of isolated neonatal rat carotid body type I cells by histamine.

The type I cells are the chemoreceptive elements of the carotid bodies and are critical in defining the ventilatory response to hypoxia and hypercapnia. Recent evidence has suggested that histamine is released by the carotid body in response to hypoxia and acts as an excitatory neurotransmitter. Here we use isolated neonatal rat type I cells to assess the presynaptic actions of histamine and define the receptor subtypes that mediate them. All four histamine receptor subtypes are expressed on the type I cells, however activation of these receptors with histamine or selective agonists caused no rise in intracellular calcium ([Ca(2+)](i)) and histamine did not augment calcium entry or modulate macroscopic currents evoked in type I cells. Thus activation of histamine receptors on type I cells is unlikely to provide a presynaptic positive feedback mechanism during chemotransduction and any excitatory role attributed to the actions of histamine is likely to come from a postsynaptic effect on the carotid sinus nerve (CSN).