R. Rigual

Oxygen and acid chemoreception in the carotid body chemoreceptors

The carotid bodies are arterial chemoreceptors that are sensitive to blood PO2, PCO2 and pH. They are the origin of reflexes that are crucial for maintaining PCO2 and pH in the internal milieu and for adjusting the O2 supply according to the metabolic needs of the organism in situations of increased demand, such as exercise and while breathing at decreased O2 partial pressures during ascent or when living at high altitude. Chemoreceptor cells of the carotid body transduce the blood-borne stimuli into a neurosecretory response that is dependent on external Ca2+. These cells have an O2-sensitive K+ current that is reversibly inhibited by low PO2. It is proposed that the depolarization produced by inhibition of this K+ current activates Ca2+ channels; Ca2+ influx and neurosecretion follow. The cells have also a potent Na(+)-Ca2+ antiporter that could be responsible for the intracellular Ca2+ rise required to trigger the release of neurotransmitters during high PCO2 or low pH stimulation.

Release of dopamine and chemoreceptor discharge induced by low pH and high PCO2 stimulation of the cat carotid body.

1. Cat carotid bodies were incubated with the precursor [3H]tyrosine to label the catecholamine deposits and then mounted in a superfusion chamber which allowed simultaneous collection of the released [3H]dopamine (DA) and recording of action potentials from the carotid sinus nerve. 2. Low pH (7.2‐6.6) superfusion of the carotid bodies for periods of 10 min produced a parallel increase in the release of [3H]DA and chemoreceptor discharge. 3. Carotid sinus nerve denervation of the carotid body 12‐15 days prior to the experiments did not modify the release of [3H]DA elicited by low pH. 4. Superfusion of the carotid bodies with Ca(2+)‐free, high‐Mg2+ (1.6 mM) media reduced basal release of [3H]DA and chemoreceptor discharge by about 30%. Release evoked by low pH was reduced by 82%. Peak and average chemoreceptor discharge recorded in response to low pH were reduced by 28%. 5. Solutions containing weak acids (sodium acetate, 10 mM), adjusted at pH 7.4, elicited release of [3H]DA and increased chemoreceptor discharge. 6. With HCO3‐CO2‐buffered superfusion media, a reduction of bicarbonate to 5.6 mM (pH 6.8), an increase in CO2 to 20% (pH 6.8), or a simultaneous increase in CO2 to 20% and bicarbonate to 90 mM (pH 7.4), resulted in all cases in a corresponding increase in [3H]DA release and chemoreceptor discharge. The most effective stimulus was 20% CO2‐pH 6.8 and the least effective 5% CO2‐5.6 mM‐HCO3‐pH 6.8. 7. Inhibition of carbonic anhydrase with acetazolamide while perfusing the carotid bodies with a 20% CO2‐equilibrated (pH 7.4) solution resulted in comparable reductions in the release of [3H]DA and chemoreceptor discharge. 8. It is concluded that the effective acidic stimulus at the carotid body chemoreceptors is an increase in hydrogen ion concentration in type I cells. It is also concluded that DA plays a critical role in the genesis of carotid sinus nerve discharges.

Caffeine inhibition of rat carotid body chemoreceptors is mediated by A2A and A2B adenosine receptors.

Caffeine, an unspecific antagonist of adenosine receptors, is commonly used to treat the apnea of prematurity. We have defined the effects of caffeine on the carotid body (CB) chemoreceptors, the main peripheral controllers of breathing, and identified the adenosine receptors involved. Caffeine inhibited basal (IC50, 210 µm) and low intensity (PO2 ≈ 66 mm Hg/30 mm K+) stimulation‐induced release of catecholamines from chemoreceptor cells in intact preparations of rat CB in vitro. Opposite to caffeine, 5′‐(N‐ethylcarboxamido)adenosine (NECA; an A2 agonist) augmented basal and low‐intensity hypoxia‐induced release. 2‐p‐(2‐Carboxyethyl)phenethyl‐amino‐5′‐N‐ethylcaboxamido‐adenosine hydrochloride (CGS21680), 2‐hexynyl‐NECA (HE‐NECA) and SCH58621 (A2A receptors agents) neither affected catecholamine release nor altered the caffeine effects. The 8‐cycle‐1,3‐dipropylxanthine (DPCPX; an A1/A2B antagonist) and 8‐(4‐{[(4‐cyanophenyl)carbamoylmethyl]‐oxy}phenyl)‐1,3‐di(n‐propyl)xanthine (MRS1754; an A2B antagonist) mimicking of caffeine indicated that caffeine effects are mediated by A2B receptors. Immunocytochemical A2B receptors were located in tyrosine hydroxylase positive chemoreceptor cells. Caffeine reduced by 52% the chemosensory discharges elicited by hypoxia in the carotid sinus nerve. Inhibition had two components with pharmacological analysis indicating that A2A and A2B receptors mediate, respectively, the low (17 × 10−9 m) and high (160 × 10−6 m) IC50 effects. It is concluded that endogenous adenosine, via presynaptic A2B and postsynaptic A2A receptors, can exert excitatory effects on the overall output of the rat CB chemoreceptors.

Chemoreception in the context of the general biology of ROS

Superoxide anion is the most important reactive oxygen species (ROS) primarily generated in cells. The main cellular constituents with capabilities to generate superoxide anion are NADPH oxidases and mitochondrial respiratory chain. The emphasis of our article is centered in critically examining hypotheses proposing that ROS generated by NADPH oxidase and mitochondria are key elements in O(2)-sensing and hypoxic responses generation in carotid body chemoreceptor cells. Available data indicate that chemoreceptor cells express a specific isoform of NADPH oxidase that is activated by hypoxia; generated ROS acting as negative modulators of the carotid body (CB) hypoxic responses. Literature is also consistent in supporting that poisoned respiratory chain can produce high amounts of ROS, making mitochondrial ROS potential triggers-modulators of the CB activation elicited by mitochondrial venoms. However, most data favour the notion that levels of hypoxia, capable of strongly activating chemoreceptor cells, would not increase the rate of ROS production in mitochondria, making mitochondrial ROS unlikely triggers of hypoxic responses in the CB. Finally, we review recent literature on heme oxygenases from two perspectives, as potential O(2)-sensors in chemoreceptor cells and as generators of bilirubin which is considered to be a ROS scavenger of major quantitative importance in mammalian cells.

Hypoxic intensity: a determinant for the contribution of ATP and adenosine to the genesis of carotid body chemosensory activity

Excitatory effects of adenosine and ATP on carotid body (CB) chemoreception have been previously described. Our hypothesis is that both ATP and adenosine are the key neurotransmitters responsible for the hypoxic chemotransmission in the CB sensory synapse, their relative contribution depending on the intensity of hypoxic challenge. To test this hypothesis we measured carotid sinus nerve (CSN) activity in response to moderate and intense hypoxic stimuli (7 and 0% O(2)) in the absence and in the presence of adenosine and ATP receptor antagonists. Additionally, we quantified the release of adenosine and ATP in normoxia (21% O(2)) and in response to hypoxias of different intensities (10, 5, and 2% O(2)) to study the release pathways. We found that ZM241385, an A(2) antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 30.8 and 72.5%, respectively. Suramin, a P(2)X antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 64.3 and 17.1%, respectively. Simultaneous application of both antagonists strongly inhibited CSN discharges elicited by both hypoxic intensities. ATP release by CB increased in parallel to hypoxia intensity while adenosine release increased preferably in response to mild hypoxia. We have also found that the lower the O(2) levels are, the higher is the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia, while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high-intensity hypoxia.

Ventilatory responses and carotid body function in adult rats perinatally exposed to hyperoxia

Hypoxia increases the release of neurotransmitters from chemoreceptor cells of the carotid body (CB) and the activity in the carotid sinus nerve (CSN) sensory fibers, elevating ventilatory drive. According to previous reports, perinatal hyperoxia causes CSN hypotrophy and varied diminishment of CB function and the hypoxic ventilatory response. The present study aimed to characterize the presumptive hyperoxic damage. Hyperoxic rats were born and reared for 28 days in 55%–60% O2; subsequent growth (to 3.5–4.5 months) was in a normal atmosphere. Hyperoxic and control rats (born and reared in a normal atmosphere) responded with a similar increase in ventilatory frequency to hypoxia and hypercapnia. In comparison with the controls, hyperoxic CBs showed (1) half the size, but comparable percentage area positive to tyrosine hydroxylase (chemoreceptor cells) in histological sections; (2) a twofold increase in dopamine (DA) concentration, but a 50% reduction in DA synthesis rate; (3) a 75% reduction in hypoxia‐evoked DA release, but normal high [K+]0‐evoked release; (4) a 75% reduction in the number of hypoxia‐sensitive CSN fibers (although responding units displayed a nearly normal hypoxic response); and (5) a smaller percentage of chemoreceptor cells that increased [Ca2+]1 in hypoxia, although responses were within the normal range. We conclude that perinatal hyperoxia causes atrophy of the CB–CSN complex, resulting in a smaller number of chemoreceptor cells and fibers. Additionally, hyperoxia damages O2‐sensing, but not exocytotic, machinery in most surviving chemoreceptor cells. Although hyperoxic CBs contain substantially smaller numbers of chemoreceptor cells/sensory fibers responsive to hypoxia they appear sufficient to evoke normal increases in ventilatory frequency.

Role of voltage‐dependent calcium channels in stimulus–secretion coupling in rabbit carotid body chemoreceptor cells

We have defined Ca2+ channel subtypes expressed in rabbit carotid body (CB) chemoreceptor cells and their participation in the stimulus‐evoked catecholamine (CA) release. Ca2+ currents (ICa) activated at –30 mV, peaked at +10 mV and were fully blocked by 200 μm Cd2+. L‐type channels (sensitive to 2 μm nisoldipine) activated at –30 mV and carried 21 ± 2% of total ICa. Non‐L‐type channels activated at potentials positive to –10 mV and carried: N channels (sensitive to 1 μmω‐conotoxin‐GVIA) 16 ± 1% of total ICa, P/Q channels (sensitive to 3 μmω‐conotoxin‐MVIIC after nisoldipine plus GVIA) 23 ± 3% of total ICa and R channels (resistant to all blockers combined) 40 ± 3% of total ICa. CA release induced by hypoxia, hypercapnic acidosis, dinitrophenol (DNP) and high K+o in the intact CB was inhibited by 79–98% by 200 μm Cd2+. Hypoxia, hypercapnic acidosis and DNP, depolarized chemoreceptor cells and eventually generated repetitive action potential discharge. Nisoldipine plus MVIIC nearly abolished the release of CAs induced by hypoxia and hypercapnic acidosis and reduced by 74% that induced by DNP. All these secretory responses were insensitive to GVIA. 30 and 100 mm K+o brought resting membrane potential (Em) of chemoreceptor cells (–48.1 ± 1.2 mV) to –22.5 and +7.2 mV, respectively. Thirty millimolar K+o‐evoked release was abolished by nisoldipine but that induced by 100 mm K+o was mediated by activation of L, N, and P/Q channels. Data show that tested stimuli depolarize rabbit CB chemoreceptor cells and elicit CA release through Ca2+ entry via voltage‐activated channels. Only L and P/Q channels are tightly coupled to the secretion of CA.

Effects of Chronic Hypoxia on Opioid Peptide and Catecholamine Levels and on the Release of Dopamine in the Rabbit Carotid Body

Abstract: Carotid body catecholamine and opioid levels were measured in rabbits exposed for 8 days to an atmosphere of 11% O2 in N2 (Po2 of ∼ 80 mm Hg) and during an identical period of recovery, i.e., after 8 days of returning to the control normoxic atmosphere. Carotid bodies show a decrease in dopamine content at day 2. Thereafter, the levels of this biogenic amine increase progressively to peak at day 10, that is, 2 days after returning to a normoxic atmosphere. Finally, dopamine levels start to decrease and reach prehypoxic control levels at day 16, that is, after 8 days of recovery. In contrast, levels of native opioid peptides remain unchanged during the whole duration of the experiment, except for a decrease at day 2 of the hypoxic exposure. Levels of total opioid peptides are also below control values at day 2 of hypoxia, increase above control values on returning to a normoxic atmosphere (maximal levels at days 10‐12), and later decrease to reach prehypoxic levels at day 16. As a result of these changes the ratios of dopamine to opioid levels show a progressive increase from day 0 to day 10 of the experiment and then return to control prehypoxic values. Carotid bodies isolated from animals that have been exposed to hypoxia for 8 days synthesize [3H]dopamine from its natural precursor [3H]tyrosine at a rate of 175 pmol/mg of protein/h, which is about double the rate of synthesis found in the carotid bodies of control animals and those allowed to recover for 8 days. The release of [3H]‐dopamine induced by mild hypoxic stimuli and by a high external K+ concentration is greater in the carotid bodies isolated from animals hypoxic for 8 days than in those of control animals (catecholamine deposits were labeled by prior incubation with [3H]tyrosine); in contrast, the carotid bodies from chronically hypoxic animals exhibit an attenuated release response to intense hypoxic stimuli and to dinitrophenol. Stimulus‐induced release of [3H]dopamine by carotid bodies isolated from animals allowed to recover for 8 days is not different from that of control animals. Our results suggest that modifications in the proportions of neurotransmitters, as well as changes in the stimulus‐secretion coupling machinery in chemoreceptor cells, contribute to the adaptative responses seen in the carotid body during high altitude acclimatization.

A revisit to O2 sensing and transduction in the carotid body chemoreceptors in the context of reactive oxygen species biology

Oxygen-sensing and transduction in purposeful responses in cells and organisms is of great physiological and medical interest. All animals, including humans, encounter in their lifespan many situations in which oxygen availability might be insufficient, whether acutely or chronically, physiologically or pathologically. Therefore to trace at the molecular level the sequence of events or steps connecting the oxygen deficit with the cell responses is of interest in itself as an achievement of science. In addition, it is also of great medical interest as such knowledge might facilitate the therapeutical approach to patients and to design strategies to minimize hypoxic damage. In our article we define the concepts of sensors and transducers, the steps of the hypoxic transduction cascade in the carotid body chemoreceptor cells and also discuss current models of oxygen- sensing (bioenergetic, biosynthetic and conformational) with their supportive and unsupportive data from updated literature. We envision oxygen-sensing in carotid body chemoreceptor cells as a process initiated at the level of plasma membrane and performed by a hemoprotein, which might be NOX4 or a hemoprotein not yet chemically identified. Upon oxygen-desaturation, the sensor would experience conformational changes allosterically transmitted to oxygen regulated K+ channels, the initial effectors in the transduction cascade. A decrease in their opening probability would produce cell depolarization, activation of voltage dependent calcium channels and release of neurotransmitters. Neurotransmitters would activate the nerve endings of the carotid body sensory nerve to convey the information of the hypoxic situation to the central nervous system that would command ventilation to fight hypoxia.

Modulation of secretion by the endoplasmic reticulum in mouse chromaffin cells

The endoplasmic reticulum (ER) has been suggested to modulate secretion either behaving as a Ca2+ sink or as a Ca2+ source in neuronal cells. Working as a Ca2+ sink, through ER‐Ca2+ pumping, it may reduce secretion induced by different stimuli. Instead, working as a Ca2+ source through the Ca2+ induced Ca2+ release (CICR) phenomenon, it may potentiate secretion triggered by activation of plasma membrane Ca2+ channels. We have previously demonstrated the presence of CICR in bovine chromaffin cells, but we now find that mouse chromaffin cells almost lack functional caffeine‐sensitive ryanodine receptors in the ER and, consistently, no CICR from the ER could be observed. In addition, inhibition of ER Ca2+ pumping with ciclopiazonic acid or thapsigargin strongly stimulated high‐K+‐evoked catecholamine secretion and cytosolic [Ca2+] ([Ca2+]c) transients. Surprisingly, 5 mm caffeine reduced high‐K+‐induced [Ca2+]c peaks but considerably potentiated secretion induced by high‐K+ stimulation. However, this potentiation was insensitive to ryanodine and additive to that induced by emptying the ER of Ca2+ with thapsigargin, suggesting that it is unrelated to the activation of ryanodine receptors. We conclude that, in mouse chromaffin cells, CICR is not functional and the ER strongly inhibits secretion by acting as a damper of the [Ca2+]c signal.