The Carotid Body Does Not Mediate the Acute Ventilatory Effects of Leptin.

Leptin is a hormone produced mostly in adipose tissue and playing a key role in the control of feeding and energy expenditure aiming to maintain a balance between food intake and metabolic activity. In recent years, it has been described that leptin might also contributes to control ventilation as the administration of the hormone reverses the hypoxia and hypercapnia commonly encountered in ob/ob mice which show absence of the functional hormone. In addition, it has been shown that the carotid body (CB) of the rat expresses leptin as well as the functional leptin-B receptor. Therefore, the possibility exists that the ventilatory effects of leptin are mediated by the CB chemoreceptors. In the experiments described below we confirm the stimulatory effect of leptin on ventilation, finding additionally that the CB does not mediate the instant to instant control of ventilation.

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.

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.

Hypoxia transduction by carotid body chemoreceptors in mice lacking dopamine D(2) receptors.

Hypoxia-induced dopamine (DA) release from carotid body (CB) glomus cells and activation of postsynaptic D(2) receptors have been proposed to play an important role in the neurotransmission process between the glomus cells and afferent nerve endings. To better resolve the role of D(2) receptors, we examined afferent nerve activity, catecholamine content and release, and ventilation of genetically engineered mice lacking D(2) receptors (D(2)(-/-) mice). Single-unit afferent nerve activities of D(2)(-/-) mice in vitro were significantly reduced by 45% and 25% compared with wild-type (WT) mice during superfusion with saline equilibrated with mild hypoxia (Po(2) approximately 50 Torr) or severe hypoxia (Po(2) approximately 20 Torr), respectively. Catecholamine release in D(2)(-/-) mice was enhanced by 125% in mild hypoxia and 75% in severe hypoxia compared with WT mice, and the rate of rise was increased in D(2)(-/-) mice. We conclude that CB transduction of hypoxia is still present in D(2)(-/-) mice, but the response magnitude is reduced. However, the ventilatory response to acute hypoxia is maintained, perhaps because of an enhanced processing of chemoreceptor input by brain stem respiratory nuclei.

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.

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 microm) and low intensity (PO2 approximately 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 x 10(-9) m) and high (160 x 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.

Hypoxia and acidosis increase the secretion of catecholamines in the neonatal rat adrenal medulla: an in vitro study.

Hypoxia elicits catecholamine (CA) secretion from the adrenal medulla (AM) in perinatal animals by acting directly on chromaffin cells. However, whether innervation of the AM, which in the rat occurs in the second postnatal week, suppresses this direct hypoxic response is the subject of debate. Opioid peptides have been proposed as mediators of this suppression. To resolve these controversies, we have compared CA-secretory responses with high external concentrations of K+ ([K+]e) and hypoxia in the AM of neonatal (1- to 2-day-old) and juvenile (14- or 15- and 30-day-old) rats subjected to superfusion in vitro. In addition, we studied the effect of hypercapnic acidosis on the CA-secretory responses in the AM during postnatal development and the possible interaction between acidic and hypoxic stimuli. Responses to high [K+]e were comparable at all ages, but responses to hypoxia and hypercapnic acidosis were maximal in neonatal animals. Suppression of the hypoxic response in the rat AM was not mediated by opioids, because their agonists did not affect the hypoxic CA response. The association of hypercapnic acidosis and hypoxia, mimicking the episodes of asphyxia occurring during delivery, generates a more than additive secretory response in the neonatal rat AM. Our data confirm the loss of the direct sensitivity to hypoxia of the AM in the initial weeks of life and demonstrate a direct response of neonatal AM to hypercapnic acidosis. The synergistic effect of hypoxia and acidosis would explain the CA outburst crucial for adaptation to extrauterine life observed in naturally delivered mammals.

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.

Single-unit recordings of arterial chemoreceptors from mouse petrosal ganglia in vitro.

A preparation was developed that allows for the recording of single-unit chemoreceptor activity from mouse carotid body in vitro. An anesthetized mouse was decapitated, and each carotid body was harvested, along with the sinus nerve, glossopharyngeal nerve, and petrosal ganglia. After exposure to collagenase/trypsin, the cleaned complex was transferred to a recording chamber where it was superfused with oxygenated saline. The ganglia was searched for evoked or spontaneous unit activity by using a glass suction electrode. Single-unit action potentials were 57 +/- 10 (SE) (n = 16) standard deviations above the recording noise, and spontaneous spikes were generated as a random process. Decreasing superfusate PO(2) to near 20 Torr caused an increase in spiking activity from 1. 3 +/- 0.4 to 14.1 +/- 1.9 Hz (n = 16). The use of mice for chemoreceptor studies may be advantageous because targeted gene deletions are well developed in the mouse model and may be useful in addressing unresolved questions regarding the mechanism of chemotransduction.

Characterization of the synthesis and release of catecholamine in the rat carotid body in vitro.

The aim of this work was to determine contents and turnover rates for dopamine (DA) and norepinephrine (NE) and to identify the catecholamine (CA) released during stimulation of the rat carotid body (CB). Turnover rates and the release of CA were measured in an in vitro preparation using a combination of HPLC and radioisotopic methods. Mean rat CB levels of DA and NE were 209 and 45 pmol/mg tissue, respectively. With [(3)H]tyrosine as precursor, rat CB synthesized [(3)H]CA in a time- and concentration-dependent manner; calculated turnover times for DA and NE were 5.77 and 11.4 h, respectively. Hypoxia and dibutyryl adenosine 3',5'-cyclic monophosphate significantly increased [(3)H]CA synthesis. In normoxia, rat CB released [(3)H]DA and [(3)H]NE in a ratio of 5:1, comparable to that of the endogenous tissue CA. Hypoxia and high K(+) preferentially released [(3)H]DA, nicotine preferentially released [(3)H]NE, and acidic stimuli released both amines in proportion to tissue content. Release of [(3)H]CA induced by hypoxia and high K(+) was nearly fully dependent on extracellular Ca(2+), whereas basal normoxic release was not altered by removal of Ca(2+) from the incubating solution. We conclude that the rat CB is an organ with higher levels of DA than NE that preferentially releases DA or NE in a stimulus-specific manner.

Developmental changes in chemoreceptor nerve activity and catecholamine secretion in rabbit carotid body: possible role of Na+ and Ca2+ currents.

In order to better understand the post-natal increase in peripheral chemoreceptor responsiveness to hypoxia, chemoreceptors of newborn (1-2 days) and older (10-12 days, 30 days, adult) rabbits were isolated and superfused, in vitro. The free tissue catecholamine concentration was measured using carbon-fiber voltammetry and pauci-fiber nerve activity was recorded from the sinus nerve during stimulation (4 min) with graded hypoxia or increased potassium. Both the peak catecholamine and peak nerve responses to stimulation with 10% and 0% oxygen increased with age, particularly between 10 and 30 days of age. In contrast, peak nerve and peak catecholamine responses to increased potassium did not significantly change with age. For a better understanding of how responsiveness increases with age, the fast Na+ and the Ca2+ currents were measured from isolated glomus cells of newborn and older rabbits, but the magnitude of the currents when normalized to membrane area was not significantly different between ages. We conclude that: (1) rabbit chemoreceptors mature in the newborn period (10-30 days) and part of this maturation is an increase in catecholamine secretion, (2) maturation of hypoxia transduction primarily occurs in steps prior to depolarization since potassium-evoked responses were not affected, and (3) an increase in the magnitude of glomus cell fast Na+ or Ca2+ currents is not a likely mechanism for the maturational change, but changes in the oxygen sensitivity of these currents cannot be excluded.

Hypoxia inhibits the synthesis of phosphoinositides in the rabbit carotid body.

Hypoxic transduction in the carotid body (CB) is regulated by several systems of second messengers, but the role of the phospholipase C system has not been studied. The aim of the present study was to characterize the turnover rate of inositol phosphates (InsPs) and phosphoinositides (PIs) and their modifications by hypoxia in the rabbit CB in vitro. In CBs, in which the PIs had been labelled previously with 3H-myo-inositol, hypoxia in the presence of LiCl did not modify the accumulation of 3H-InsPs, whilst exposure to hypoxia during the loading period in the presence of LiCl reduced the accumulation of 3H-InsPs by more than 50%. Endogenous levels of inositol 1,4,5-trisphosphate were unaltered by hypoxia. Synthesis of 3H-PIs from 3H-myo-inositol was markedly inhibited by hypoxia in the CB, but not in the rat superior cervical ganglion used as control tissue. Levels of 3H-phosphatidylinositol (3H-PtdIns), 3H-phosphatidylinositol 4-monophosphate and 3H-phosphatidylinositol 4,5-bisphosphate were similarly decreased, indicating that inhibition occurs at a step prior to PtdIns synthesis. It is concluded that the phospholipase C system of second messengers does not play a significant role in the short-term regulation of hypoxic transduction cascade. It can be speculated that the decrease in PI availability produced by hypoxia might be involved in the functional changes observed in the CB on chronic hypoxic exposure.

Cholera and pertussis toxins reveal multiple regulation of cAMP levels in the rabbit carotid body.

It is known that hypoxia (PO2 approximately equal to 66-18 mm Hg), acting via unknown receptors, increases carotid body cAMP levels in Ca(2+)-free solutions, indicating that low PO2 activates adenylate cyclases independently of the action of the released neurotransmitters. The aim of the present work was to investigate the involvement of G proteins in the genesis of the basal level of cAMP and on the increase in cAMP induced by low PO2. In carotid body homogenates, cholera toxin- and pertussis toxin-induced [32P]ADP-ribosylation of two protein bands of approximately equal to 42 and 45 kDa, and approximately equal to 39 and 40 kDa respectively; in both cases, prior incubation of the carotid bodies with the toxins reduced [32P]ADP-ribosylation by > 90%. In intact carotid bodies, cholera toxin treatment increased cAMP levels more in normoxic than in hypoxic organs, indicating that hypoxia releases neurotransmitters acting on receptors negatively coupled to adenylate cyclases. Cholera toxin-treated carotid bodies incubated in Ca(2+)-free solution had identical cAMP levels in normoxia and in hypoxia. In pertussis toxin-treated normoxic carotid bodies the cAMP level was close to control, but in pertussis toxin-treated hypoxic carotid bodies cAMP rose to a level similar to those seen in normoxic cholera toxin-treated organs, indicating that low PO2 releases neurotransmitters acting on receptors positively coupled to adenylate cyclases. Pertussis toxin-treated carotid bodies incubated in Ca(2+)-free solution lost their capacity to increase cAMP in response to hypoxia, indicating that a G protein sensitive to pertussis toxin is needed for this response. This implies that the carotid bodies express a pertussis toxin-sensitive G protein positively coupled to adenylate cyclases, or that a Gs protein requiring the cooperative action of Go/Gi donated beta gamma subunits mediates the increase in cAMP level produced by hypoxia.

Effects of fluoride and cholera and pertussis toxins on sensory transduction in the carotid body.

The regulation of the chemoreceptor cell function by G proteins has been studied by measuring the release of 3H-labeled catecholamines ([3H]CA) in carotid bodies (CBs) treated with fluoride, cholera toxin (CTX), and pertussis toxin (PTX). Fluoride augmented the basal release of [3H]CA in a dose- (5-20 mM) and Ca(2+)-dependent manner. Nisoldipine (1 microM) and ethylisopropyl amiloride (EIPA; 10 microM) inhibited this effect by approximately 60%, and both drugs combined inhibited it in full. BAY K 8644 (1 microM) doubled the effect of fluoride. The effects of fluoride on the stimulus-evoked release of [3H]CA varied with the type of stimulus and the duration of the treatment. Simultaneous application of fluoride with the stimulus increased by five times the release evoked by hypoxia and by two times that by K+ and dinitrophenol (DNP). Preincubation with fluoride for 1 h caused an inhibition (approximately 70%) of the release evoked by high K+ and veratridine, whereas that evoked by DNP and low PO2 was still augmented (approximately 2 times). Preincubation (4 h) of the CBs with CTX (3 micrograms/ml) reduced by 54% the release of [3H]CA evoked by 35 mM K+ but did not affect that evoked by low PO2 or DNP. A similar treatment with PTX (1 microgram/ml) affected only the release of [3H]CA evoked by DNP, reducing it by 65%. The data show that fluoride, CTX, and PTX have different effects on the release of [3H]CA evoked by high external K+, DNP, and low PO2, indicating that the stimulus-secretion coupling process for each stimulus is differently regulated by G proteins.

Oxygen sensing in the carotid body.

In the present article we review in a concise manner the literature on the mechanisms of O2 chemoreception in the carotid body of adult mammals. In the first section we describe the basic structure of the carotid body, and define this organ as a secondary sensory receptor. In the second section is presented the most relevant literature on the O2 metabolism in the carotid body to define the parameters of O2 chemoreception, including hypoxic thresholds and P50 of the hypoxic responses. The final section is devoted to the mechanisms of detection of the hypoxic stimulus. We provide the data in favor and against each of the current three models on O2 chemoreception: the membrane model, the metabolic hypothesis with its different versions and the NAD(P)H oxidase model.