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.

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.

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.

Effects of almitrine on the release of catecholamines from the rabbit carotid body in vitro.

1. Almitrine increases ventilation by stimulating the carotid body (CB) arterial chemoreceptors but neither its intraglomic target nor its mechanism of action have been elucidated. 2. We have tested the hypothesis that chemoreceptor cells are targets for almitrine by studying its effects on the release of 3H-catecholamines in an in vitro rabbit CB preparation. 3. It was found that almitrine (0.3 and 1.5 x 10(-6) M; i.e. 0.2 and 1 mg ml-1) increases the resting release of 3H-catecholamines from CBs (previously loaded with [3H]-tyrosine) incubated in a balanced 95% O2/5% CO2-equilibrated solution. 4. Almitrine at a concentration of 3 x 10(-6) M (2 mg l-1) also augmented the release of 3H-catecholamines elicited by incubating the CBs in a hypoxic solution (equilibrated with 7% O2/5% CO2 in N2), by high external K+ (35 mM) and by veratridine (2 x 10(-5) M), but did not modify release induced by dinitrophenol (7.5 x 10(-5) M). 5. At the same concentration (3 x 10(-6) M), almitrine increased the rate of dopamine synthesis and was ineffective in modifying the cyclic AMP levels in either normoxic or hypoxic CBs. 6. It is concluded that chemoreceptor cells are the intraglomic targets for almitrine. The mechanisms of action of almitrine on chemoreceptor cells are discussed.

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.

Effect of low O2 on glucose uptake in rabbit carotid body.

Glucose consumption in the rabbit carotid body was studied in vitro by measuring phosphorylation rates of tracer concentrations of 2-[3H]deoxy-glucose. The rate of glucose consumption measured in 100% O2-equilibrated modified Tyrode medium was 61 nmol.g tissue-1 x min-1 and was linear for up to 30 min. Incubation of carotid bodies for 5 or 10 min in moderately hypoxic solution (20% O2-80% N2) resulted in a 44% increase in the rate of glucose consumption. The glucose consumption of the nodose ganglion was not affected during similar incubation with low-O2 medium. High-resolution autoradiography of freeze-dried tissues revealed that the type I parenchymal cells are the principal site of glucose consumption in both 100% O2- and 20% O2-incubated carotid bodies. This metabolic response of the carotid body to hypoxia was not secondary to neurotransmitter release, because similar elevations in glucose utilization were observed with low-O2 medium containing zero Ca2+, a condition in which the release of neurotransmitters from type I cells is inhibited. Lowering the pH of the incubation medium from 7.4 to 7 or 6.8 markedly reduced the rate of glucose utilization by both the carotid body and the nodose ganglion. Ouabain (2 x 10(-4) and 1 x 10(-3) M) reduced by 20% the glucose consumption of carotid bodies incubated in 100% O2-equilibrated solution and abolished the metabolic response produced by low-O2 medium. The results suggest that the utilization of metabolic energy is an integral component of the chemoreceptor response to hypoxia.

Effects of low pH on synthesis and release of catecholamines in the cat carotid body in vitro.

The rates of dopamine and noradrenaline synthesis in the cat carotid body (c.b.) are 5.9 +/- 0.58 pmol/c.b./2 h and 0.3 +/- 0.02 pmol/c.b./2 h, respectively. The synthesis is doubled when the organs are incubated at pH 7. Similarly, low pH induces a release of dopamine from the c.b. which is proportional to increased activity in the carotid sinus nerve.

Synthesis and release of catecholamines by the cat carotid body in vitro: effects of hypoxic stimulation.

The role of catecholamines (CAs) in cat carotid body chemoreception has been controversial. On the basis of pharmacological experiments, it would appear that endogenous dopamine (DA) may act either as an inhibitory or excitatory transmitter. Neurochemical studies on the effects of natural stimulation on the release of carotid body CAs in the cat have also been inconclusive. In the present study, we have characterized the synthesis and release of CAs in the in vitro cat carotid body preparation in response to different levels of hypoxic stimulation and have correlated these measures with the chemosensory activity of the carotid sinus nerve. The synthesis of [3H]DA and [3H]norepinephrine was linear for at least 4 h in carotid bodies incubated with their natural precursor [3H]tyrosine. Synthesis of both [3H]CAs plateaued when the [3H]tyrosine concentration in the media reached 40 microM, which is a concentration similar to that found in cat plasma. Exposure of the animals to an atmosphere of 10% O2 in N2 for 3 h prior to removal and incubation of the carotid bodies with [3H]tyrosine resulted in an approximately 100% increase in the rate of [3H]DA synthesis but no change in [3H]norepinephrine synthesis. This selective increase in [3H]DA synthesis was not detected when [3H]dihydroxyphenylalanine was used as precursor. Carotid bodies first incubated with [3H]tyrosine and later superfused with solutions equilibrated with different gas mixtures (0-100% O2 in N2) exhibited an increase in [3H]DA release and carotid sinus nerve discharge which were inversely related to the oxygen concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of the pituitary-adrenocortical axis in control of the native and cryptic enkephalin levels and proenkephalin mRNA in the sympathoadrenal system of the rat.

The effects of hypophysectomy (HPX) and dexamethasone (DEX) on the levels of Met5-enkephalin (ME), ME precursors, and the abundance of proenkephalin (pEK) mRNA, were examined in the adrenal medulla (AM) and superior cervical ganglia (SCG). To assess possible changes in enkephalin processing, both cryptic (after trypsin and carboxypeptidase B digestions) and native (without enzyme digestions) ME-like immunoreactivity (ME-LI) was measured. Three weeks after HPX the proportion of pEK mRNA to the total RNA content in the AM was not significantly changed when compared to sham-operated (SO) animals. Total (native + cryptic) ME-LI was decreased by 45% in the AM of HPX rats. This decrease was paralleled by a 58% depletion of AM proteins. Cryptic ME-LI was also reduced by 43%. In contrast, native ME-LI was not altered after HPX, indicating enhanced processing of ME precursors. Treatment with DEX (5 daily injections--1 mg/kg, i.p.) increased the relative abundance of pEK mRNA (+27%) and total ME-LI in the AM of HPX group, but not in SO group. Native ME-LI, cryptic ME-LI, and their ratio were not significantly affected by DEX in the AM of HPX or SO rats. In SCG, the relative abundance of pEK mRNA decreased by 25% after hypophysectomy. Total and cryptic ME-LI in the SCG of HPX rats were not changed when compared to SO rats. In contrast, HPX reduced native ME-LI suggesting decreased processing of ME precursors. Similarly, as in AM, DEX produced increase in the SCG pEK mRNA only in HPX (+68%) and not in the SO rats. In SCG, DEX produced decreases in total ME-LI which could be attributed to an increased enkephalin release. An overall reduction of cryptic ME-LI was also observed after DEX, whereas native ME-LI remained unchanged suggesting increased processing of enkephalins. Our findings indicate that the pituitary adrenocortical axis controls the relative proportions of ME to its precursors, and that this control involves both glucocorticoid-dependent (SCG) and glucocorticoid-independent (AM) mechanisms. In contrast, our studies do not suggest specific control of pEK synthesis by the pituitary adrenocortical axis. The pituitary adrenocortical axis may also influence the relative contents of ME and catecholamines in the AM and SCG. The ratio of ME/catecholamines increased after HPX (AM and SCG) and after DEX (SCG). Such regulation may contribute to the control of co-transmitter output in the sympathoadrenal system.

Regulation of tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA levels in the sympathoadrenal system by the pituitary-adrenocortical axis.

The pituitary-adrenocortical axis plays a complex role in the regulation of the levels of enzymes of the catecholamine biosynthetic pathway. In this report we have explored molecular mechanisms of these regulations, by examining the effects of hypophysectomy (HPX) and dexamethasone (DEX) on tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels in the adrenal medulla (AM) and superior cervical ganglia (SCG). Three weeks after hypophysectomy weights (-48%), total RNA (-49%), and DNA (-22%) contents in AM were significantly reduced, when compared to sham-operated animals (SO). In SCG decreases in weight (-23%) and in the ratio of RNA/DNA (-25%) were also found. TH mRNA contents paralleled decreases in total RNA levels and no significant change in the relative abundance of TH mRNA was found. When HPX rats were injected for 5 days with DEX (1 mg/kg, i.p.), TH mRNA levels in the SCG (+51%) and in the AM (+74%) were significantly increased when compared to saline-treated HPX animals. DEX given to SO rats increased TH mRNA in SCG (+49%); a 27% increase in TH mRNA in the AM was also observed. The relative abundance of PNMT mRNA in the AM was reduced after hypophysectomy (-64%). This decrease was completely reversed by DEX. In contrast, DEX did not affect PNMT mRNA levels in the AM of SO rats. PNMT mRNA was not detected in SCG of saline- or DEX-treated rats. In conclusion, our findings suggest that the pituitary-adrenocortical axis is involved in the regulation of the steady-state levels of TH and PNMT mRNAs. This regulation involves: (1) induction of TH mRNA contents in AM and SCG by increased plasma glucocorticoid levels; and (2) maintenance of the steady-state levels of PNMT mRNA in AM by glucocorticoid-dependent mechanisms.

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.

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.

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.