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Sleep apnea with periodic cessation of breathing during sleep is a highly prevalent respiratory disorder affecting an estimated 10% of adults. Patients with sleep apnea exhibit several co-morbidities including hypertension, stroke, disrupted sleep, and neurocognitive and metabolic complications. Emerging evidence suggests that a hyperactive carotid body (CB) chemo reflex is an important driver of apneas in sleep apnea patients. Gasotransmitters carbon monoxide (CO) and hydrogen sulfide (H 2 S) play important roles in oxygen sensing by the CB. We tested the hypothesis that an augmented CB chemo reflex stemming from disrupted CO-H 2 S signaling may lead to sleep apnea. This possibility was tested in mice deficient in hemeoxygenase-2 (HO-2), an enzyme involved in CO synthesis, which were shown to exhibit hyperactive CB activity due to high H 2 S levels. We found that HO-2 -/- mice exhibit a high incidence of apneas during sleep compared to wild type mice. Blocking the CB hyperactivity with L-propargylglycine, an inhibitor of cystathionine-γ-lyase (CSE), which catalyzes H 2 S synthesis, prevented apneas in HO-2 -/- mice. These findings suggest that targeting CB with inhibitors of CSE might be a novel therapeutic strategy for preventing sleep apnea.

Parkinsonism is underlain by dopamine (DA) deficiency in the mid-brain, a neurotransmitter innately involved with respiratory regulation. However, the state of respiration in parkinsonism is an unsettled issue. In this study we seek to determine ventilation and its responses to hypoxia in a reserpine--alpha-methyl-tyrosine model of parkinsonism in the rat. We also attempted to differentiate between the role of discrete brain and carotid body DA stores in the modulation of the hypoxic ventilatory response (HVR). To this end we used domperidone, a peripheral D2 receptor antagonist, and levodopa, a central D2 receptor agonist. The HVRs to acute 12% and 8% hypoxia were studied in a whole body plethysmograph in the same rats before and after the induction of parkinsonic symptoms in conscious rats. We found that resting ventilation and the HVR were distinctly reduced in parkinsonism. The reduction was particularly evident in the peak hypoxic hyperpneic augmentation. Domperidone, which enhanced ventilation in the control healthy condition, failed to reverse the reduced parkinsonic HVR. In contrast, levodopa, which did not appreciably affected ventilation in the healthy condition, caused the parkinsonic HVR to return to and above the baseline healthy level. The findings demonstrate the predominance of a lack of the central DA stimulatory element and minimize the role of carotid body DA in the ventilatory impediment of parkinsonism. In conclusion, the study provides the pathophysiological savvy concerning the respiratory insufficiency of parkinsonism, a sequela which carries a risk of chronically impaired blood oxygenation, which may drive the disease worsening.

Premature infants are vulnerable to infections and have unstable breathing (Di Fiore JM, Martin RJ, Gauda EB, Respir Physiol Neurobiol 189:213-222, 2013). Inflammation adversely modifies carotid body (CB) structure and chemosensitivity in adult animals. We determined the effect of inflammation on CB structure and function in newborn rat pups. Pups were given LPS (0.1 mg/kg; IP) or saline at postnatal day 2 (P2). At P9-10 (1 week after exposure) various studies were done including ventilation, carotid sinus nerve (CSN) activity and histology. Using whole body plethysmography, we found that LPS exposure attenuates the change in interbreath (IBI) interval in response to changes in oxygen tension 1 week after LPS exposure. The response of the CSN to hypoxia was attenuated and delayed in onset in LPS-treated animals as compared to controls. Histological sections of the CB were examined for inflammatory cells at P4 (n = 7) and P9-12 (n = 6). After LPS exposure, only mast cells were seen, often encircling the CB, and clustered within the CSN as it entered the CB. Mast cells per section (mean ± SEM) were higher at P9-12 in LPS (7.4 ± 1.5) vs saline (5.4 ± 1.4) exposed animals (p = 0.04). Surprisingly, more mast cells were seen at 7-10 days vs 48 h after LPS exposure. In a newborn model of inflammation, breathing is altered which is associated with changes in structure and function of the carotid body.

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, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.Initial data indicated several previously unreported artefacts associated with using the CamKKβ 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 CamKKβ or via an off target action of STO609 on the channel itself.

GAL-021 and GAL-160 are alkylamino triazine analogues, which stimulate ventilation in rodents, non-human primates and (for GAL-021) in humans. To probe the site and mechanism of action of GAL-021 and GAL-160 we utilized spirometry in urethane anesthetized rats subjected to acute bilateral carotid sinus nerve transection (CSNTX) or sham surgery. In addition, using patch clamp electrophysiology we evaluated ionic currents in carotid body glomus cells isolated from neonatal rats. Acute CSNTX markedly attenuated and in some instances abolished the ventilatory stimulant effects of GAL-021 and GAL-160 (0.3 mg/kg IV), suggesting the carotid body is a/the major locus of action. Electrophysiology studies, in isolated Type I cells, established that GAL-021 (30 μM) and GAL-160 (30 μM) inhibited the BK(Ca) current without affecting the delayed rectifier K(+), leak K(+) or inward Ca(2+) currents. At a higher concentration of GAL-160 (100 μM), inhibition of the delayed rectifier K(+) current and leak K(+) current were observed. These data are consistent with the concept that GAL-021 and GAL-160 influence breathing control by acting as peripheral chemoreceptor modulators predominantly by inhibiting BK(Ca) mediated currents in glomus cells of the carotid body.

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.

Adenosine is a key excitatory neurotransmitter at the synapse between O(2)-sensing chemoreceptor cells-carotid sinus nerve (CSN) endings in the carotid body (CB). Herein, we have investigated the significance of adenosine, through the blockade of its receptors with caffeine, on the CB hypoxic sensitization induced by chronic intermittent hypoxia (CIH) in the rat. CIH animals were obtained by submitting rats during 15 days from 8:00 to 16:00 to 10 %O(2) for 40 s and 20 % O(2) for 80 s (i.e., 30 episodes/h). Caffeine (1 mM) was tested in spontaneous and 5 %O(2) evoked-CSN chemosensory activity in normoxic and CIH animals. CIH decreased basal spontaneous activity but increased significantly CSN activity evoked by acute hypoxia. Caffeine did not modify basal spontaneous activity in normoxic rats, but decreased significantly by 47.83 % basal activity in CIH animals. In addition, acute application of caffeine decreased 49.31 % and 56.01 % the acute hypoxic response in normoxic and CIH animals, respectively. We demonstrate that adenosine contributes to fix CSN basal activity during CIH, being also involved in hypoxic CB chemotransduction. It is concluded that adenosine participates in CB sensitization during CIH.

Carotid body (CB) glomus cells respond to hypoxia by releasing neurotransmitters, such as ATP, which are believed to stimulate excitatory receptors on apposed nerve endings of the carotid sinus nerves as well as bind to autoreceptors on the glomus cell membrane to modulate response magnitude. The CB response to hypoxia is small at birth and increases during postnatal maturation in mammals. As ATP has been shown to inhibit the glomus cell response to hypoxia via an autoreceptor mechanism, we hypothesized that ATP-mediated inhibition may vary with age and play a role in postnatal development of the hypoxia response magnitude. The effects of ATP on CB glomus cell intracellular calcium ([Ca(2+)](i)) responses to hypoxia were studied at two ages, P0-1 and P14-18. The inhibitory effect of ATP or a stable ATP analog on the glomus cell response to hypoxia was greater in newborn rats compared to the more mature age group. Use of selective P2Y receptor agonists and antagonists suggests that the inhibitory effect of ATP on the glomus cell [Ca(2+)](i) response to hypoxia may be mediated by a P2Y12 receptor. Thus, developmental changes in ATP-mediated glomus cell inhibition may play a role in carotid chemoreceptor postnatal maturation.

During hypoxic or hypoxemic conditions, tissue oxygenation and arterial O(2) carrying capacity are upregulated by two complementary systems, namely the neural respiratory network (central and peripheral) that leads to increased minute ventilation thereby increasing tissue oxygenation, and erythropoietin (Epo) release by the kidney that activates erythropoiesis in bone marrow to augment arterial blood O(2) carrying capacity. Despite the fact that both neural respiratory control and Epo-mediated elevation of red blood cells are responsible for keeping arterial O(2) content optimal, no interaction between these systems has been described so far. Here we review data obtained in our laboratory demonstrating that ventilatory and erythropoietic systems are tightly connected. We found Epo is the key factor mediating this relationship through modulation of the chemoreflex pathway. Moreover, we showed that this interaction occurs in a sex-dependent manner.

The possible roles of different potassium channels in oxygen sensing mechanisms of the carotid body are still not well defined. It has been suggested that leak potassium (K(+)) channels, voltage gated K(+) channels (Kv) and Ca(2+) dependent large conductance K(+) channels (BK) play important roles in the peripheral chemo-sensing mechanisms of the carotid body. In the present study, we have made an attempt to clarify the extent to which, these channels are involved in such mechanisms using in vitro model of isolated rabbit carotid body. Selective TASK-1 channel blocker, anandamide (3 microM) induced stimulation of the carotid sinus nerve (CSN) activity, in a very similar pattern to normal hypoxic responses, with peak discharge rates of the CSN up to 63 +/- 21% of the hypoxic responses (n = 33), and no additional increase in the CSN activity was observed during the hypoxic stimulation with the co-application of anandamide (n = 6). On the other hand, inhibition of BK channels by TEA (5 mM, n = 6), was sub-maximum and typical hypoxic responses were preserved during the increased CSN activity induced by TEA (n = 7). Maximal stimulation of the CSN activity was obtained by blocking Kv channels with 4AP (2.5 mM, n = 15), which was refractory to the hypoxic response. However the hypoxic response reappeared during hyperpolarization (n = 12). We have found that the leak K(+) channels (TASK-1) seem to be importantly involved in the initiation of the oxygen sensing mechanisms of the rabbit carotid body.

We recently reported that volatile anaesthetics directly depress the isolated glomus cell response to hypoxia, halothane more so than sevoflurane, in a manner mimicking the action of these agents on the human hypoxic ventilatory response. We wished to extend these investigations to action of another agent (isoflurane), and we planned to examine the effects of this agent and halothane on background K(+) channels. In an isolated rat pup glomus cell preparation intracellular calcium [Ca(2+)]i (measured using indo-1 dye), halothane and isoflurane (0.45-2.73 MAC) depressed the Ca(2+) transient response to hypoxia (p = 0.028), halothane more than isoflurane (p < 0.001). Evaluating the effects of halothane, isoflurane (both 2.5 MAC) and hypoxia on the open probability of background TASK-like K(+) channels in cell attached patch recordings, halothane in euoxia strongly increased channel activity (2 fold) but isoflurane only increased activity by 50% (p < 0.001). In the presence of hypoxia halothane also increased channel activity (3 fold) while isoflurane again only had weak effects (p = 0.004). Thus there were marked differences between these agents on K(+) channel activity, comparable to their effects on the hypoxia induced Ca(2+) transient. When glomus cells were exposed to a depolarising stimulus using 100 mM K(+), both halothane and isoflurane modestly reduced the magnitude of the resulting Ca(2+) transient (by 44% and 10% respectively, p < 0.001). We conclude that the effect of volatile anaesthetics on the glomus cell response to hypoxia is mediated at least in part by their effect on background K(+) channels, and that this plausibly explains their whole-body effect. An additional effect on voltage-gated Ca(2+) is also possible.

The purpose of this study was to ascertain if effects of halothane and sevoflurane (0.18-1.45 MAC) on the magnitude of the rise in intracellular calcium ([Ca(2+)]i with approximately 90s hypoxia (measured using indo-1 dye) in rat pup carotid body type I glomus cells. paralleled their known effects on the human hypoxic ventilatory response, where halothane is more depressive. We also assessed these agents' effect on [Ca(2+)]i response to 100 mM K(+). Halothane depressed the [Ca(2+])i transient in hypoxia more than sevoflurane (p = 0.036). Both agents also depressed the [Ca(2+)]i response to K+ - halothane more than sevoflurane (p = 0.004). These actions reflect their known influence on human hypoxic ventilatory response, consistent with the notion that the cellular process underlies the whole-body effect. The responses to K(+), which depolarises the cell membrane, indicates that in addition to a putative effect on K(+) channels, voltage-activated Ca(2+) channels may also be involved in the anaesthetic effect.

Hydrogen sulfide (H(2)S) is produced endogenously in many types of mammalian cells. Evidence is now accumulating to suggest that H(2)S is an endogenous signalling molecule, with a variety of molecular targets, including ion channels. Here, we describe the effects of H(2)S on the large conductance, calcium-sensitive potassium channel (BK(Ca)). This channel contributes to carotid body glomus cell excitability and oxygen-sensitivity. The experiments were performed on HEK 293 cells, stably expressing the human BK(Ca) channel alpha subunit, using patch-clamp in the inside-out configuration. The H(2)S donor, NaSH (100microM-10 mM), inhibited BK(Ca) channels in a concentration-dependent manner with an IC(50) of ca. 670microM. In contrast to the known effects of CO donors, the H(2)S donor maximally decreased the open state probability by over 50% and shifted the half activation voltage by more than +16mV. In addition, although 1 mM KCN completely suppressed CO-evoked channel activation, it was without effect on the H(2)S-induced channel inhibition, suggesting that the effects of CO and H(2)S were non-competitive. RT-PCR showed that mRNA for both of the H(2)S-producing enzymes, cystathionine-beta-synthase and cystathionine-gamma-lyase, were expressed in HEK 293 cells and in rat carotid body. Furthermore, immunohistochemistry was able to localise cystathionine-gamma-lyase to glomus cells, indicating that the carotid body has the endogenous capacity to produce H(2)S. In conclusion, we have shown that H(2)S and CO have opposing effects on BK(Ca)channels, suggesting that these gases have separate modes of action and that they modulate carotid body activity by binding at different motifs in the BK(Ca)alphasubunit.

Calcium sensitivity of petrosal ganglion neurons (PGNs) to chemical stimuli with and without PC-12 cells in co-culture instead of glomus is not known- the idea being that two types of unusual cells could form synapse and provide a model for studies of chemotransduction. Calcium levels in the PGNs were measured in the presence of different chemical stimuli in the bath medium. Remarkably, the PGNs alone were not sensitive to hypoxia (10 torr), PCO ( approximately 300 torr in normoxa) nor to ATP (100microM) but they developed the sensitivity to these stimuli in synaptic contact with PC-12 cells. The sharp rise in calcium level was suppressed (2/3) by suramin (100microM), a purinergic blocker, and the remaining 1/3 was blocked by hexomethonium, a cholinergic blocker. Taken together, these observations suggest that PGNs developed neurotransmission when in contact with PC-12 cells, as if the latter substituting for glomus cells, thus providing a model for chemotransduction studies. The reason for the insensitivity of PGNs alone to the chemical stimuli is unknown at this time.

Chemoreceptor cells from rabbit carotid body (CB) exhibit transient outward currents reversibly inhibited by low P(o2). Molecular and functional dissection of the components of these outward currents indicates that at least two different channels (Kv4.3 and Kv3.4) contribute to this current. Furthermore, several lines of evidence support the conclusion that Kv4 channel subfamily members (either Kv4.3 alone or Kv4.3/Kv4.1 heteromultimers) are the oxygen sensitive K channels (K(o2)) in rabbit CB chemoreceptor cells. However, the pharmacological characterization of these currents shows that they are almost completely blocked by high external TEA concentrations, while Kv4 channels have been shown to be TEA-insensitive. We hypothesized that the expression of regulatory subunits in chemoreceptor cells could modify TEA sensitivity of Kv4 channels. Here, we explore the presence and functional contribution of DPPX to K(o2) currents in rabbit CB chemoreceptor cells by using DPPX functional knockdown with siRNA. Our data suggest that DPPX proteins are integral components of K(o2) currents, and that their association with Kv4 subunits modulate the pharmacological profile of the heteromultimers.

Research on arterial chemoreceptors, particularly on the carotid body, has been fruitful in the last fifty years, to which this review is addressed. The functional anatomy of the organ appears to be well established. The biophysical bases by which glomus cells transduce chemical changes in the milieu intérieur (hypoxia, hypercapnia, acidosis) into electrical and biochemical changes in glomus cells have received much attention. Physical changes (in temperature, flow and osmolarity) are also detected by the carotid body. Electrical coupling between glomus cells themselves appears as very extensive. Sustentacular cells classically considered as ensheathing glia for glomus cells and nerve endings now appear to behave as stem cells precursors for glomus cells under chronic hypoxic conditions. Many papers have been devoted to transmitters released from glomus cells (acetylcholine, dopamine, ATP) and well as to their effects upon chemosensory nerve activity. Chemosensory neurons have been explored from generation of action potentials at peripheral nerve endings, passing to properties of perikarya at petrosal ganglia and finally at characterization of synaptic transmission at solitary tract nuclei. There is abundant literature on ventilatory and cardiovascular reflexes elicited from arterial chemoreceptors. The transient effects of sudden and brief withdrawal of chemosensory discharges by hyperoxia also provide clues on the role played by carotid bodies in the homeostasis of full organisms.

In the present article we review in a concise manner the literature on the general biology of adenosine signalling. In the first section we describe briefly the historical aspects of adenosine research. In the second section is presented the biochemical characteristics of this nucleoside, namely its metabolism and regulation, and its physiological actions. In the third section we have succinctly described the role of adenosine and its metabolism in hypoxia. The final section is devoted to the role of adenosine in chemoreception in the carotid body, providing a review of the literature on the presence of adenosine receptors in the carotid body; on the effects of adenosine at presynaptic level in carotid body chemoreceptor cells, as well as, its metabolism and regulation; and at postsynaptic level in carotid sinus nerve activity. Additionally, a review on the effects of adenosine in ventilation was done. This review discusses evidence for a key role of adenosine in the hypoxic response of carotid body and emphasizes new research likely to be important in the future.

The non-specific cAMP phosphodiesterase (PDE) inhibitor isobutyl- methylxanthine (IBMX) has been used to manipulate cAMP levels in carotid body (CB) preparations but the characterization of different PDE isoforms in CB has never been performed. PDE4 is one of the PDE families that uses cAMP as a specific substrate and changes its activity and affinity for drug inhibitors according to the degree of its phosphorylation. We investigated the effects of hypoxia on cAMP accumulation induced by different PDE4 inhibitors in the CB based on the hypothesis that acute changes in O(2) could interfere with their affinity.Concentration-response curves for the effects of the PDE4 selective inhibitors, rolipram and Ro 20-1724 and IBMX on cAMP were obtained in CBs, removed from rats and incubated in normoxia (20%O(2)) or hypoxia (5%O(2)).No differences were found between cAMP concentrations in normoxic and hypoxic conditions in the absence of PDE inhibitors. In both conditions, the E(max) calculated for IBMX was similar to that of the specific PDE4 inhibitors. Hypoxia shifted the concentration response curves to the left with the following rank order of potency IBMX> RO 20-1724=rolipram and increased E(max) by about 25%.This pharmacological approach supports the hypothesis that there is PDE4 activity in CBs that is enhanced by acute hypoxia although the low potency of the PDE4 inhibitors to increase cAMP do not support an important role for PDE4 activation in the O(2)-sensing machinery at the CB.

Beginning with the pioneering work of Heymans and collaborators in the 1930's, investigations into the role of the mammalian carotid body (CB) in the control of ventilation have attracted much attention. Progress for many years was restricted to the whole animal and organ level, resulting in characterization of the stimulus-response characteristics of the CB with its afferent nerve supply during exposure to chemostimuli such as low PO(2) (hypoxia), elevated PCO(2) (hypercapnia), and low pH (acidity). Major advances on the cellular and molecular mechanisms of chemotransduction occurred 20 years ago with the use of freshly-dissociated CB preparations and single cell studies using patch clamp and spectrofluorimetric techniques. This review will focus on more recent advances based on novel preparations including co-cultures of isolated CB receptor clusters and dispersed sensory or autonomic neurons, thin CB tissue slice preparations, and transgenic models. These preparations have contributed significantly, not only to our understanding of the transduction and neurotransmitter mechanisms that operate in the CB during sensory processing, but also to the identification and characterization of novel CB stimuli such as hypoglycemia. Though the complexity of this remarkable organ still belies its tiny size, these recent advances are slowly unraveling the intricacies surrounding its ability to act as a polymodal detector of blood-borne chemicals and to alter its sensitivity to patterned stimuli.

Mechanisms involved in carotid body (CB) chemoreceptor cells O(2)-sensing and responses are not fully understood. So far, it is known that hypoxia depolarizes chemoreceptor cells via O(2)-sensitive K(+)-channel inhibition; calcium influx via voltage-gated channels and neurotransmitter secretion follow. Presence of high voltage activated (HVA) calcium channels in rat CB chemoreceptor cells is well documented, but the presence of low voltage activated (LVH) or T-type calcium channels has not been reported to date. The fact that O(2)-sensitive PC12 cells express T-type channels and that they are inducible by chronic hypoxia (CH) lead us to hypothesize they could be present and play a role in the genesis of the hypoxic response in rat CB chemoreceptor cells. We have analyzed the expression of the three isoforms of T-type calcium channels (alpha1G, alpha1H and alpha1I) and the isoforms alpha1C and alpha1D of L-type calcium channels in rat CB by RT-PCR. We found that rat CB expresses alpha1G and alpha1C subunits. After chronic hypoxic treatment of adult rats (10 degrees O(2), 8 days), expression of alpha1G seems to be down-regulated whereas alpha1C expression is up-regulated. Functionally, it was found that the release of catecholamine induced by hypoxia and high external K({+}) from CB chemoreceptor cells was fully sensitive to L-type channel inhibition (nisoldipine, 2 microM), while specific inhibition of T-channels (mibefradil, 2 microM) inhibited exclusively hypoxia-induced release (50 degrees ). As a whole, present findings demonstrate the presence of T-type as well as L-type calcium channels in rat CB and suggest a selective participation of the T-type channels in the hypoxic activation of chemoreceptor cells.

Benzodiazepines (BZs) suppress ventilation possibly by augmenting the GABA(A) receptor activity in the respiratory control system, but precise sites of action are not well understood. The goals of this study were: (1) to identify GABA(A) receptor subunits in the carotid body (CB) and petrosal ganglion (PG); (2) to test if BZs exert their effects through the GABA(A) receptor in the CB chemosensory unit. Tissues were taken from euthanized adult cats. RNA was extracted from the brain, and cDNA sequences of several GABA(A) receptor subunits were determined. Subsequent RT-PCR analysis demonstrated the gene expression of alpha2, alpha3, beta3, and gamma2 subunits in the CB and the PG. Immunoreactivity for GABA and for GABA(A) receptor beta3 and gamma2 subunits was detected in chemosensory glomus cells (GCs) in the CB and neurons in the PG. The functional aspects of the GABA-GABA(A) receptor system in the CB was studied by measuring CB neural output using in vitro perfusion setup. Two BZs, midazolam and diazepam, decreased the CB neural response to hypoxia. With continuous application of bicuculline, a GABA(A) receptor antagonist, the effects of BZs were abolished. In conclusion, the GABA-GABA(A) receptor system is functioning in the CB chemosensory system. BZs inhibit CB neural response to hypoxia by enhancing GABA(A) receptor activity.

Experiments using live dissociated carotid body (CB) cells for patch clamping, [Ca(++)](i) or other measurements require positive identification of the cell being recorded. At present, cell morphology is usually employed, but several cell types within the carotid body evidence similar morphologic characteristics. Therefore, we sought to develop a method utilizing a vital dye to identify glomus cells before and during experiments that require live cells, such as patch clamp studies. It was previously reported that the binding sites for peanut agglutinin (PNA) were highly expressed by all neuroendocrine-derivatives of the sympathoadrenal neural crest, including glomus cells, small, intensely fluorescent cells, PC-12 cells, and adrenal chromaffin cells in situ (katz et al. 1995). By utilizing the binding characteristics of galactose-specific lectin peanut agglutinin (PNA) on the outer cell membrane, we tested the possibility that the fluoresceinated PNA may preferentially bind to CB glomus cells. The results to date show: (1) Rhodamine tagged PNA (Rhod-PNA) binds to the live dissociated glomus cells in less than one hour incubation and can be visualized in superfused cells; (2) Rhod-PNA labeled cells are perfectly matched with tyrosine hydroxylase (TH) positive glomus cells; (3) Rhod-PNA did not interfere with Fura-2 for Ca(++) imaging; (4) Rhod-PNA bound to glomus cells in [Ca(++)](i) studies does not affect O(2) response of glomus cells. Thus fluoresceinated PNA may be a useful marker for live CB glomus studies, without adversely affecting their physiologic response.

Polyamines modulate many biological functions. Here we report a novel inhibitory modulation by spermine of catecholamine release by the rat carotid body and have identified the molecular mechanism underpinning it. We used molecular (RT-PCR and confocal microscopy) and functional (i.e., neurotransmitter release, patch clamp recording and calcium imaging) approaches to test the involvement of: (i) voltage-dependent calcium channels, and; (ii) the extracellular calcium-sensing receptor, CaR, a G protein-coupled receptor which is also activated by polyamines. RT-PCR and immunohistochemistry of isolated carotid bodies revealed that only Ca(v)1.2 and Ca(v)2.2 were expressed in type 1 cells while Ca(v)1.3, Ca(v)1.4, Ca(v)2.1, Ca(v)2.3 and Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3, could not be detected. CaR expression was detected exclusively in the nerve endings. In isolated carotid bodies, the hypoxia-dependent (7% O(2) for 10 minutes) and depolarization-evoked catecholamine release were partially suppressed by pre- (and co)-incubation with 500microM spermine. In dissociated type 1 glomus cells intracellular calcium concentration did not change following spermine treatment, but this polyamine did inhibit the depolarisation-evoked calcium influx. Whole-cell patch clamp recordings of HEK293 cells stably transfected with Ca(v)1.2 demonstrated that spermine inhibits this calcium channel. Interestingly, this inhibition was not apparent if the extracellular solution contained a concentration of Ba(2) above 2 mM as the charge carrier. In conclusion, spermine attenuates catecholamine release by the carotid body principally via inhibition of Ca(v)1.2. This mechanism may represent a negative feedback, which limits transmitter release during hypoxia.

Unlabelled: Peripheral arterial chemoreceptors in the carotid body (CB) are modulated by pH/CO(2). Soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate ions (HCO(3)). Because CO(2)/HCO(3) mediates depolarization in chemoreceptors, we hypothesized that sAC mRNA would be expressed in the CB, and its expression and function would be regulated by CO(2)/HCO(3).Sprague-Dawley rats at postnatal days 16-17 were used to compare sAC mRNA gene expression between CB and non-chemosensitive tissues: superior cervical (SCG), petrosal (PG) and nodose ganglia (NG) by quantitative real time-PCR. Rat sAC gene expression was standardized to the expression of GAPDH (housekeeping gene) and the data were analyzed with the Pfaffl method. Gene and protein expression, and sAC regulation in the testis was used as a positive control. To determine the regulation of sAC mRNA expression and activity, all tissues were exposed to increasing concentrations of bicarbonate (0, 24, 44 mM, titrated with CO(2) and maintained a constant pH of 7.40)., Results: sAC mRNA expression was between 2-11% of CB expression in the SCG, PG and NG. Furthermore, only in the CB did HCO(3) upregulate sAC gene expression and increase cAMP levels., Conclusion: sAC mRNA and protein expression is present in peripheral arterial chemoreceptors and non-chemoreceptors. In the CB, CO(2)/HCO(3) not only activated sAC but also regulated its expression, suggesting that sAC may be involved in the regulation of cAMP levels in response to hyper/hypocapnia.

We evaluate in rats the role of NO in the solitary tract nucleus (STn) after an anoxic stimulus to carotid body chemoreceptor cells (CChrc) with cyanide (NaCN), on the hyperglycemic reflex with glucose retention by the brain (BGR) and FOS expression (FOS-ir) in the STn. The results suggest that nitroxidergic pathways in the STn may play an important role in glucose homeostasis. A NO donor such as sodium nitroprusside (NPS) in the STn before CChrc stimulation increased arterial glucose level and significantly decreased BGR. NPS also induced a higher FOS-ir expression in STn neurons when compared to neurons in control rats that only received artificial cerebrospinal fluid (aCSF) before CChrc stimulation. In contrast, a selective NOS inhibitor such as Nomega-nitro-L-arginine methyl ester (L-NAME) in the STn before CChrc stimulation resulted in an increase of both, systemic glucose and BGR above control values. In this case, the number of FOS-ir positive neurons in the STn decreased when compared to control or to NPS experiments. FOS-ir expression in brainstem cells suggests that CChrc stimulation activates nitroxidergic pathways in the STn to regulate peripheral and central glucose homeostasis. The study of these functionally defined cells will be important to understand brain glucose homeostasis.

We have recently demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A(2B) receptors. Here, we have investigated the hypothesis that this control is exerted via an interaction between adenosine A(2B) and dopamine D(2) receptors present in chemoreceptor cells and if it is, the location of this interaction on the CB hypoxic transduction cascade. Experiments were performed in vitro in CB from 3 months rats. The effect of adenosine A(2B) and dopamine D(2) receptor agonists applied alone or conjunctly, was studied on the basal and evoked release (10% O(2) and ionomycin) of CA from CB. We have observed that the inhibitory action of propylnorapomorphine, a D(2) selective agonist, on the normoxic and 10%O(2)-evoked release of CA was abolished by NECA, an A(2) agonist, meaning that an interaction between the D(2) and A(2B) receptors controls the release of CA from CB. Further, propylnorapomorphine inhibits the release of CA evoked by ionomycin, being this effect totally reversed by NECA. The present results provide direct pharmacological evidence that A(2B) and D(2) receptors interact to modulate the release of CA from rat CB between the steps of Ca(2+) entry and increase in intracellular free Ca(2+), and the activation of exocytosis and neurotransmitter release, of the stimulus-secretion coupling process.

The carotid body (CB) plays important roles in cardiorespiratory changes in intermittent hypoxia (IH). Erythropoietin (EPO), a hypoxia-inducible factor (HIF)-1 target gene, is present in the chemoreceptive type-I cells in the CB but its expression and role in IH resembling sleep apnoeic conditions are not known. We hypothesized that IH upregulates the expression of EPO and its receptor (EPOr) in the rat CB. The CB expressions of EPO and EPOr were examined in rats breathing 10% O(2) (in isobaric chamber for CH, 24 hour/day) or in IH (cyclic between air and 5% O(2) per minute, 8 hour/day) for 3-28 days. Immunohistochemical studies revealed that the EPO and EPOr proteins were localized in CB glomic clusters. The proportional amount of cells with positive staining of EPO immunoreactivities was significantly increased in both IH and CH groups when compared with the normoxic control. The EPO expression was more markedly increased in the CH than that of the IH groups throughout the time course, reaching a peak level at day 14. The positive EPOr immunostaining was increased significantly in the 3-day CH group. By day 14, the EPOr expression elevated considerably at peak levels in both IH and CH rats, whereas the elevation was greater in the CH rats. These results suggest an upregulation of EPO and its receptor expression in the rat CB under IH and CH conditions, presumably mediated by the activation of HIF-1 pathway. The increased EPO binding to its receptor might play a role in the enhancement of CB excitability during the early pathogenesis in patients with sleep-disordered breathing.

It has been proposed that histamine is an excitatory transmitter between the glomus cells of the carotid body (CB) and the nerve endings of the petrosal ganglion (PG) neurons. The histamine biosynthetic pathway and the presence of histamine H1, H2 and H3 receptors have been reported in the CB. Thus, histamine meets some of the criteria to be regarded as a transmitter. However, there is no evidence that glomus cells contain histamine, or whether its application produces chemosensory excitation. Therefore, we studied its immunocytochemical localization on cat CB and its effects on chemosensory activity. Using perfused and superfused in vitro CB and PG preparations, we assessed the effects of histamine hydrochloride on chemosensory discharges and of histamine H1, H2 and H3 receptor blockers. We found the presence of histamine immunoreactivity in dense-core vesicles in glomus cells. In an in vitro CB preparation we performed pharmacological experiments to characterize histamine effects. The application of histamine hydrochloride (0.5-1,000 microg) to the CB produces a dose-dependent increase in the carotid sinus nerve activity. The H1 receptor blockade with pyrilamine 500 nM produces partial decrease of the histamine-induced response, whereas the H2 receptor blockade (ranitidine 100microM) fail to abolish the histamine excitatory effects. Antagonism of the H3 receptor results in an increase in carotid body chemosensory activity. On the other hand, application of histamine to the isolated PG had no effect on the carotid nerve discharge. Our results suggest that histamine is a modulator of the carotid body chemoreception through H1 and H3 receptor activation.

Although controversial, animal and tissue studies indicate that carotid bodies are sensitive to changes in glucose as well as in oxygen, thereby functioning as metabolic sensors. This study was designed to test the hypothesis that carotid bodies in humans participate in the counter-regulatory response to insulin-induced hypoglycemia.Dopamine and hyperoxia were used to suppress the carotid bodies' responsiveness in 16 normal subjects. Insulin and glucose infusions were used to clamp the plasma glucose in a step-wise decrease to 2.5,mmol/l over 4 hours while counter-regulatory hormones were measured.The hypoglycemic trajectories were similar under all three interventions (dopa-mine, hyperoxia and control), but the total glucose infused was significantly larger for hyperoxia than for dopamine. Cortisol and epinephrine both showed the expected increase with hypoglycemia, but there was no difference among interventions. Glucagon and norepinephrine levels were increased by dopamine, but only the normalized increase in glucagon was lower with dopamine and hyperoxia than control.The decrease in total glucose required for the dopamine experiments was most likely due to the higher baseline glucagon and norepinephrine levels. Hyperoxia did require more infused glucose, indicating some increased insulin sensitivity, but it was not clearly due to a decrease in cortisol or epinephrine responses. Thus, we did not find direct evidence of the carotid bodies' role in glucose homeostasis in humans.

This study was a systematic review of the anesthetic effect on carotid body response to hypoxia. We undertook a systematic literature search (electronic plus manual) for full-paper articles in English that used methodologies enabling any anesthetic effect to be located to the carotid body. We found just 7 articles that met our inclusion criteria, incorporating 16 separate studies. Anesthetic (mean dose +/- SD 0.70 +/- 0.33 MAC) significantly depressed carotid body response by 24% (p = 0.041). There were no differences between individual agents (halothane, enflurane, isoflurane) and no influence of the use of neuromuscular blockade or of species (although the data were sufficiently sparse to interpret such sub-group analysis with caution).

The carotid bodies are the major peripheral chemoreceptors that detect changes in arterial blood oxygen level. PACAP-deficient mice are prone to sudden neonatal death and have reduced respiratory response to hypoxia and hypercapnia. To investigate whether PACAP contributes to the chemotransduction in carotid body, we studied the action of PACAP on glomus cells isolated from rat carotid body. We found that PACAP triggered cytosolic [Ca2+] ([Ca2+]i) rise in glomus cells. Removal of extracellular Ca2+ reversibly inhibited the PACAP-mediated [Ca2+]i rise. Under voltage clamp conditions and in the presence of tetraethylammonium (TEA) and Cd2+, PACAP reduced the outward current evoked at positive potentials. We suggest that the inhibition of a TEA-insensitive current is a mechanism underlying the PACAP-mediated [Ca2+]i rise in glomus cells. The loss of the stimulatory action of PACAP in glomus cells may partly account for the reduction in chemoresponse in the PACAP-deficient mice.