Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells.

Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O2 and CO2/H+ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 µM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 µM), an effect opposed by the D2/3R antagonist, sulpiride (1-10 µM). As expected, acute hypercapnia (10% CO2; pH 7.4), or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.

Fast neurogenesis from carotid body quiescent neuroblasts accelerates adaptation to hypoxia.

Unlike other neural peripheral organs, the adult carotid body (CB) has a remarkable structural plasticity, as it grows during acclimatization to hypoxia. The CB contains neural stem cells that can differentiate into oxygen-sensitive glomus cells. However, an extended view is that, unlike other catecholaminergic cells of the same lineage (sympathetic neurons or chromaffin cells), glomus cells can divide and thus contribute to CB hypertrophy. Here, we show that O2-sensitive mature glomus cells are post-mitotic. However, we describe an unexpected population of pre-differentiated, immature neuroblasts that express catecholaminergic markers and contain voltage-dependent ion channels, but are unresponsive to hypoxia. Neuroblasts are quiescent in normoxic conditions, but rapidly proliferate and differentiate into mature glomus cells during hypoxia. This unprecedented "fast neurogenesis" is stimulated by ATP and acetylcholine released from mature glomus cells. CB neuroblasts, which may have evolved to facilitate acclimatization to hypoxia, could contribute to the CB oversensitivity observed in highly prevalent human diseases.

Role of ATP and adenosine on carotid body function during development.

The carotid body is the main peripheral oxygen sensor involved in cardio-respiratory control under both normoxic and hypoxic conditions. This review focuses on data from newborn animals related to the involvement of the purinergic system in carotid body function during development. We describe the potential effects mediated by ATP and adenosine receptors on ventilation, chemoreceptor activity and their influence on respiratory instability, such as apnea. The conclusions that appear from this review is that in newborn rats, activation of ATP receptors increases the carotid body function although with no age dependent manner, regulates breathing under normoxia, and enhances the initial increase in ventilation in response to hypoxia (likely reflecting carotid body responses). However, activation of adenosine receptors may play a role on carotid body function under chronic conditions, such as intermittent hypoxia or exposure to the adenosine receptor antagonist caffeine. Under the later conditions, an indirect effects involving the carotid body dopaminergic system are observed.

Structural and neurochemical changes in the maturation of the carotid body.

Functional maturation of the carotid body in the postnatal period relies partly on structural and neurochemical changes, which are reviewed here. Structural changes include changes in cytological composition, and increases in glomic tissue volume, dense-cored granules of type I cells, synapses of type I cells with type II cells and afferent nerve fibres. Vascular volume also increases, but in the same proportion as extravascular volume. During maturation, the carotid body also shows higher density and hypoxic sensitivity of K(+)-channels and an increased hypoxic [Ca(2+)](i) response. Modulation of content and release of catecholamine occurs, together with decreased expression of tyrosine hydroxylase and dopamine ß-hydroxylase and increased expression of choline acetyltransferase. Expression of dopamine 2 receptor and nicotinic α3 and α7 receptor subunits increases, and muscarinic M1 receptor protein, nicotinic α4 and ß2 receptor subunits and adenosine receptor 1 decrease. Maturation of the carotid body may also be explained with reference to the developmentally regulated expression of trophic factors and their receptors.

Antagonism of progesterone receptor suppresses carotid body responses to hypoxia and nicotine in rat pups.

We tested the hypothesis that antagonism of progesterone receptor (PR) in newborn rats alters carotid body and respiratory responses to hypoxia and nicotinic receptor agonists. Rats were treated with the PR antagonist mifepristone (daily oral gavage 40 µg/g/d) or vehicle between postnatal days 3 and 15. In 11-14-day-old rats, we used in vitro carotid body/carotid sinus nerve preparation and whole body plethysmography to assess the carotid body and ventilatory responses to hypoxia (65 mmHg in vitro, 10% O2 in vivo) and to nicotinic receptor agonists (as an excitatory modulator of carotid body activity-nicotine 100 µM for in vitro studies, and epibatidine 5 µg/kg, i.p., which mainly acts on peripheral nicotinic receptors, for in vivo studies). The carotid body responses to hypoxia and nicotine were drastically reduced by mifepristone. Compared with vehicle, mifepristone-treated rats had a reduced body weight. The ventilatory response to epibatidine was attenuated; however, the hypoxic ventilatory response was similar between vehicle and mifepristone-treated pups. Immunohistochemical staining revealed that mifepristone treatment did not change carotid body morphology. We conclude that PR activity is a critical factor ensuring proper carotid body function in newborn rats.

Adenosine A2a receptors and O2 sensing in development.

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O2 sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5'-nucleotidase and the resulting activation of adenosine A(2A) receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A(2A) receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A(2A) receptors mediate hypoxic inhibition of breathing and rapid eye movements. A(2A) receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A(2A) receptors play virtually no role in O2 sensing by the carotid bodies, but brain A(2A) receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A(2A) receptors have been implicated in O2 sensing by carotid glomus cells, while central A(2A) receptors likely blunt hypoxic hyperventilation. In conclusion, A(2A) receptors are crucially involved in the transduction mechanisms of O2 sensing in fetal carotid bodies and brains. Postnatally, central A(2A) receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O2 sensing in carotid chemoreceptors, particularly in developing lambs.

Alteration of carotid body chemoreflexes after neonatal intermittent hypoxia and caffeine treatment in rat pups.

In human neonates, caffeine therapy for apnoea of prematurity, especially when associated with hypoxemia, is maintained for several weeks after birth. In the present study, we used newborn rats and whole-body plethysmography to test whether chronic exposure to neonatal caffeine treatment (NCT), alone or combined with neonatal intermittent hypoxia (n-IH) alters: (1) baseline ventilation and response to hypoxia (12% O(2), 20 min); and (2) response to acute i.p. injection of caffeine citrate (20 mg/kg) or domperidone, a peripheral dopamine D2 receptor antagonist (1 mg/kg). Four groups of rats were studied as follows: raised under normal conditions with daily gavage with water (NWT) or NCT, or exposed to n-IH (n-IH+NWT and n-IH+NCT) from postnatal days 3 to 12. In n-IH+NCT rats, baseline ventilation was higher than in the other groups. Caffeine or domperidone enhanced baseline ventilation only in NWT and n-IH+NWT rats, but neither caffeine nor domperidone affected the hypoxic ventilatory response in these groups. In n-IH+NWT rats, the response during the early phase of hypoxia (<10 min) was higher than in other groups. During the late response phase to hypoxia (20 min), ventilation was lower in n-IH+NWT and n-IH+NCT rats compared to NWT or NCT, and were not affected by caffeine or domperidone injection. NCT or caffeine injection decreased baseline apnoea frequency in all groups. These data suggest that chronic exposure to NCT alters both carotid body dopaminergic and adenosinergic systems and central regulation of breathing under baseline conditions and in response to hypoxia.

Position and blood supply of the carotid body in a Kenyan population / Posición y suministro sanguíneo del cuerpo carotídeo en una población de Kenia

Position and source of blood supply to the human carotid body displays population variations. These data are important during surgical procedures and diagnostic imaging in the neck but are only scarcely reported and altogether missing for the Kenyan population. The aim of this study was to describe the position and blood supply of the carotid body in a Kenyan population. A descriptive cross-sectional study at the Department of Human Anatomy, University of Nairobi, was designed. 136 common carotid arteries and their bifurcations were exposed by gross dissection. The carotid body was identified as a small oval structure embedded in the blood vessel adventitia. Position and source of blood supply were photographed. Data are presented by tables and macrographs. 138 carotid bodies were identified. Commonest position was carotid bifurcation (75.4 percent) followed by external carotid artery (10.2 percent), internal carotid artery (7.2 percent) and ascending pharyngeal artery (7.2 percent). Sources of arterial blood supply included the carotid bifurcation (51.4 percent), ascending pharyngeal (21.0 percent), external carotid (17.4 percent) and internal carotid (10.2 percent) arteries. Position and blood supply of the carotid body in the Kenyan population displays a different profile of variations from those described in other populations. Neck surgeons should be aware of these to avoid inadvertent injury.

La posición y la fuente de suministro sanguíneo del cuerpo carotídeo humano muestra variaciones en la población. Estos datos son importantes durante los procedimientos quirúrgicos y de diagnóstico por imagen en el cuello, pero son poco informados e inclusive faltan por completo en la población de Kenia. El objetivo de este estudio fue describir la posición y el aporte sanguíneo del cuerpo carotídeo en una población de Kenia. Se diseñó un estudio descriptivo de corte transversal en el Departamento de Anatomía Humana de la Universidad de Nairobi. 136 arterias carótidas comunes y sus bifurcaciones fueron expuestas mediante disección simple. El cuerpo carotídeo fue identificado como una pequeña estructura oval ubicada en la adventicia del vaso sanguíneo. La posición y la fuente de suministro sanguíneo fueron fotografiados. Los datos obtenidos fueron presentados en las tablas y fotomacrografías. 138 cuerpos carotídeos fueron identificados. La posición más frecuente fue la bifurcación carotídea (75,4 por ciento), seguida de la arteria carótida externa (10,2 por ciento), arteria carótida interna (7,2 por ciento) y la arteria faríngea ascendente (7,2 por ciento). Las fuentes de suministro sanguíneo arterial incluyeron la bifurcación carotídea (51,4 por ciento), arteria faríngea ascendente (21,0 por ciento), arteria carótida externa (17,4 por ciento) y arterias carótidas internas (10,2 por ciento). La posición y el suministro sanguíneo del cuerpo carotídeo en la población de Kenia muestra un perfil de variaciones diferente a las descritos en otras poblaciones. Los cirujanos de cuello deben conocer estas variaciones para así evitar lesiones accidentales.

Developmental profile of cholinergic and purinergic traits and receptors in peripheral chemoreflex pathway in cats.

This study describes the developmental profile of specific aspects of cholinergic and purinergic neurotransmission in key organs of the peripheral chemoreflex: the carotid body (CB), petrosal ganglion (PG) and superior cervical ganglion (SCG). Using real time RT-PCR and Western blot analyses, we assessed both mRNA and protein expression levels for choline-acetyl-transferase (ChAT), nicotinic receptor (subunits alpha3, alpha4, alpha7, and beta2), ATP and purinergic receptors (P2X2 and P2X3). These analyses were performed on tissue from 1- and 15-day-old, 2-month-old, and adult cats. During development, ChAT protein expression level increased slightly in CB; however, this increase was more important in PG and SCG. In CB, mRNA level for alpha4 nicotinic receptor subunit decreased during development (90% higher in 1-day-old cats than in adults). In the PG, mRNA level for beta2 nicotinic receptor subunit increased during development (80% higher in adults than in 1-day-old cats). In SCG, mRNA for alpha7 nicotinic receptor levels increased (400% higher in adults vs. 1-day-old cats). Conversely, P2X2 receptor protein level was not altered during development in CB and decreased slightly in PG; a similar pattern was observed for the P2X3 receptor. Our findings suggest that in cats, age-related changes in cholinergic and purinergic systems (such as physiological expression of receptor function) are significant within the afferent chemoreceptor pathway and likely contribute to the temporal changes of O2-chemosensitivity during development.

Expression of sex-steroid receptors and steroidogenic enzymes in the carotid body of adult and newborn male rats.

This study describes the localization and pattern of expression of estradiol and progesterone receptors as well as key enzymes for steroid synthesis (i.e. P450 side-chain-cleavage--P450scc, and P450 aromatase--P450Aro) in the carotid body (CB) and superior cervical ganglion (SCG) of adult, newborn and late fetal male rats, using immunohistochemistry, Western blot and real-time RT-PCR. Our results show a constitutive expression of the beta estradiol receptor (Erbeta) and the 80 kDa and 60 kDa progesterone receptors (PR-A and PR-C) isoforms in the CB, while in the SCG Eralpha, Erbeta, PR-A and PR-C are expressed. While P450Aro staining was negative, P450scc staining was strong both in the SCG and CB. In late fetal and newborn rats, Eralpha was not detected in the CB or SCG, but a slight staining appeared for P450 aromatase in the CB, and to a lesser extent in SCG. P450scc was strongly expressed in CB and SCG of late fetal and newborn rats. We conclude that the carotid body shows a constitutive expression of Erbeta and PR and may be able to synthesize steroids, including estradiol during late fetal life.

Neurotransmitters in carotid body development.

This review examines the possible role of neurotransmitters present in the carotid body on the functional expression of chemosensory activity during postnatal development. In particular, dopamine, acetylcholine, adenosine and neuropeptides are reviewed. Evidence to date shows involvement of these transmitters in signal transmission from the chemoreceptor cells to chemosensory afferent fibers of the sinus nerve, with clear age- or maturation-dependence of some aspects. However, it remains unresolved whether these neurotransmitters, some of which are expressed in the carotid body before birth, are directly involved in the maturation of the functional properties of the carotid chemoreceptors in sensing oxygen or other stimuli during postnatal development.

Differential expression of a(2a), A(1)-adenosine and D(2)-dopamine receptor genes in rat peripheral arterial chemoreceptors during postnatal development.

The sensitivity of peripheral arterial chemoreceptors in the carotid body to hypoxia increases with postnatal maturation. Carotid sinus nerve activity is augmented by adenosine binding to A(2a)-adenosine receptors and attenuated by dopamine binding to D(2)-dopamine receptors. In this study, we used in situ hybridization histochemistry to determine the change in the levels of mRNA expression for A(2a) and A(1)-adenosine receptors and D(2)-dopamine receptors in the rat carotid body. We also investigated the cellular distribution and possible colocalization of these receptor mRNAs and tyrosine hydroxylase (TH) mRNAs during the first 2 weeks of postnatal development. By using immunohistocytochemistry, we detected A(2a)-adenosine receptor protein in the carotid body and petrosal ganglion. We found that A(2a)-adenosine receptor mRNA and protein are expressed in the carotid body in animals at 0, 3, 6 and 14 postnatal days. The level of A(2a)-adenosine receptor mRNA expression significantly decreased by 14 postnatal days (P<0.02 vs. day 0) while D(2)-dopamine receptor mRNA levels significantly increased by day 3 and remained greater than D(2)-dopamine receptor mRNA levels at day 0 (P<0.001 all ages vs. day 0). TH mRNA was colocalized in cells in the carotid body with A(2a) adenosine receptor and D(2)-dopamine receptor mRNAs. A(1)-adenosine receptor mRNA was not expressed in the carotid body at any of the ages examined. In the petrosal ganglion, A(1)-adenosine receptor mRNA was abundantly expressed in numerous cells, A(2a)-adenosine receptor mRNA was expressed in a moderate number of cells while D(2)-dopamine receptor mRNA was seen in a few cells in the rostral petrosal ganglion. In conclusion, using in situ hybridization histochemistry, we have shown that mRNA for both the excitatory, A(2a)-adenosine receptor, and the inhibitory, D(2)-dopamine receptor, is developmentally regulated in presumably type I cells in the carotid body which may contribute to the maturation of hypoxic chemosensitivity. Furthermore, the presence A(1)-adenosine receptor mRNAs in cell bodies of the petrosal ganglion suggests that adenosine might also have an inhibitory role in hypoxic chemotransmission.

Peptidergic innervation in the amphibian carotid labyrinth.

The amphibian carotid labyrinth, which corresponds to the mammalian carotid body and carotid sinus, is innervated by nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and somatostatin (SOM). SP, CGRP, VIP, and NPY immunoreactive varicose fibers are more densely distributed in the peripheral portion of the carotid labyrinth than FMRF and SOM fibers. The time of appearance of SP, CGRP, and VIP is different for each. First CGRP fibers, then SP fibers appear at an early stage of larval development, and finally VIP fibres are detected at a later stage of larval development. Most SP fibres show coexistence with CGRP, and some SP fibres which show coexistence with NPY immunoreactivity are assumed to be continuous with those demonstrating VIP immunoreactivity. This indicates the possibility of coexistence of four different peptides in the same nerve fibers within the labyrinth. In various vasculatures of mammals, it has been shown that SP, CGRP, VIP, and NPY have a vasoactive nature in relation to the vascular smooth muscle cells. On this basis, it seems that the target of the peptidergic innervation in the amphibian carotid labyrinth is the smooth muscle cells which are abundantly distributed in the intervascular stroma. Accordingly, the peptidergic innervation may be involved in the vascular regulatory function of the labyrinth, although the possibility that these peptides participate in the chemoreception cannot be ruled out. In addition, the vascular regulatory function of the labyrinth may be modulated by the interaction of multiple neuropeptides.