Postnatal maturation of neuroepithelial bodies and carotid body innervation: a quantitative investigation in the rabbit

The intrapulmonary airways contain oxygen-sensitive chemoreceptors which may be analogous to the arterial chemoreceptors: the neuroepithelial bodies (NEB). While the NEB are prominent in the neonatal lung, physiological studies indicate that the carotid bodies are still relatively inactive at birth. This points to an unequal degree of development of both during the early neonatal period. As a reflexogenic chemoreceptor function depends on a well-developed innervation, we undertook a comparative investigation of the development of the NEB and the carotid body glomus cell innervation. Two morphological aspects of the innervation of NEB and carotid body glomus cells were quantified in rabbits of different age groups. The total sectional area of intracorpuscular and intraglomerular nerve endings per NEB or glomus cell group, respectively, was measured and the area percentage of mitochondria and synaptic vesicles was determined. In the NEB, no significant difference in total sectional area of the nerve endings between the age groups was observed, while in the carotid body there was a significant increase in the adult age group. In addition, the area percentage of mitochondria and synaptic vesicles of the nerve endings did not change significantly with age in the NEB, while in the carotid body these increased and decreased, respectively, with age. These observations point to a shift from morphologically efferent nerve endings, rich in synaptic vesicles, to morphologically afferent nerve endings, rich in mitochondria. Our interpretation of these findings is that, at birth, the NEB innervation is more mature than the carotid body glomus cell innervation and that the latter matures at a later time than the former. These findings support the theory that the NEB may act as complementary chemoreceptors to the carotid body during the early postnatal period.     

Immunohistochemical evidence for species-specific coexistence of catecholamines, serotonin, acetylcholine and nitric oxide in glomus cells of rat and guinea pig aortic bodies.

The aortic bodies are small paraganglia distributed along the vagus nerve and its branches in the vicinity of the aortic arch which, like the carotid bodies, act as arterial chemoreceptors. In the rat carotid body, corelease of ATP and acetylcholine (ACh) from glomus cells is considered to be the main mechanism mediating fast hypoxic chemotransmission while dopamine, serotonin, and nitric oxide (NO) exert modulating effects. The present study was aimed at determination of the endogenous sources of serotonin, ACh and NO within rat and guinea pig aortic bodies by immunohistochemical double- and triple-labeling approaches, utilizing antibodies to serotonin, the NO and ACh synthesizing enzymes neuronal NO synthase (nNOS) and choline acetyltransferase (ChAT), respectively, as well as to the vesicular acetylcholine transporter (VAChT). Additional marker antibodies were directed against the rate-limiting enzyme of catecholamine synthesis, i.e. tyrosine hydroxylase (TH), and the vesicular protein, synaptophysin (SYN). In both species, all aortic body glomus cells were immunoreactive to serotonin and cholinergic markers. In the rat, all glomus cells were additionally catecholaminergic, as indicated by TH-immunoreactivity, whereas this applied only to a subgroup of guinea pig glomus cells. On the other hand, all guinea pig glomus cells were nNOS-immunoreactive, whereas only nerve fibers but not glomus cells exhibited nNOS-immunoreactivity in the rat. These data support the concept that the chemoexcitatory transmitters ACh and serotonin are involved in hypoxic excitation of aortic chemoreceptor terminals in both species. The production of the inhibitory modulators, dopamine and NO, however, appears to be species-specifically regulated.     

Ultrastructure of the primate carotid body: a morphometric study of the glomus cells and nerve endings in the monkey (Macaca fascicularis)

Summary The carotid body of the monkey (Macaca fascicularis) was studied at both the light and electron microscopic levels in an effort to provide a detailed quantitative characterization of this chemoreceptor organ in the primate. Structurally, the monkey carotid body was organized into lobules of from three to eight glomus cells (in section) and their ensheathing supporting cells. Interspersed among the lobules was abundant connective tissue stroma, fibroblasts and mast cells. Fenestrated capillaries, small arterioles and venules also permeated the organ. Each supporting cell partially ensheathed about three glomus cells and could be easily differentiated from glomus cells by their darker cytoplasmic staining, lack of dense-core vesicles and angular nuclear profile. Glomus cells exhibited an intense catecholamine histofluorescence and contained abundant dense-core vesicles. On the basis of dense-core vesicle size, shape and numerical density, four types of glomus cells were identified. The most common type (62% of all glomus cells) contained vesicles with an average diameter of 219 nm and a density of 8 vesicles per m² of cytoplasm. The second type possessed larger vesicles (264 nm in diameter) and accounted for about 14% of all glomus cells. A third type of glomus cell contained smaller (167 nm) and fewer (5 vesicles per m²) dense-core vesicles. The fourth type of glomus cell contained pleomorphic-shaped vesicles with a maximal diameter of 232 nm. Each of these last two types accounted for about 12% of all glomus cells. All four types of glomus cells were innervated, averaging 1.43 nerve endings per glomus cell (in sections). Nerve endings were primarily of the bouton-like variety averaging 2 m² in sectional area and containing 34.3 clear-core synaptic vesicles (average size 73.5 nm in diameter) per m² of cytoplasm. Of the 57 nerve endings examined in single sections, 16% displayed junctions typical of synaptic specializations and most of these were presynaptic to glomus cells. Glomus cell-glomus cell synapses were not observed. Based on these quantitative observations and on previous studies of carotid body cytoarchitecture in other laboratory species, it appears that the primate organ most closely resembles the cat carotid body, although several differences exist.     

Fine structure of the carotid body of the midterm human fetus

Summary The human fetal carotid body was studied using both histochemical and electron microscopic methods. The glomus cells of a mid term fetal carotid body evidently contain catecholamines. This was demonstrated both by formaldehyder-induced fluorescence of the cells and by the presence of typical dense-cored vesicles (diameter 1430–3200 Å) in the cytoplasm of the chief cells. The glomus cells were densely innervated and the synapses found on their surface were probably cholinergic in type, containing agranular synaptic vesicles measuring 400–700 Å in diameter with a few dense-cored vesicles measuring 900 to 1300 Å. Synapses were not found in any other cell type within the glomus caroticum. The prominent feature of the glomus cell cytoplasm was the presence of the dense-cored vesicles. The density of the vesicular core varied only slightly from cell to cell. There were no perceptible differences in vesicular size between the different cells. The glomus cells were mostly surrounded by the processes of the sustentacular cells, which usually also surrounded the capillary walls. No glomus cells were ever found in direct contact with the capillary wall. The capillaries were wide and very numerous over the restricted area of the organ. They formed sinusoidal loops, probably anastomosing with each other. Finally, the features of the fine structure are discussed, correlating the present findings with our knowledge about the adult functional carotic body.     

Restoration of reflex ventilatory response to hypoxia after removal of carotid bodies in the cat

In mammals there are two sets of peripheral arterial chemoreceptors, the carotid bodies innervated by the sinus branch of the glossopharyngeal nerve and the aortic bodies innervated by the vagus nerves. The afferent impulse discharge from both receptors increases during hypoxia and there is a reflexly mediated increase in ventilation (hypoxic hyperventilation). In the present study we tested this response by exposing anesthetized cats to decreased inspired O₂ concentration before and up to 315 days after bilateral resection of the carotid bodies. Acutely after removing the carotid bodies, hypoxic hyperventilation was abolished. This observation supports the view that the reflex pathway from the aortic body receptors normally contributes minimally to hypoxic hyperventilation. Subsequently, there was a restoration of hypoxic hyperventilation. Restoration was significant 30–43 days after removing the carotid bodies, it reached 70% of the preoperative value at 93–111 days and was essentially complete in terminal experiments 260–315 days after carotid body resection. In terminal experiments, hypoxic hyperventilation was not affected by recutting the regenerated carotid sinus nerves but was abolished completely by bilateral transection of the cervical vagosympathetic trunks. The restored ventilatory response was due predominantly to an increase in rate of breathing while an increase in tidal volume was predominant before carotid body resection. Resting ventilation breathing room air was not consistently decreased after carotid body resection while expired CO₂ was elevated from day 20 to day 111 and at the preoperative level in terminal experiments.It is concluded that restoration of hypoxic hyperventilation in the cat after carotid body resection is mediated by the reflex pathway from aortic body chemoreceptors. The possible contribution of chemo-receptive regenerated carotid sinus nerve axons was excluded. It is suggested that restoration may be a consequence of the central reorganization of chemoreceptor afferent pathways consequent to interruption of the carotid body reflex pathway and that as a result the ‘gain’ of the aortic body ventilatory chemoreflex is enhanced.     

Light,fluorescence and electron microscopic studies of rabbit subclavian glomera

The subclavian glomera (aortic bodies) of young New Zealand white rabbits were studied with the light, fluorescence, and electron microscopes. Two cell types were identified: type I, granule-containing (chief) cells, and type II, agranular (sustentacular) cells. The type I cells possessed large nuclei, the normal complement of cytoplasmic organelles and numerous electron-opaque cytoplasmic granules. The type II cells were agranular with attenuated cytoplasmic processes which partially or completely ensheathed the type I cells. The glomera were well vascularized. Capillary endothelial cells contained numerous pinocytotic vesicles, but few fenestrae. Two profiles of nerve terminals were observed. One, apposing the type I cells, contained numerous electron-lucent vesicles, several dense-cored vesicles, mitochondria and possessed membrane specializations resembling those usually observed in synaptic zones. The other profile contained abundant mitochondria and a few electron-lucent and densecored vesicles. Structural specializations were not observed on the apposed membranes of these terminals or adjacent to type II cells. Fluorescence histochemistry revealed an intense yellow-green fluorescence in the glomera, which indicated the presence of biogenic amines, possibly primary catecholamines or an indolamine. The electron-opaque granules observed in the type I cells were believed to be the storage sites for these amines. The subclavian glomera were found to be morphologically similar to the carotid body which is a known chemoreceptor.     

Inhibition in carotid body chemoreceptors mediated by D-2 dopaminoceptors: Antagonism by benzamides

Inhibition of chemosensory nerve impulses in the cat is evoked by dopamine (DA) applied to carotid body chemoreceptors. Pharmacological characterization of the dopaminoceptors involved in this action was determined through their blockade with benzamides, selective antagonists of D-2 receptors. Both metoclopramide and sulpiride were effective blockers of DA-induced chemosensory inhibition. Furthermore, both drugs induced an immediate increase in the frequency of carotid nerve chemosensory impulses, suggesting the presence of previous tonic inhibition of chemoreceptor discharges by endogenous DA released from glomus cells.     

Postnatal development of carotid body glomus cell O2 sensitivity

In mammals, the main sensors of arterial oxygen level are the carotid chemoreceptors, which exhibit low sensitivity to hypoxia at birth and become more sensitive over the first few days or weeks of life. This postnatal increase in hypoxia sensitivity of the arterial chemoreceptors, termed "resetting", remains poorly understood. In the carotid body, hypoxia is transduced by glomus cells, which are secretory sensory neurons that respond to hypoxia at higher P(O2) levels than non-chemoreceptor cell types. Maturation or resetting of carotid body O2 sensitivity potentially involves numerous aspects of the O2 transduction cascade at the glomus cell level, including glomus cell neurotransmitter secretion, neuromodulator function, neurotransmitter receptor expression, glomus cell depolarization in response to hypoxia, [Ca2+]i responses to hypoxia, K+ and Ca2+ channel O2 sensitivity and K+ channel expression. However, although progress has been made in the understanding of carotid body development, the precise mechanisms underlying postnatal maturation of these numerous aspects of chemotransduction remain obscure.     

Dopamine beta-hydroxylase-like immunoreactivity in the rat and cat carotid body: a light and electron microscopic study

Summary Immunocytochemical localization of dopamine -hydroxylase (DBH) was used to study the synthesis and storage sites of norepinephrine (noradrenaline) in the rat and cat carotid bodies. In the rat carotid body some parenchymal cells exhibited strong DBH-like immunoreactivity (DBH-I), while others displayed only faint DBH-I. In a typical parenchymal cell cluster, most cells with strong DBH-I were irregular in shape and appeared to partially surround those with weak DBH-I which usually were rounded in contour. In the cat carotid body most parenchymal cells showed a strong to moderate DBH-I. In both the rat and cat carotid bodies varicose nerve fibres with DBH-I were associated primarily with blood vessels. All autonomic ganglion cells examined, which were associated with the rat carotid body, showed DBH-I. Electron microscopy revealed that most DBH-I in the strongly positive cells of the rat carotid body was associated with dense granules (possibly corresponding to dense-cored vesicles of various sizes), although some was found in other sites. In oval cells with less DBH-I, reactivity resided in some of the large granules. In the cat carotid body the glomus cells contained more granules of various sizes and shapes than did those of the rat carotid body. Most of the cat glomus cell granules exhibited DBH-I activity. Our results indicate that some of glomus cells in the rat and most of the glomus cells in the cat contain DBH and therefore may be sites of norepinephrine synthesis.     

Evidence for central reorganization of ventilatory chemoreflex pathways in the cat during regeneration of visceral afferents in the carotid sinus nerve

There are two sets of peripheral arterial chemoreceptors in the cat, the carotid bodies innervated by the carotid sinus nerve and the aortic bodies with afferents in the aortic depressor nerves. Reflex stimulation of ventilation in response to hypoxia is abolished acutely after interrupting the sensory pathway from the carotid body chemoreceptors in the cat even though the reflex pathway from the aortic body chemoreceptors is intact. However, in chronically maintained preparations, there is a restoration of the hypoxic response which is mediated by the aortic chemoreflex pathway. It was proposed that restoration was due to a ‘central reorganization’ of chemoreflex pathways which followed interruption of the sensory pathway from the carotid bodies and that the reorganization enhanced the efficacy of the aortic ventilation chemoreflex. This proposal was tested in the present experiments by measuring reflex ventilatory and cardiovascular responses to electrical stimulation of the sensory nerves containing aortic and carotid chemoreceptor afferents following bilateral interruption of carotid sinus nerves and carotid body resection. Responses measured acutely (1–6 h) after interruption were compared with those measured 60–80 and 110–140 days later. At 60–80 days, a chemoreflex response (increase in tidal volume of ventilation) to stimulation of the interrupted carotid sinus sensory pathway was markedly attenuated while the response to stimulation of the uninterrupted pathway in aortic depressor nerves was enhanced. At 110–140 days, the tidal volume response to carotid sinus nerve stimulation was greatly enhanced while the aortic depressor nerve response declined from the elevated level. There were significant but less pronounced changes in the response of other ventilatory and cardiovascular variables to aortic depressor nerve and carotid sinus nerve stimulation.The results support the idea that there is a ‘central reorganization’ of chemoreflex pathways which is reflected functionally by changes in the efficacy of reflexes evoked from aortic depressor nerve and carotid sinus nerve. The changes are analagous to those occurring in somatic reflexes during regeneration of sensory nerves. It is suggested that the changes in efficacy of carotid sinus nerve reflexes are due to a degenerative loss of synapses of the central projections of interrupted carotid sinus nerve sensory axons (degenerative atrophy) and subsequent regenerative like changes (regenerative proliferation) in the central projections. The changes in the efficacy of aortic depressor nerve reflexes may be attributed to formation of new synapses by converging central projections of this uninterrupted pathway (reactive synaptogenesis) and subsequent regression of the newly formed synapses.     

Stimulation of carotid body chemoreceptors does not influence the discharge of A1 neurons projecting to the forebrain

Stimulation of carotid body chemoreceptors activates putative vasopressin neurons in the supraoptic nucleus, an effect which has been abolished by lesions in the caudal ventrolateral medulla. Stimulation within the A1 catecholamine cell group in the ventrolateral medulla also activates supraoptic neurons and releases vasopressin. Therefore the A1 catecholamine neurons may be the means by which carotid body chemoreceptors influence the supraoptic nucleus and other parts of the forebrain. To test this possibility the influence of carotid body chemoreceptors on the discharge of rostrally-projecting neurons in the A1 region of the caudal ventrolateral medulla has been assessed in rats anaesthetized with a mixture of urethane and sodium pentobarbitone. Tests were performed on 131 neurons, 23 of which were antidromically invaded following electrical stimulation within the supraoptic nucleus, the medial forebrain bundle or the ventral noradrenergic bundle. The positions of all antidromically invaded neurons were marked with dye and in six animals subsequent fluorescence histochemistry showed that the blue spots were in the proximity of one or more catecholamine-containing cell bodies in the ventrolateral medulla. The recorded neurons were therefore presumed to be part of the A1 group of catecholamine-containing neurons. All neurons located were tested for their responses to specific stimulation of ipsilateral carotid body chemoreceptors and also to general baroreflex activation. Not one of the antidromically invaded neurons was affected by chemoreceptor stimulation and only one was activated by baroreflex activation. Of the non-antidromically invaded neurons, seven were activated and 13 were depressed following chemoreceptor stimulation but in many cases the latency to onset was very long.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine-containing glomus cells in the rabbit carotid body. II. Immunocytochemical evidence of dopamine-beta-hydroxylase and norepinephrine.

Summary The presence of noradrenergic glomus cells in the rabbit carotid body was investigated at the light and electron microscope levels, using dopamine-ß-hydroxylase and norepinephrine immunocytochemistry as well as the chromaffin reaction.Frozen and semi-thin plastic sections showed some dopamine-ß-hydroxylase immunoreactive glomus cells either isolated in the connective tissue or, more frequently, mixed with unreactive cells. At the ultrastructural level, immunopositive cells differed from immunonegative ones by the larger size of most of their dense-cored vesicles. Similar observations were made after using anti-norepinephrine antibodies. Immunoreactive cells to anti-dopamine-ß-hydroxylase and anti-norepinephrine antibodies were relatively few although their number varied from carotid body to carotid body. The immunolabelling intensity was very variable from cell to cell. Consecutive frozen sections processed for norepinephrine- and dopamine-immunocytochemistry showed many cell clusters containing both norepinephrine and dopamine-immunoreactive glomus cells.Some chromaffin glomus cells were clearly identifiable by the very strong electron opacity of their dense-cored vesicles; most of these vesicles were characterized by their large size, as the dense-cored vesicles observed in dopamine-ß-hydroxylase- and norepinephrine-immunopositive cells.These results demonstrated that dopamine-ß-hydroxylase and norepinephrine-immunopositive, as well as chromaffin cells, were identical to the cells which take up exogeneous norepinephrine, described in part I of this study. However, many intermediate levels were found between norepinephrine-immunonegative and strongly norepinephrine-immunopositive glomus cells, suggesting that the distinction between these two kinds of cells is not clearcut.     

Cardiovascular responses in apnoeic asphyxia: role of arterial chemoreceptors and the modification of their effects by a pulmonary vagal inflation reflex

1. In the spontaneously breathing anaesthetized dog, the systemic circulation was perfused at constant blood flow; there was no pulmonary blood flow and the systemic arterial blood P(O2) and P(CO2) were controlled independently by an extracorporeal isolated pump-perfused donor lung preparation. The carotid and aortic bodies were separately perfused at constant pressure with blood of the same composition as perfused the systemic circulation.2. Apnoeic asphyxia, produced by stopping the recipient animal's lung movements and, at the same time, making the blood perfusing the systemic circulation and the arterial chemoreceptors hypoxic and hypercapnic by reducing the ventilation of the isolated perfused donor lungs, caused an increase in systemic vascular resistance.3. While the systemic arterial blood was still hypoxic and hypercapnic, withdrawal of the carotid and aortic body ;drive' resulted in a striking reduction in systemic vascular resistance. Re-establishing the chemoreceptor ;drive' immediately increased the vascular resistance again.4. Apnoeic asphyxia carried out while the carotid and aortic bodies were continuously perfused with oxygenated blood of normal P(CO2) had little or no effect on systemic vascular resistance.5. The systemic vasoconstrictor response produced by apnoeic asphyxia was reduced or abolished by re-establishing the recipient animal's lung movements, and this effect occurred in the absence of changes in the composition of the blood perfusing the systemic circulation and arterial chemoreceptors. This abolition of the vasoconstriction was due to a pulmonary reflex.6. Apnoeic asphyxia slowed the rate of the beating atria due to excitation of the carotid and aortic body chemoreceptors. This response can be over-ridden by an inflation reflex arising from the lungs.7. It is concluded that the cardiovascular responses observed in apnoeic asphyxia are due, at least in part, to primary reflexes from the carotid and aortic body chemoreceptors engendered by arterial hypoxia and hypercapnia. The appearance of these responses is, however, dependent upon there being no excitation of a pulmonary (inflation) vagal reflex.     

Ultrastructure of the glomus cells in the carotid body of chronically hypoxic rats: With special reference to the similarity of amphibian glomus cells

The ultrastructural characteristics of the glomus cells in the rat carotid body exposed to extremely long-term hypoxia (10–12 weeks) were investigated. The glomus cells could be classified into four distinct types according to the shape of dense-cored vesicles in the glomus cell cytoplasm: (1) small vesicle cells (SVCs, 50 nm in mean diameter), (2) large vesicle cells (LVCs, 80 nm in mean diameter), (3) dilated eccentric vesicle cells (EVCs, 400–800 nm in diameter), and (4) mixed vesicle cells (MVCs, large and eccentric vesicles). Many clusters of glomus cells were found to contain all four categories of cell types. The appearance of EVCs was a unique and common characteristic of glomus cells in this long-term hypoxia model. We also noted other ultrastructural features with chronic hypoxia which are characteristic of the amphibian carotid labyrinth glomus cells: (1) incomplete covering of glomus cells with the supporting cell missing over a wide area, (2) long thin cytoplasmic projections in the intervascular stroma, and (3) intimate apposition of the glomus cells and pericytes (g-p connection), endothelial cells (g-e connection), plasma cells, and fibrocytes. Because arterial PO₂ is generally low in amphibia, these may be general features of hypoxic adaptation and facilitate both uptake of oxygen from blood and release of catecholamine into the blood. The g-p and g-e connections may take part in the regulation of the microcirculation in the enlarged carotid body. © 1993 Wiley-Liss Inc.     

The contribution of the arterial chemoreceptors to the stimulation of respiration by adrenaline and noradrenaline in the cat

1. Intravenous infusions of adrenaline and noradrenaline in doses averaging 0.8 mug/kg.min increased the respiratory minute volume of anaesthetized cats breathing room air. The mean increase in respiratory minute volume was 14% during adrenaline infusion and 8% during noradrenaline infusion.2. In a small group of decerebrate cats infusions of adrenaline and noradrenaline increased ventilation by 19 and 27% respectively.3. Intravenous catecholamine infusions also increased the respiratory responses of anaesthetized animals to the inhalation of 5% or 10% O(2) in N(2) and to the inhalation of 5% CO(2) in air.4. Adrenaline and noradrenaline infusions had no significant effect on the ventilation of animals breathing 100% O(2), nor did they significantly alter the respiratory response to the inhalation of 5% CO(2) in O(2).5. After section of the carotid sinus and aortic nerves, a blood-pressure compensator being used to minimize changes in arterial pressure, catecholamines had no effect on the respiration of cats breathing air.6. An increase in carotid body chemoreceptor discharge accompanied the increase in ventilation during catecholamine infusion.7. Intravenous catecholamine infusions still produced an increase in ventilation and carotid body chemoreceptor discharge after both aortic nerves and both cervical sympathetic nerves had been cut.8. Intra-arterial infusions into one carotid artery of 0.2 mug/kg.min of adrenaline or 0.1 mug/kg.min of noradrenaline led to mean increases in respiratory minute volume of 9.9 and 11.5% respectively. No increase occurred after section of the corresponding carotid sinus nerve. Such infusions also evoked an increase in carotid body chemoreceptor discharge.9. It is concluded that the hyperpnoea produced by adrenaline and noradrenaline infusions in the cat is predominantly reflex in origin and is mediated by the arterial chemoreceptors.10. The increase in ventilation produced by adrenaline appears to have a component additional to its effect upon the chemoreceptors though the nature of this action has not been identified.     

Low glucose effects on rat carotid body chemoreceptor cells' secretory responses and action potential frequency in the carotid sinus nerve

Glucose deprivation (hypoglycaemia) is counterbalanced by a neuroendocrine response in order to induce fast delivery of glucose to blood. Some central neurons can sense glucose, but nevertheless the most important glucose sensors/glycaemia regulators are located outside the brain. Some recent experimental evidence obtained in carotid body (CB) slices and isolated chemoreceptor cells in culture supports a role for the CB in glucose sensing and presumably glucose homeostasis, but this role has been questioned on the basis of a lack of effect of low glucose on the carotid sinus nerve activity. This work was performed in an attempt to clarify if low glucose is or is not a stimulus for the rat CB chemoreceptors. Using freshly isolated intact CB preparations we have monitored the release of catecholamines (CAs) and ATP from chemoreceptor cells in response to several concentrations of glucose, as indices of chemoreceptor cell sensitivity to glycaemia, and the electrical activity in the carotid sinus nerve (CSN), as an index of reflex-triggering output of the CB. We have observed that basal (20% O₂) and hypoxia (7 and 10% O₂)-evoked release of CAs was identical in the presence of normal (5.55 m m ) and low (3, 1 and 0 m m ) glucose concentrations. 0 m m glucose did not activate the release of ATP from the CB, while hypoxia (5% O₂) did. Basal and hypoxia (5% O₂)-induced CSN action potential frequency was identical with 5.55 and 1 m m glucose. Our results indicate that low glucose is not a direct stimulus for the rat carotid body chemoreceptors.     

Calcium handling by the cat carotid body--a pyroantimonate study

Subcellular regulation mechanisms of calcium concentrations related to oxygen sensing in the carotid body are unclear. In the present study, we investigated the ultrastructural distribution patterns of calcium in carotid body cells and its changes evoked by hypoxia. Carotid bodies were dissected from anesthetized cats exposed in vivo to normoxic or acute hypoxic conditions. We used the oxalate-pyroantimonate technique that yields an electron-opaque calcium precipitate. X-ray microanalysis and appropriate controls confirmed the presence of calcium in the precipitate. Calcium precipitates were found in all types of cells in carotid body parenchyma: chemoreceptor cells, sustentacular cells, and nerve endings. In normoxic chemoreceptor cells, the precipitate was localized in dense core vesicles, mitochondria, and nuclei, but rarely in the cytoplasm. The most apparent effect of hypoxia was disappearance of the precipitate from dense core vesicles and was associated with its appearance in the cytoplasm. The amount of precipitate throughout the carotid body parenchyma was decreased overall due to hypoxia. These results indicate the involvement of subcellular calcium trafficking in hypoxia-sensing in the carotid body. The redistribution pattern of granular calcium deposits from organelles to the cytoplasm of chemoreceptor cells agrees with biochemical data of calcium release from intracellular stores during hypoxia.     

Chemoreceptor A-fibres in the human carotid body contain tyrosine hydroxylase and neurofilament immunoreactivity.

Previous retrograde tracing studies on rat and guinea-pig showed a projection of sensory tyrosine hydroxylase-immunoreactive neurons to the region of the carotid bifurcation via the carotid sinus nerve. In the present study, focussing on the sensory innervation of the human carotid body, antisera to tyrosine hydroxylase and other catecholamine synthesizing enzymes were applied for an immunohistochemical investigation of carotid bodies obtained at autopsy. In addition, an array of antisera directed to non-enzyme antigens known to be present in viscero-afferent neurons were incorporated in the study. The glomic lobules consisting of glomus cells and sustentacular cells contained a variable number of enzyme-immunoreactive glomus cells. Arteries were supplied by nerve fibres displaying the full phenotype of sympathetic noradrenergic axons, i.e. immunoreactivity to tyrosine hydroxylase, aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. The glomic lobules, however, were densely innervated by tyrosine hydroxylase-immunoreactive axons lacking immunoreactivity to aromatic-L-amino-acid-decarboxylase and dopamine-beta-hydroxylase. These fibres reacted with neurofilament 160kD-antibody but were devoid of immunoreactivity to all neuropeptides tested (calcitonin gene-related peptide, somatostatin, substance P). Ultrastructurally, tyrosine hydroxylase/neurofilament 160kD-immunoreactive axons gave rise to large axonal swellings filled with mitochondria and vesicles, and established extensive contacts to glomus cells. Nerve bundles surrounded by a perineural sheath contained both myelinated (2.0-2.8 microns in diameter) and unmyelinated (0.14-3.0 microns) tyrosine hydroxylase-immunoreactive axons. Most of the unmyelinated immunoreactive axons were running singularly within a Schwann cell-sheath. Judged from the pattern of immunoreactivities as well as their preterminal and terminal ultrastructure, tyrosine hydroxylase-immunoreactive axons innervating glomus cells are of sensory origin. Although final proof by retrograde tracing cannot be presented in man, this conclusion is supported by experimental evidence in laboratory animals. The myelinated immunoreactive axons correspond to chemoreceptor A-fibres whereas the classification of the large unmyelinated immunoreactive axons has yet to be established. The lack of immunoreactivity to the dopamine-synthesizing enzyme, aromatic-L-amino-acid-decarboxylase, in this fibre type does not support the view of dopamine being the primary transmitter of chemoreceptor afferents.     

Demonstration of choline acetyltransferase activity in the caotid body of the cat

1. The distribution of choline acetyltransferase in the carotid body of the cat has been investigated with the electron microscope to determine sites of enzymic activity. This is of relevance to the possible role of acetylcholine as a transmitter in the carotid body.2. Tissues were fixed for short periods and incubated by the method of Kasa, Mann & Hebb, for the fine structural localization of choline acetyltransferase.3. The enzyme was found in the cytoplasm of the type I cells and seemed to be associated with vesicles. No enzyme was found in the large nerve endings synapsing with the type I cell.4. Whole carotid bodies were assayed for their choline acetyltransferase activity and significant amounts were found.5. It is concluded that acetylcholine may be a transmitter in the carotid body and that it is synthesized in type I cells. A possible mode of initiation of chemoreceptor afferent impulses is suggested.