Carbonic anhydrase in the carotid body and the carotid sinus nerve

Summary It is well known that carbonic anhydrase plays an important role in the physiological responses of carotidbody chemoreceptors to hypercapnia. Nevertheless the precise location of the enzyme within the carotid body has been a matter of controversy for many years. Using the Hansson method we found histochemical evidence that this enzyme is localized in type I cells. Type II cells and nerve terminals did not show enzymatic activity. These results allow us to define the carotid body as a secondary receptor in the context of the acidic hypothesis of transduction in the carotid body.     

Adenosine triphosphatase activity in the carotid body of the cat

Summary Three kinds of nucleoside phosphatases were demonstrated histochemically in the cat carotid body with nucleoside triphosphate, nucleoside disphosphate and nucleoside monophosphate as substrates. Each of these enzyme activities exhibited the substrate specificity respectively. The nucleoside triphosphatase activity showed specific localization in association with the parenchymal cells of the carotid body.The electronmicroscopy revealed that the reaction product was located on and between the two apposing plasma membranes of type I and type II cells, of a type II cell and its wrapping axons and of the intricate basal infolding of a type II cell itself.Some possible functions of the adenosine triphosphatase in the carotid body are discussed.     

Adenine nucleotides in the carotid body

Summary Remarkably large amounts of adenine nucleotides are identified in type I-cells of the carotid body by fluorescence microscopy (labelling with quinacrine) and electron microscopy (uranaffin reaction). At the fine-structural level the matrix material of specific granules displays enhanced electron density after fixation with uranium ions. It is suggested that ATP is stored within specific granules in addition to catecholamines and proteins. Adenine nucleotides should be considered as one of the secretion products of the chief cells in the carotid body, being capable locally of influencing vascular flow and/or chemoreceptor terminals.Histochemical analysis of the activities leading to a splitting of adenine nucleotides shows a high reactivity with ATP or ADP as substrates. Reaction products are confined to the entire vascular bed of the carotid body. Using AMP or -glycerophosphate as substrate, practically no phosphohydrolytic activity could be detected within the carotid body. Thus, the phosphatases are adequate to remove ATP and ADP, but not to form adenosine.This study was supported by a grant from the Deutsche Forschungsgemeinschaft, Nr. Bo 525/3     

Evidence for histamine as a transmitter in rat carotid body sensor cells

Carotid bodies harboring sensor cells for oxygen have a strategic location at the bifurcation of the carotid artery, which supplies the brain. Upon arterial hypoxia they transmit signals to the respiratory center, which increases the frequency of breathing. Dopamine is considered as the predominant transmitter of the rat carotid body sensor cells. Here we show that the rat carotid body sensor cells are the first cell type known to have the complete apparatus to synthesize, store and release both dopamine and histamine. The tyrosine hydroxylase positive dopaminergic sensor cells of juvenile rats express the histamine biosynthesis enzyme, histidine decarboxylase. Moreover, the sensor cells have not only vesicular monoamine transporter 1 (VMAT1) transporting catecholamines but also VMAT2, which is highly specific for histamine. Additionally, we found that these cells possess components of the neuroendocrine exocytosis apparatus, synaptosome-associated protein of 25 kDa (SNAP 25) and syntaxin1. The amount of histamine determined in the rat carotid body (164 pmol/carotid body) is more than 10-fold higher compared with that of dopamine. As a main effect, hypoxia significantly increased histamine release from isolated rat carotid bodies as it has been shown for dopamine. Finally, RT-PCR experiments indicate the presence of histamine receptors H1, H2 and H3 in the carotid body. Our data suggest that histamine is synthesized, stored and released upon hypoxia by dopaminergic sensor cells of the rat carotid body.     

Ultrastructure of calcitonin gene-related peptide-and substance P-like immunoreactive nerve fibres in the carotid body and carotid sinus of the guinea pig

Summary Previous studies have demonstrated that substance P-(SP) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) coexist in sensory nerve fibres in the guinea-pig carotid body and carotid sinus. In the present study the ultrastructure of these nerve fibres was investigated by means of single-and double-labelling immunocytochemistry. In both, carotid body and carotid sinus immunoreactive fibres were unmyelinated axons of small dianeter (0.12–0.56 m). At the subcellular level, SP-and CGRP-LI were colocalized in intra-axonal dense core vesicles, suggesting corelease and simultaneous action of these two compounds. SP/CGRP-LI nerve fibres within the carotid body were mainly found in the interparenchymal connective tissue, but also occurred in relationship to blood vesslesl and nests of glomus cells. Neither in the carotid body not in the carotid sinus, SP/CGRP-LI axons corresponded to the large terminals which are generally considered to represent the main chemoreceptor and baroreceptor endings, respectively. Thus, SP/CGRP-LI fibres either belong to the chemo-and baroreceptors of the C-fibre class or constitute a fibre population not directly involved in conduction of baro-and chemoreflexes.This study was supported by the DFG, grant He 919/6-2     

The occurrence and distribution of certain polypeptides within the human carotid body

Summary Both carotid bodies from 26 patients coming to necropsy were fixed in 10% neutral buffered formalin and sections 4 m thick were stained for various peptides by use of the immunogold technique. The results show that the human carotid body contains met- and leu-enkephalin, substance P, vasoactive intestinal peptide (VIP), neurotensin and bombesin. The distribution of these six peptides within the carotid body differs. Thus met- and leu-enkephalin are both present predominantly within glomic chief cells but with a marked tendency to favour the dark variant of these cells. Substance P and VIP both show a weak immunoreactivity in comparison to the enkephalins and are present in all three variants of chief cell. Neurotensin shows the weakest immunoreactivity of all and is restricted to a few glomic chief cells in a minority of cases. Bombesin also shows a weak immunoreactivity in glomic chief cells but a strong reaction in glomic arteries and arterioles. In these vessels bombesin appears to be confined to smooth muscle cells in the media but we cannot say whether it is secreted by them or merely bound to receptor sites on their membranes. These findings are related to quantitative data on the concentration of peptides in the human carotid body from a previous paper with which we were associated.     

Loss of histochemically demonstrable catecholamines in the glomus cells of the carotid body after alpha-methyl-para-tyrosine treatment.

A statistically significant decrease in the intensity of catecholamine fluorescence of some carotid body glomus cells was observed after inhibition of the enzyme tyrosine hydroxylase by injection of 80 mg/kg alpha-methyl-paratyrosine. The intensity of the formaldehyde-induced fluorescence was measured in individual glomus cells. The maximum decrease in the intensity was observed 4 to 6 hr after the alpha-methyltyrosine injection. This suggests a rapid turnover in the catecholamines of the carotid body.     

Immunohistochemical and histochemical evidence for the presence of noradrenaline,serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and -aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.     

Poly(ADP-ribose) polymerase activity in the cat carotid body in hypoxia and hyperoxia.

Reactive oxygen species (ROS) induce DNA damage with the ensuing activation of the chromosomal repair enzyme poly(ADP-ribose) polymerase (PARP). ROS also interact with the function of carotid body chemoreceptor cells. The possibility arises that PARP is part of the carotid chemosensing process. This study seeks to determine the presence of PARP and its changes in response to contrasting chemical stimuli, hypoxia and hyperoxia, both capable of generating ROS, in cat carotid bodies. The organs were dissected from anesthetized cats exposed in vivo to acute normoxic (PaO2 approximately 90 mmHg), hypoxic (PaO2 approximately 25 mmHg), and hyperoxic (PaO2 > 400 mmHg) conditions. Carotid body homogenate was the source of PARP and [adenine 14C] NAD was the substrate in the assay. Specimens of the superior cervical ganglion and brainstem were used as reference tissues. We found that PARP activity amounted to 27 pmol/mg protein/min in the normoxic carotid body. The activity level more than doubled in both hypoxic and hyperoxic carotid bodies. Changes of PARP in the reference tissues were qualitatively similar. We conclude that PARP is present in the carotid body but the augmentation of the enzyme activity in both hypoxia and hyperoxia reflects DNA damage, induced likely by ROS and being universal for neural tissues, rather than a specific involvement of PARP in the chemosensing process.     

Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat

Summary The cellular localization of carbonic anhydrase (CAH) in the carotid body of the rat was investigated by means of Hansson's cobalt-precipitation technique in cultures of dissociated cells. In both young (2-day-old) and old (77-day-old) cultures, the parenchymal glomus (type-I) cells were selectively stained by this technique, and in addition expressed tyrosine hydroxylase and neuron-specific enolase as revealed by immunofluorescence. Enzymic reaction product of CAH appeared to be predominantly intracellular since staining was more intense and occurred more rapidly following permeabilization of the cell membranes with Triton X-100; its formation was inhibited by the CAH-inhibitor acetazolamide (1–10 M) or by increasing the pH from 5.8 to 7.5. Cryostat sections of the carotid bifurcation revealed intense CAH-reaction product in cell clusters of the carotid body, in a few cells of the nodose ganglion, and in red blood cells. Neuronal cell bodies of the petrosal ganglion and superior cervical ganglion (SCG) were largely non-reactive. The SCG is known to contain clusters of small intensely fluorescent (SIF) cells, which were also non-reactive when grown in dissociated cell culture. Thus, although glomus and SIF cells are often considered to be similar cell types, functional CAH-activity appears unique to glomus cells, and this may be important for the physiological response of the carotid body to certain chemosensory stimuli.     

Distribution of carotid body type I cells and other periadventitial type I cells in the carotid bifurcation regions of the rabbit

Summary The distribution of carotid body type I and periadventitial type I cells in the carotid bifurcation regions was investigated unilaterally in seven and bilaterally in two New Zealand White rabbits. Carotid body type I cells occurred in close proximity to the wall of the internal carotid artery immediately rostral to the carotid bifurcation, within a division of connective tissue with defineable but irregular borders. Caudally, and separate from the main mass of carotid body type I cells, isolated groups of periadventitial type I cells lay freely in the connective tissue around the internal carotid artery and alongside the carotid bifurcation and common carotid artery. A overall picture of the carotid body in the rabbit was reconstructed and the occurrence and significance of periadventitial type I cells discussed.The authors are indebted to Mr. Stephen Jones of the Department of Histopathology, St Bartholomew's Hospital, for expert assistance in the preparation of the material, and to Mr. A.J. Aldrich of the Department of Anatomy for photography. This work was supported by a grant from the Wellcome Trust to one of us (M. de B.D.)     

Ontogeny of the carotid body and glomus cells distributed in the wall of the common carotid artery and its branches in the chicken

Summary Developmental patterns of immunoreactivity for serotonin and neuropeptide Y were investigated immunohistochemically in the carotid body and glomus cells in the wall of the common carotid artery and around its branches of chickens at various developmental ages. The development of peptidergic nerve fibers was also studied. Serotonin immunoreactivity began to appear in the glomus cells of the carotid body and around arteries at 10 days of incubation and became very intense from 12 days onwards. Neuropeptide Y immunoreactivity also appeared in these cells at 10 days, became intense at 14 days, and was sustained until 20 days. After hatching, neuropeptide Y immunoreactivity in the carotid body rapidly decreased with age and almost cisappeared at posnatal day 10. However, it persisted for life in the glomus cells distributed in the wall of the common carotid artery. Substance P- and calcitonin gene-related peptide (CGRP)-immunoreactive fibers first penetrated into the carotid body parenchyma at 12 days of incubation. These peptidergic nerve fibers in the carotid body and glomus cell groups in and around arteries gradually increased with age, and approached the adult state at 18 days of incubation. Only a few galanin-and vasoactive intestinal peptide (VIP)-immunoreactive fibers were observed in the late embryonic carotid bodies. They rapidly developed after hatching and reached adult numbers at postnatal day 10. During late embryonic and neonatal development, considerable numbers of met-enkephalin-immunoreactive fibers were detected in the connective tissue encircling the carotid body.     

Accessory carotid body within the parathyroid gland III of the chicken

In the chicken, the cranial and caudal parathyroid glands (parathyroid gland III and IV), which are connected to each other, are located adjacent to the carotid body. In the present study, we found that a mass of glomus cells surrounded by a thick layer of connective tissue was frequently distributed within the parathyroid gland III. The glomus cells in the parathyroid III, as well as those of the carotid body, expressed intense immunoreactivity for serotonin, chromogranin A, and tyrosine hydroxylase but no immunoreactivity for neuropeptide Y. The cells possessed long cytoplasmic processes containing dense-cored vesicles of 70–220 nm in diameter, and were in close association with sustentacular cells. In and around the glomus cell clusters of the parathyroid III, dense networks of varicose fibers showed immunostaining with the monoclonal antibody TuJ1 to a neuronspecific class III -tubulin isotype, c4. Furthermore, the distribution was also detected of numerous galanin-, vasoactive intestinal peptide (VIP)-, substance P-, and calcitonin gene-related peptide (CGRP)-immunoreactive fibers.     

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO₂ and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.     

Mash1 is required for glomus cell formation in the mouse carotid body

The carotid body consists of chemoreceptive glomus cells, sustentacular cells and nerve endings. The murine carotid body, located at the carotid bifurcation, is always joined to the superior cervical ganglion of the sympathetic trunk. Glomus cells and sympathetic neurons are immunoreactive for the TuJ1, PGP9.5, tyrosine hydroxylase (TH) and neuropeptide Y (NPY) markers. Glomus cells are also immunoreactive for serotonin (5-HT). A targeted mutation of Mash1, a mouse homolog of the Drosophila achaete-scute complex, results in the elimination of sympathetic ganglia. In Mash1 null mutant mice, the carotid body primordium forms normally in the wall of the third arch artery at embryonic day (E) 13.0 and continues to develop, although the superior cervical ganglion is completely absent. However, no cells in the mutant carotid body display the TuJ1, PGP 9.5, TH, NPY and 5-HT markers throughout development. The absence of glomus cells was also confirmed by electron microscopy. The carotid body of newborn null mutants is composed of mesenchymal-like cells and nerve fibers. Many cells immunoreactive for the S-100 protein, a sustentacular cell marker, appear in the mutant carotid body during fetal development. The Mash1 gene is thus required for the genesis of glomus cells but not for sustentacular cells.     

Neuropeptide-Y-immunoreactive chief cells in the carotid body of young rats

The immunolight- and electron-microscopic study revealed neuropeptide Y (NPY) immunoreactivity in the chief cells and a few nerve fibers of the carotid body of young rats. NPY-immunoreactive chief cells were often seen as a single cell or a group of a few chief cells in the carotid body of young rats. The immunoreaction deposits were mainly seen in the dense-cored vesicles and diffusely in the cytoplasm of the chief cells. These findings suggest that NPY may be stored in the core of granules of the chief cells of the carotid body in young rats.     

Synaptic morphology of the carotid body of the domestic fowl

Efferent and reciprocal synapses have been demonstrated in the carotid body of the domestic fowl (Gallus gallus domesticus). Synapses were also found with purely afferent morphology, but were probably components of reciprocal synapses. The general morphology of the endings suggested the presence of two types of axon, afferent axons making reciprocal and perhaps afferent synapses with Type I cells, and efferent axons making efferent synapses with Type I cells. A few axo-dendritic synapses were also found. The dense-cored vesicles associated with the afferent components of reciprocal synapses and with the possible true afferent synapses varied in diameter and core but could belong to one population of pre-synaptic vesicles. These observations are consistent wtih a new theory for the carotid body receptor mechanism. This proposes a spontaneously discharging afferent axon inhibited by an inhibitory transmitter substance released by the Type I cell via the "afferent" component of its reciprocal synapse, the "efferent" component inhibiting this release. Besides this chemoreceptor modulation of its afferent axon, the Type I cell may also have a general secretory function.     

Origin and development of the catecholamine-storing cells of the human fetal carotid body

Summary The carotid bifurcation areas of 25 human fetuses aged from 8 to 22 weeks were studied using the formaldehyde-induced fluorescence method. A long process from the sympathetic trunk reached the area at the age of 8 weeks contacting with the carotid body primordium. Brightly fluorescent cells can be seen both in the carotid body and in the ganglionic process. Migration of these cells from the sympathetic trunk to the carotid body is suggested. The connection from the sympathetic trunk to the carotid body totally disappeared after the tenth week, leaving no fluorescent elements between these two. Control electron microscopy and light microscopy were performed to identify the fluorescent and nonfluorescent components of the human fetal carotid body.     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Innervation of the carotid body. An experimental quantitative study

The innervation of the carotid body in the cat was studied by means of light- and electron-microscopic techniques. Sinus nerve resection, glossopharyngeal resection, bilateral cervical sympathectomy, excisions of two nerves, and injection of 6-hydroxydopamine (6-OH-DA) were performed in different groups of animals. It was found that resection of the sinus nerve produces a rapid phase of degeneration of intralobular fibers and synaptic boutons, followed by a reinnervation with a progressive reappearance of these elements. This reinnervation is retarded by sympathectomy and prevented by 6-OH-DA. It is therefore concluded that reinnervation is due to collateral regeneration of nearby sympathetic fibers. Resection of the sinus nerve produces an increase in the number of argentaffin cells and dense-cored vesicles in the cytoplasm of principal cells. These findings suggest the existence of efferent synaptic contacts between this nerve and principal cells. Part of the intralobular fibers and synaptic boutons degenerate after bilateral sympathectomy demonstrating that sympathetic axons connect synaptically to the principal cells. Sympathetic fibers reach the carotid body, not only from branches of the cervical plexuses but also from fibers running in the adventitia of the common carotid artery, and via glossopharyngeal and sinus nerves. The vagus nerve contributes a few fibers to the parenchymal lobules of the carotid body.