A comparative study of the distribution of carotid body type-I cells and periadventitial type-I cells in the carotid bifurcation regions of the rabbit,rat, guinea-pig and mouse

Summary The bilateral distribution of carotid body type-I cells was investigated in five rabbits, rats, guinea-pigs and mice by serially sectioning the carotid bifurcation regions. Carotid body type-I cells occurred bilaterally in close proximity to the wall of the internal carotid artery in the rabbit, rat and mouse and to the wall of the ascending pharyngeal artery in the guinea-pig. The rat carotid body was sometimes recessed into the lateral aspect of the superior cervical ganglion and was the most easily defined organ in the four animals studied. Caudally, and separate from the principal mass of carotid body type I cells, isolated groups of periadventitial type-I cells were observed in the connective tissues around the internal carotid artery and adjacent to the carotid bifurcation and common carotid artery in the rabbits only. An overall picture of the carotid body in the four animals was constructed. In all specimens rostral-caudal dimensions were recorded and compared bilaterally.The authors are indebted to Mr. Stephen Jones and Miss Alison Field of the Department of Histopathology, St Bartholomew's Hospital, for expert assistance in the preparation of the material; Miss J. McClelland and Miss C. Slatter for illustrations, and Mr. A. J. Aldrich and Mr. P.S. Hazell for photography. This work was supported by a grant from the Wellcome Trust to one of us (M. de B. D.)     

Sympathetic innervation of the carotid bifurcation in the rabbit and cat: Blood vessels,carotid body and carotid sinus

Summary Two postganglionic branches of the superior cervical ganglion enter the area of the carotid bifurcation in the rabbit and the cat. The common and external carotid arteries receive a rich adrenergic nerve supply, which can be demonstrated by fluorophores of biogenic amines appearing after formaldehyde treatment. The internal carotid artery is only sparsely innervated; however, it shows a dense sympathetic supply at the site of pressor receptors. Following removal of the superior cervical ganglion, a total loss of fluorescent adrenergic nerves occurs and degeneration of nerve endings possessing dense core vesicles is conspicuous. These nerve terminals are situated mainly subendothelially in the carotid body sinusoids; they only rarely terminate on type I cells.     

Carotid body of Camelus dromedarius.

The carotid body of the camel is located between a mass of loose connective tissue at the point of separation of the internal carotid artery from the carotid trunk. A capsule-like connective tissue sheath sends strands in between the parenchyme of this organ and separates lobes and lobules, making it disseminated in type, as in man and in the horse. Two distinct types of cells were found in the parenchyma. Type I cells with specific electton-dense, cored vesicles, and type II cells with protoplasmic extensions. Unlike the previously reported arrangement in the carotid body of some species, the type I cells have direct contact with the basement membrane of glomi and capillaries. Synaptic contacts were seen on both cell types.     

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.     

The structure of the carotid bifurcation in the lizards Tiliqua occipitalis and Trachysaurus rugosus

The origin of the internal carotid artery in the lizards Tiliqua occipitalis and Trachysaurus rugosus has been shown to be both extremely variable and complex. In the dense connective tissue investing the junction of the internal carotid artery and the summit of the carotid arch are groups of cells associated with sinusoids derved from arterioles arising from the neighboring great vessels. The conspicuous epithelioid cells in these groups resemble the mammalian carotid body glomus and amphibian α cells. Nerve fibers showing no obvious synaptic specializations pass to and over these cell groups. It is suggested that these cell groups and sinusoids are comparable to mammalian carotid body glomeruli. A similar group of cells has been described in the concavity of the summit of the fourth aortic arch. This cell group, however, gives a distinct chromaffin reaction.     

Immunofluorescent and structural features of cells in the intervascular stroma of the amphibian carotid labyrinth

Summary The amphibian carotid labyrinth consists of a pars cavernosa, the main chamber of which is in communication with both the base of the external carotid artery, and the vessels of the labyrinthine pars capillaris. On the walls of the main chamber is a network of thick strands of connective tissue and modified smooth muscle cells surrounding the openings into the p. capillaris. These openings lead into wide-diameter atrial vessels, which in turn branch to form the short narrow-diameter vessels. The short vessels form the major component of the labyrinth. A few extremely narrow-diameter vessels are also present. The short vessels open into the roots of the internal carotid artery on the ventral aspect of the carotid labyrinth. The intervascular stroma of the p. capillaris contains numerous stellate and bipolar cells. These cells give a positive response to an immunofluorescent technique specific for smooth muscle myosin and tropomyosin. As the ultrastructural features of these cells are comparable in many respects to smooth muscle, they have been designated as modified smooth muscle cells. It is proposed that these cells act in both an active and passive fashion in maintaining the luminal dimensions of the short vessels relatively constant.     

Quantitative studies of rat carotid body type I cells

The general structure and results of quantitative studies of rat carotid body type I cells are described. In contrast to previous reports, there was no change in mitochondrial V/v% on stimulating the carotid body with 10% oxygen. The volume of cytoplasm occupied by electron-dense cored vesicles was significantly increased, whilst their density per square micrometre of cytoplasm was decreased during hypoxia. Thus, the size of vesicles is increased by hypoxic stimulation. On the basis of vesicle diameter and density we were unable to find evidence of more than one variety of type I cell.     

FRS2 alpha 2F/2F mice lack carotid body and exhibit abnormalities of the superior cervical sympathetic ganglion and carotid sinus nerve

The docking protein FRS2α is an important mediator of fibroblast growth factor (FGF)-induced signal transduction, and functions by linking FGF receptors (FGFRs) to a variety of intracellular signaling pathways. We show that the carotid body is absent in FRS2α^2F/2F mice, in which the Shp2-binding sites of FRS2α are disrupted. We also show that the carotid body rudiment is not formed in the wall of the third arch artery in mutant embryos. In wild-type mice, the superior cervical ganglion of the sympathetic trunk connects to the carotid body in the carotid bifurcation region, and extends thick nerve bundles into the carotid body. In FRS2α^2F/2F mice, the superior cervical ganglion was present in the lower cervical region as an elongated feature, but failed to undergo cranio-ventral migration. In addition, few neuronal processes extended from the ganglion into the carotid bifurcation region. The number of carotid sinus nerve fibers that reached the carotid bifurcation region was markedly decreased, and baroreceptor fibers belonging to the glossopharyngeal nerve were absent from the basal part of the internal carotid artery in FRS2α^2F/2F mutant mice. In some of the mutant mice (5 out of 14), baroreceptors and some glomus cells were distributed in the wall of the common carotid artery, onto which the sympathetic ganglion abutted. We propose that the sympathetic ganglion provides glomus cell precursors into the third arch artery derivative in the presence of sensory fibers of the glossopharyngeal nerve.     

Distribution and ontogeny of chromogranin A and tyrosine hydroxylase in the carotid body and glomus cells located in the wall of the common carotid artery and its branches in the chicken

Summary Development and distribution of chromogranin A and tyrosine hydroxylase in the carotid body and glomus cells located in and around arteries were examined in chickens at various developmental stages by an immunohistochemical staining. In 9-day-old embryos, numerous cells immunoreactive for tyrosine hydroxylase were already detected in the connective tissue surrounding the carotid body. Some of these cells also showed immunoreactivity for chromogranin A. At 10 days of incubation, a few cells immunoreactive for tyrosine hydroxylase and chromogranin A were detected within the carotid body parenchyma. At 12 days of incubation, almost all glomus cells of the carotid body were intensely immunoreactive for these substances. Furthermore, numerous tyrosine hydroxylase- and chromogranin A-immunoreactive cells were observed in the wall of the common carotid artery, along the whole length of the carotid body artery, and around the roots of the inferior thyroid artery, the ascending esophageal artery and the esophagotracheobronchial artery; the cells already exhibited adult pattern of distribution at this stage of development. Thereafter, glomus cells immunoreactive for both substances gradually increased in number and in intensity of immunoreactivity with age, although the cells located in the wall of the common carotid artery lost immunoreactivity for tyrosine hydroxylase after hatching.     

Distribution and ontogeny of chromogranin A and tyrosine hydroxylase in the carotid body and glomus cells located in the wall of the common carotid artery and its branches in the chicken

Development and distribution of chromogranin A and tyrosine hydroxylase in the carotid body and glomus cells located in and around arteries were examined in chickens at various developmental stages by an immunohistochemical staining. In 9-day-old embryos, numerous cells immunoreactive for tyrosine hydroxylase were already detected in the connective tissue surrounding the carotid body. Some of these cells also showed immunoreactivity for chromogranin A. At 10 days of incubation, a few cells immunoreactive for tyrosine hydroxylase and chromogranin A were detected within the carotid body parenchyma. At 12 days of incubation, almost all glomus cells of the carotid body were intensely immunoreactive for these substances. Furthermore, numerous tyrosine hydroxylase- and chromogranin A-immunoreactive cells were observed in the wall of the common carotid artery, along the whole length of the carotid body artery, and around the roots of the inferior thyroid artery, the ascending esophageal artery and the esophagotracheobronchial artery; the cells already exhibited adult pattern of distribution at this stage of development. Thereafter, glomus cells immunoreactive for both substances gradually increased in number and in intensity of immunoreactivity with age, although the cells located in the wall of the common carotid artery lost immunoreactivity for tyrosine hydroxylase after hatching.     

Distribution of CGRP-, somatostatin-, galanin-, VIP-, and substance P-immunoreactive nerve fibers in the chicken carotid body

Summary An immunoperoxidase method was used to investigate and compare the distribution of neuropeptide-immunoreactive (ir) nerve fibers and neurofilament-ir fibers in chick carotid body. The vagus nerve and its branches were intensely immunoreactive with an antiserum against chick neurofilaments. The branches from the vagus and the recurrent laryngeal nerves anastomosed within the connective tissue encircling the carotid body, and then entered the organ to form a network of neurofilament-ir fibers. Immunoreactivities for CGRP, somatostatin, galanin, VIP and substance P were found in the carotid body; they were located within varicose fibers. Immunoreactivity for each peptide was discretely and characteristically distributed. Dense networks of varicose CGRP-ir nerve fibers were found throughout the carotid body in close proximity to clusters of carotid body cells and to blood vessels. Substance P-ir fibers were distributed similarly to CGRP-ir fibers. Somatostatin-ir fibers appeared as patches distributed around chief cells. Numerous galanin- and VIP-ir nerve fibers were observed in the connective tissue surrounding the carotid body, but they occurred in only moderate densities in the parenchyma.     

The carotid sinus in lizards with an anatomical survey of the ventral neck region

The anatomy of the ventral neck region of the scincid lizards Chalcides ocellatus and Scincus scincus is presented and is found to be similar to that of other lizards as described in the literature. The internal carotid artery arises by 3-5 roots from the dorsal side of the ascending limb of the carotid arch. During its first part, the internal carotid artery is completely divided into two nearly equal channels. The carotid sinus is more complicated in Chalcides than in Scincus. In lizards, it may be homologous to the carotid labyrinth of fishes and amphibians. Around the origin of the internal carotid artery are two kinds of epithelioid cells scattered in the adventitial connective tissue: a- large cells with rounded, faintly stained nuclei, and little, clear cytoplasm; b- cells with small darkly stained nuclei. Both kinds of cells appear to represent different levels of secretory activity. The number of the large cells increases with greater complexity of the carotid sinus. The cells also increase in size and number during summer (sexual period); this is especially true in younger animals. The epithelioid cells are considered to be homologous to the carotid body of higher vertebrates. The carotid sinus and epithelioid cells together form a closely interrelated system which may be intermediate between the carotid labyrinth of fishes and amphibians, and the carotid body of birds and mammals.     

Quantitative studies of the cat carotid body. III. The type I cells

The work reported here examines the quantitative ultrastructure of cat carotid body type I cells, and the effects of hypoxia on the cells. On the basis of size and density of electron-dense cored vesicle (EDCV) we were unable to identify more than one major population of type I cells. Prior ventilation of the animals with 10% O2 resulted in an increase in the volume percentage (Vv%) of mitochondria and a decrease in the Vv% of EDCV as compared to the values after ventilation with 100% O2. The lack of effect of prior denervation of the carotid body on the hypoxic changes suggests that the effects are not mediated via an efferent pathway.     

Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.     

Metabolic activation of carotid body glomus cells by hypoxia

The effects of low O2 on glucose consumption in the rabbit carotid body were studied using the in vitro 2-deoxyglucose technique. Metabolically active structures within the tissue were localized autoradiographically after freeze-drying and vacuum fixation/embedding of selected incubated tissue samples. In 100% O2-equilibrated media, the mean basal glucose consumption calculated from the rate of 2-[1,2-3H]deoxy-D-glucose phosphorylation and its specific activity in the incubation media was 61 nmol.g tissue-1.min-1 in the carotid body and 42 nmol.g tissue-1.min-1 in parallel experiments with nodose ganglia. Low PO2 (20% O2-equilibrated media in vitro) increased glucose consumption in the carotid body by 44% but did not alter glucose metabolism of nodose ganglia. Autoradiographic data showed that preneural type I parenchymal cells are the principal site of glucose consumption in carotid chemosensory tissue. The mechanisms responsible for the hypoxia-induced increase in glucose consumption by the type I cells are discussed in relation to sensory transduction by the carotid body chemoreceptors.     

Movement of neurosecretory product through the anatomical compartments of the neural lobe of the pituitary gland

Summary Electron microscopic studies of the carotid body of the domestic fowl (Gallus gallus domesticus) have shown Type I and Type II cells combined with axons into compact groups. The many Type I cells in the depths of the organ had a body, containing the nucleus, and an elongated, flared process. Some of the Type I cells in the superficial regions tended to be spindle-shaped. Type I cells were characterised by membrane-bound, dense-cored vesicles about 120 nm in diameter. Type II cells invested the Type I cells and had axons embedded in them as in Schwann cells.The fine structure of the carotid body in the domestic fowl resembles that of the Lovebird (Uroloncha domestica) and of various amphibia and mammals. The possibility is discussed that the Type I cells may have a chemoreceptor or a general secretory function, or even both pathway for functions together. The main role of the Type II cells seems to be to provide a of these axons leading to or from Type I cells.The authors are grateful to Mr. R. P. Gould of the Department of Anatomy, Middlesex Hospital Medical School for permission to use some of his and Dr. Hodges' original material in the illustrations. Dr. Hodges also wishes to thank the A.R.C. and the University of London Central Research Fund for financial assistance. We are also most appreciative of the photographic assistance of J. Geary.     

Quantitative studies of rat carotid body type I cell nerve endings

The results of a stereological and morphometric analysis of rat carotid body type I cell nerve endings are described. 66.9% of endings possessed symmetrical junctions. Of the remaining endings, 3.6% were presynaptic and 26% were postsynaptic to type I cells; 3.6% of endings had a reciprocal configuration. Apart from membrane specialisations, no other ultrastructural criteria were found to distinguish the different types of endings. Ventilation with 100% and 10% oxygen showed that the hypoxic mixture reduced synaptic vesicle concentration in the nerve endings; this effect was independent of the innervation to 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.     

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.     

Interactions between hypoxia and hypercapnic acidosis on calcium signaling in carotid body type I cells

The effects of hypercapnic acidosis and hypoxia on intracellular Ca(2+) concentration ([Ca(2+)](i)) were determined with Indo 1 in enzymatically isolated single type I cells from neonatal rat carotid bodies. Type I cells responded to graded hypoxic stimuli with graded [Ca(2+)](i) rises. The percentage of cells responding was also dependent on the severity of the hypoxic stimulus. Raising CO(2) from 5 to 10 or 20% elicited a significant increase in [Ca(2+)](i) in the same cells as those that responded to hypoxia. Thus both stimuli can be sensed by each individual cell. When combinations of hypoxic and acidic stimuli were given simultaneously, the responses were invariably greater than the response to either stimulus given alone. Indeed, in most cases, the response to hypercapnia was slightly potentiated by hypoxia. These data provide the first evidence that the classic synergy between hypoxic and hypercapnic stimuli observed in the intact carotid body may, in part, be an inherent property of the type I cell.