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The carotid body is an arterial chemoreceptor organ that senses arterial pO(2) and pH. Previous studies have indicated that both reactive oxygen species (ROS) and nitric oxide (NO) are important potential mediators that may be involved in the response of the carotid body to hypoxia. However, whether their production by the chemosensitive elements of the carotid body is indeed oxygen-dependent is currently unclear. Thus, we have investigated their production under normoxic (20% O(2)) and hypoxic (1% O(2)) conditions in slice preparations of the rat carotid body by using fluorescent indicators and confocal microscopy. NO-synthesizing enzymes were identified by immunohistochemistry and histochemistry, and the subcellular localization of the NO-sensitive indicator diaminofluorescein was determined by a photoconversion technique and electron microscopy. Glomus cells of the carotid body responded to hypoxia by increases in both ROS and NO production. The hypoxia-induced increase in NO generation required (to a large extent, but not completely) extracellular calcium. Glomus cells were immunoreactive to endothelial NO synthase but not to the neuronal or inducible isoforms. Ultrastructurally, the NO-sensitive indicator was observed in mitochondrial membranes after exposure to hypoxia. The data show that glomus cells respond to exposure to hypoxia by the enhanced production of both ROS and NO. NO production by glomus cells is probably mediated by endothelial NO synthase, which is activated by calcium influx. The presence of NO indicator in mitochondria suggests the hypoxic regulation of mitochondrial function via NO in glomus cells.

Fenestrated capillaries represent the basic structural unit in the carotid body. They mediate a characteristic hyperpermeability state in this organ. Endothelial fenestrae and plasmalemmal vesicles are of particular importance in this respect. The present electron microscopic study of the capillaries of the mouse carotid body demonstrates prominent endothelial cell structures that are suggested to be closely related to endothelial fenestrae and plasmalemmal vesicles. These structures include: (1) Vesiculo-vacuolar organelles formed by fusion and intercommunication of vesicles and vacuoles of variable dimensions. (2) Pockets in the form of fenestrated membrane-bound vacuoles that communicate either with the capillary lumen, pericapillary space or both via multiple apertures or fenestrae. (3) Multi-layered fenestrated Lamellae where the endothelial cytoplasm is divided into multiple attenuated sheets provided with several fenestrae. The latter two structures are preferentially located in the thick perinuclear region of the endothelial cell. Their fenestrae are always distributed in linear series and show close similarity to the usual chains of fenestrae in the attenuated periphery of the endothelial cells. The individual apertures of the fenestrated vacuoles and multi-layered fenestrated lamellae are closely similar to the stomata of fully opened plasmalemmal vesicles suggesting a relationship between them. Morphological and morphometrical analysis of a series of fenestrae belonging to these structures revealed that they are identical to the usual chains of fenestrae in the attenuated periphery of the endothelial cells.

The structural relationship between type I glomus cells and the vascular smooth muscle was investigated by electron microscopy in the mouse carotid body. A close apposition (<0.1 microm) between the glomus parenchyma and the neighbouring arterioles was regularly present. Profiles of type I glomus cells were found to be exposed to the vascular smooth muscle without any supporting cell investment. In circumscribed areas of these profiles, type I glomus cells and the vascular smooth muscle cells made contact by fusion of their basal laminae. These glomus-cell-myocyte junctions structurally resemble vascular neuromuscular junctions of sympathetic nerve terminals. In addition to the occurrence of such glomus cell-myocyte contacts, myoendothelial junctions also appeared frequently. On the basis of these observations, it is suggested that type I glomus cells play a role in the regulation of the vascular tone in the carotid body and that a physiological interaction between the endothelial cells, the vascular smooth muscle cells and the type I glomus cells exists.

The specific tissue of the carotid body is built up of groups of glomus cells, enveloped by glial-type sustentacular cells, and innervated by sensory nerve fibers. These units sense arterial pO(2) and respond to hypoxia by a variety of reactions that include initiation of the arterial chemoreflex, i.e., increasing firing activity in the carotid sinus nerve. Until now, neither the cellular localization of the initial events that lead to stimulation of chemoreceptor afferents nor the molecular mechanism of oxygen sensing in the carotid body have been unequivocally identified. Proposed molecular candidates for the mechanism of oxygen sensing include: 1). components of the mitochondrial respiratory chain, 2). NADPH oxidases generating reactive oxygen species in an oxygen-dependent manner, 3). oxygen-regulated plasmalemmal K(+)-channels, and 4). nonoxidase iron-proteins. Our still limited knowledge on their cellular distribution within the carotid body is reviewed here. It is evident that: 1). the distribution of at least some oxygen sensor candidates is not ubiquitous but cell-type-specific, and 2). each specific parenchymal cell type of the carotid body contains at least one of the proposed oxygen sensor candidates. This applies also for the glial-type sustentacular cells that exhibit immunoreactivity to the two-pore domain K(+)-channel, TASK-1. These observations fit best with the assumption that each cell type within the carotid body is principally responsive to hypoxia. The differential equipping of glomus cells, nerve endings, and sustentacular cells with sensor proteins might serve to determine different thresholds of sensitivity and/or to connect the process of oxygen sensing to different signaling pathways. It also favors the assumption that several mechanisms of oxygen sensing may act simultaneously. The cellular identification of the cell type initiating the chemoreceptor reflex, however, has to await the molecular identification of the particular oxygen sensor molecule that initiates increased carotid sinus nerve activity., (Copyright 2002 Wiley-Liss, Inc.)

In the present study, we investigated the immunohistochemical localization of two-pore K(+)-channels TASK-1, TASK-2, TASK-3 and TRAAK in the rat carotid body. Type I cells were positive for TASK-1, TASK-2, TASK-3 and TRAAK. Intrinsic nerve cell bodies were also strongly positive for TASK-1, TASK-2 and TRAAK, but negative for TASK-3. In addition, some type II cells, Schwann cells in the nerve bundles and fibroblast between type I cell clusters were also immunostained for TASK-1. Smooth muscle cells of the carotid body artery were intensely positive for TASK-3. Our results indicate that TASK-1 immunoreactivity was ubiquitously distributed in many cell types and immunoreactivities for TASK-2, TASK-3 and TRAAK were cell type specific distribution patterns in the rat carotid body.

The carotid body monitors arterial oxygen tension. Spectrophotometric recording of the intact organ has revealed a cytochrome aa3 and a cytochrome b558 as potential oxygen sensor candidates. The latter is known as part of the NADPH oxidase system generating superoxide anions in the "respiratory burst" defense mechanism, and glomus cells have been found to exhibit immunoreactivity against this phagocyte cytochrome b558. Using a monoclonal antibody against the large cytochrome b558 subunit, gp91phox, and other antibodies serving as neural (PGP 9.5) and monocyte/macrophage markers (ED1, ED2), we here demonstrate at light and electron microscopical level that monocytes/macrophages are abundantly present in the rat carotid body and represent the major source of cytochrome b558 in this organ. Their presence has profound implications on the interpretation of spectrophotometric recordings aimed to elucidate the mechanisms of oxygen sensing since their high cytochrome b558 content will obscure possible contributions of cell types involved in the oxygen sensor process.

We demonstrate, by means of immunohistochemistry, that type I cells of human, guinea pig, and rat carotid bodies react with antisera raised against the subunits p22phox, gp91phox, p47phox, and p67phox of the NAD(P)H oxidase isolated from human neutrophil granulocytes. The findings support previous photometric studies that indicate that carotid body type I cells possess a putative oxygen sensor protein that is similar to the neutrophil NAD(P)H oxidase and consists of a hydrogen peroxide generating low-potential cytochrome b558 with cofactors regulating the electron transfer to oxygen.

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.

The distribution of serotonin immunoreactivity (-IR) was studied in adult human carotid bodies, obtained at post-mortem, using both the peroxidase-antiperoxidase method on paraffin sections and a double-labelling immunofluorescence on frozen sections. Antibodies against synaptophysin and protein gene product (PGP) 9.5 were used for identification of serotonin-IR cells. Serotonin-IR was demonstrable in the carotid bodies of adult humans and it was coexpressed mostly with synaptophysin or PGP 9.5 in type I cells. Some serotonin immunopositive type I cells were located in close proximity to capillaries. Serotonin-IR was also observed in a few endothelial cells.

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.

The distribution of substance P- (SP-) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) was studied in the human carotid body at the light and electron microscopic level. Of the different compartments of the carotid body, the glomic lobules received the densest innervation by SP/CGRP double-labelled axons, followed by interlobular arteries and interlobular connective tissue. Ultrastructurally, preterminal SP/CGRP-LI axons are unmyelinated and measure 0.09-1.02 micron in diameter with a median diameter of 0.28 micron (calculated conduction velocity: 0.5 m/s). Within the glomic lobules, vesicle-containing varicosities are numerous around glomus cells, but only few of them directly contact (non-synaptically) glomus cells. The findings are consistent with the existence of two populations of SP/CGRP-LI C-fibers, one of them probably representing chemoreceptor C-fibers, the other belonging to the ubiquitous vascular sensory innervation. In addition to its localization in nerve fibers, CGRP-LI also occurs in dense core vesicles of a subpopulation of glomus cells. In this regard, the human carotid body is unique among mammalian species studied so far, which severely hampers the interpretation of pharmacological data obtained in laboratory animals in terms of human physiology.

The peptidergic innervation of arterial chemoreceptor organs (the rat carotid body and vagal paraganglia; guinea pig carotid body) was studied immunohistochemically. Five different populations of nerve fibres in the guinea pig carotid body could be discriminated according to their origin and their chemical coding. The innervation pattern of the rat carotid body differed in some aspects. Comparison of the rat carotid body and vagal paraganglia suggested that autonomic neuropeptide Y-like immunoreactive fibres act primarily via vascular mechanisms rather than directly on the chemoreceptor tissue. Sensory fibres were shown to contain immunoreactivities for substance P, calcitonin gene-related peptide (rat and guinea pig) and somatostatin (guinea pig). The functional role of the identified peptide-containing sensory fibres remains to be established.

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 diameter (0.12-0.56 microns). 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 vessels 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.

Current investigations on the immunohistochemical occurrence and co-occurrence of biogenic polypeptides in the mammalian carotid body were reviewed and extended by our own recent findings. The family of chromogranins and related peptides in glomus cells appears to have a widespread interspecies distribution, whereas other peptides investigated occur in a species-specific pattern. Immunoreactivity to antisera against opioids, which derive from the proenkephalin sequence, appears to be present in glomus cells of the rabbit, cat, dog, and a shrew. Conversely, glomus cells of pig and guinea pig predominantly are immunoreactive to cleavage products of prodynorphin, which co-occur in some cells with substance P and met-enkephalin-arg-phe, respectively. In the rat and Callithrix jacchus, opioid immunoreactivity is present in nerve fibres but not in glomus cells. Immunoreactivity to other peptides, such as neurotensin, cholecystokinin, neuropeptide Y, and galanin, is found only in one or two particular species. Neurotensin immunolabelling occurs in beagle dog glomus cells, which are known to lack substance P. Cholecystokinin immunoreactivity is present in glomus cells of dog and Callithrix, and co-exists with chromogranin A, neuropeptide Y, and substance P. Substance P appears to exist in both carotid body glomus cells and nerve fibres. Substance P immunoreactivity is present in glomus cells of all species investigated, except dog. Coexistence of substance P and calcitonin gene-related peptide (CGRP) is demonstrated in nerve fibres of the guinea pig carotid body, which originate in the petrosal and jugular ganglia. Other peptides visualized immunohistochemically in mammalian carotid body nerve fibres are vasoactive intestinal peptide and neuropeptide Y. The functional significance of the various peptides present in the carotid body is discussed.