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Histamine has been proposed to be an excitatory transmitter between the carotid body (CB) chemoreceptor (glomus) cells and petrosal ganglion (PG) neurons. The histamine biosynthetic pathway, its storage and release, as well as the presence of histamine H1, H2 and H3 receptors have been found in the CB. However, there is only indirect evidence showing the presence of histamine in glomus cells, or weather its application produces chemosensory excitation. Thus, we studied the histamine immunocytochemical localization in the cat CB, and the effects of histamine, and H1, H2 and H3 receptor blockers on carotid sinus nerve (CSN) discharge, using CB and PG preparations in vitro. We found histamine immunoreactivity in dense-cored vesicles of glomus cells. Histamine induced dose-dependent increases in CSN discharge in the CB, but not in the PG. The H1-antagonist pyrilamine reduced the CB responses induced by histamine, the H2-antagonists cimetidine and ranitidine had no effect, while the H3-antagonist thioperamide enhanced histamine-induced responses. Present data suggests that histamine plays an excitatory modulatory role in the generation of cat CB chemosensory activity.

Since the dye- and electronic couplings between the carotid body chief cells have been demonstrated, the detection and localization of the gap junctions in the carotid body is crucial to understanding the functional mechanism of chemoreception. However, conventional electron microscopy has been unsuccessful in unquestionably detecting ultrastructural features equivalent to the gap junctions, such as close (2 nm in width) membrane appositions in ultrathin sections and aggregations of intramembranous particles in freeze-fracture replicas of the carotid body. We previously reported using a modified electron microscopic study by chemically fixed and subsequent rapid freezing and freeze-substitution method a number of close membrane appositions comparable to the gap junctions. However, we later found that the freeze-substitution also induces numerous close apposition of the membrane in sites where the gap junctions are not known to occur, indicating that the modified electron microscopy by freeze-substitution is not always confirmative in the detection of the gap junction. With regard to the molecular evidence for the gap junction in the carotid body, there have so far been few data on the immunohistochemical demonstration on connexin 32 and 43 in cultured chief cells, but not in the in situ cells., (Copyright 2002 Wiley-Liss, Inc.)

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.

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.

The existence and extent of the perineurial cell envelope in the carotid body was clearly demonstrated by the immunoreactivity for nerve growth factor receptor (NGFR) and the freeze-fracture analysis. Consequently the chemoreceptor is regarded as multiple units, each of which consists of certain numbers of chief and sustentacular cells and nerves covered as a whole by a sheath of the perineurial cells.

We have studied the kinetic properties of the O2-sensitive K+ channels (KO2 channels) of dissociated glomus cells from rabbit carotid bodies exposed to variable O2 tension (PO2). Experiments were done using single-channel and whole-cell recording techniques. The major gating properties of KO2 channels in excised membrane patches can be explained by a minimal kinetic scheme that includes several closed states (C0 to C4), an open state (O), and two inactivated states (I0 and I1). At negative membrane potentials most channels are distributed between the left-most closed states (C0 and C1), but membrane depolarization displaces the equilibrium toward the open state. After opening, channels undergo reversible transitions to a short-living closed state (C4). These transitions configure a burst, which terminates by channels either returning to a closed state in the activation pathway (C3) or entering a reversible inactivated conformation (I0). Burst duration increases with membrane depolarization. During a maintained depolarization, KO2 channels make several bursts before ending at a nonreversible, absorbing, inactivated state (I1). On moderate depolarizations, KO2 channels inactivate very often from a closed state. Exposure to low PO2 reversibly induces an increase in the first latency, a decrease in the number of bursts per trace, and a higher occurrence of closed-state inactivation. The open state and the transitions to adjacent closed or inactivated states seem to be unaltered by hypoxia. Thus, at low PO2 the number of channels that open in response to a depolarization decreases, and those channels that follow the activation pathway open more slowly and inactivate faster. At the macroscopic level, these changes are paralleled by a reduction in the peak current amplitude, slowing down of the activation kinetics, and acceleration of the inactivation time course. The effects of low PO2 can be explained by assuming that under this condition the closed state C0 is stabilized and the transitions to the absorbing inactivated state I1 are favored. The fact that hypoxia modifies kinetically defined conformational states of the channels suggests that O2 levels determine the structure of specific domains of the KO2 channel molecule. These results help to understand the molecular mechanisms underlying the enhancement of the excitability of glomus cells in response to hypoxia.

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

After the surgical removal of the postsynaptic sensory sinus nerve, the presynaptic specializations in the chief cell of the rat carotid body chemoreceptor rapidly disappear. When the sinus nerve is allowed to regenerate, the presynaptic specializations reappear almost simultaneously with the re-establishment of apposition of the nerve to chief cells and the number of the synapses is restored to a normal range by the third week after deafferentation. This finding indicates that the maintenance of the presynaptic specializations in the chief cell depends on the persistence of the postsynaptic element, and further suggest the neurotrophic maintenance of the presynaptic specializations may be exerted by the postsynaptic sinus nerve. The sustentacular cells may play a role in governing the specificity of reinnervation by the sinus nerve.

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.

Paraganglia associated with the ninth and tenth cranial nerves have been observed in several locations in the head and neck region. The most studied of these organs, both morphologically and functionally, is the carotid body, a chemoreceptor organ located in the carotid bifurcation. In the present study a microglomus (microparaganglion) composed of islands of carotid-body-like cells was found in the endoneural space of the recurrent laryngeal nerve of rats, 5 to 10 mm from the nerve entrance into the larynx. This microglomus was surrounded by bundles of myelinated and unmyelinated nerve fibers. Fenestrated blood vessels were observed between cells. The cells were characterized by their cytoplasmic membrane-bound granules and dense-core vesicles. Ultrastructurally, these cells seemed to be identical to carotid body chemoreceptor cells and thus to indicate that the cells of the microglomus store catecholamines. Nerve terminals impinging on the cells, classified as both afferent and efferent, were also identified. The recurrent laryngeal endoneural microglomus may have a chemosensitive function influencing the laryngeal physiologic reflex mechanisms.

Recent studies have implicated an abnormality in carotid body structure and function in the pathogenesis of sudden infant death syndrome (SIDS). In the present investigation, the light and electron microscopic findings in carotid bodies from ten victims of SIDS were compared with those in six control infants and five infants dying of congenital heart disease. The cross-sectional area of carotid body chemoreceptor cells and the frequency, distribution, and size of neurosecretory granules were assessed morphometrically. The area of carotid body occupied by chemoreceptor cells (the functional area) was comparable in SIDS victims, control infants, and infants with congenital heart disease. By electron microscopy, the carotid body chief cells from all groups contained numerous electron-dense neurosecretory granules. Distribution, frequency, and size of neurosecretory granules in SIDS victims and control infants did not differ significantly. Morphology of carotid bodies from SIDS victims was found to be normal. The presence of neurosecretory granules in chemoreceptor cells of SIDS victims suggests that the cellular mechanism of neurotransmitter synthesis and storage is not altered.

The functionally first of all considerable components of the carotid bodies are glomus cells and nerve fibres. The glomus cells form two groups. The chemoreceptor cells belong in one of these, the capsular cells in the other. The chemoreceptor cells are polygonal bodies. in their cytoplasm, organella are expressed. The cell nucleus is round, chromatin is homogeneous. Cells are characterized by the osmiophilic bodies. The capsular cells are long-shaped bodies with processes, they are associated with the chemoreceptor cells and sometimes lap over these, like veils. A number of the nerve fibres take place in Schwann's cells wrapped in a mesaxon membrane. The larger part, surrounds the round cell-nests consisting of chemoreceptor cells with a close-meshed network. From the network some fibres draw to the chemoreceptor cells and terminate on these in the form of boutons. Nerve endings form synapses with the chemoreceptor cells. Three forms of these have been found. One of these is the conventional synapsis, the other the saccular synapsis and the third one the parallel contact. Conventional synapses contain all the components identified in the neurons of the central nervous system. Saccular synapses are the magnified pictures of boutons. They consist of axolemma, axoplasm, synaptic vesicles, and mitochondria. In the parallel contacts, the axolemma and cytolemma touch each other. In the axoplasm, there are synaptic vesicles and mitochondria. Conventional synapses conduct peripherally, saccular synapses and parallel contacts centrally. On chemoreceptor cells, there are no neuronal marks, consequently, they are no interneurons. For reciprocal synapses the morphological bases are missing. The chemoreceptor cells are sensory cells.

Several neural and vascular mechanisms regulate the sensitivity of carotid body chemoreceptors to hypoxia, hypercapnia, and acidosis. Factors that control blood flow and oxygen delivery in the carotid body along with those that augment or diminish catecholamine release from glomus cells can have major effects on chemoreceptor function. In addition, the sensory nerves themselves may participate in the regulation of chemoreceptor sensitivity. A portion of the carotid body's sensory nerves are presynaptic to glomus cells. In response to stimulation, the sensory nerve terminals exhibit ultrastructural changes that resemble changes associated with increased release of transmitter from motor nerves: 1) the number of small (synaptic) vesicles decreases; and 2) coated vesicles and coated regions of cisternal membrane increase in number during stimulation. If sensory nerves of the carotid body release a neurotransmitters, sensory nerve activity could influence glomus cell secretion of catecholamines or other substances tha modify chemoreceptor sensitivity. Such an effect could be produced in the carotid body by hypoxia and other conditions that stimulate the sensory nerves or it could result from antidromic activity evoked in the sensory nerves by primary afferent depolarization of their terminals in the CNS.

The carotid body chemoreceptor has been investigated in the normal mouse and in the wobbler mutant. Electron microscopic studies show that the synaptic vesicle-containing nerve endings on type I cells are reduced in number in the mutant to 4.2% of the normal number. The function of the chemoreceptors was examined by recording ventilation and testing the response to different inspired gases and to the intra-arterial injection of cyanide. In addition recordings of chemoreceptor activity were made from the sinus nerve. No distinction could be drawn between the normal and the mutant mouse. No relationship was found between chemoreceptor activity and the respiratory cycle. If the synaptic vesicle-containing endings are afferent then only 4.2% of the normal number appear to be needed for normal function.