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.     

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 dense-cored 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.     

Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies

Substance P-immunoreactive (SP-1) structures in the carotid bodies of rats and cats were examined with the light and electron microscopes. In both species SP-I varicose nerve fibers were located singly in the interstitial connective tissue in close association with blood vessels. They were small unmyelinated fibers enveloped in a common Schwann cell sheath with other SP-negative fibers. Some of SP-I fibers contained large dense-cored granules and small clear vesicles in addition to microtubules and mitochondria and probably represented nerve fiber varicosities. The latter often were found incompletely invested by Schwann cell sheaths. SP-fibers were found occasionally in the envelopes of supporting cells at the periphery of parenchymal cell groups. However, none of the nerve terminals making synaptic contacts with glomus cells exhibited SP-like immunoreactivity. In cat carotid bodies some glomus cells showed moderate to intense SP-like immunoreactivity. The intense SP-I glomus cells displayed numerous dense-cored vesicles of 85 to 140 nm in diameter and frequently showed synaptic contacts with SP-negative nerve terminals. In rat carotid bodies we were unable to detect consistent SP-immunoreactivity in glomus cells. Our results do not favor the hypothesis that SP is a neurotransmitter/modulator in the chemoreceptor afferents synapsing on glomus cells in either the cat or rat carotid body. However our results support the hypothesis that SP in cat glomus cells may play a role in the modulation of chemoreceptor activity.     

Observations on the fine structure of the baroreceptors and adrenergic innervation of the guinea-pig carotid sinus

We examined the fine structure of the baroreceptors and the adrenergic innervation of the guinea-pig carotid sinus. The tunica adventitia contained many nerve bundles whose perineuria enclosed unmyelinated nerve fibers, alone or together with myelinated nerve fibers. Baroreceptors, which lay close to elastic and collagen fibers in the adventitia and media, were surrounded by “terminal” cells with ultrastructural features characteristic of Schwann cells and contained inclusions of various types. Morphologic features of the baroreceptors included densely packed mitochondria, osmiophilic lamellated and homogeneous bodies, clear and granular vesicles, lamellar systems, glycogen granules, neurofilaments, neurotubuli, and vacuolated mitochondria. In animals that had been treated with 6-hydroxydopamine, occasional electrondense endings (or fibers) were observed in the adventitial layer. The baroreceptors in the guinea-pig carotid sinus appear to have most of the morphologic features reported for other species.     

Autophagy in Tobacco Suspension-Cultured Cells in Response to Sucrose Starvation

The response of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2) to nutrient starvation was investigated. When the cells that were grown in Murashige-Skoog medium containing 3% (w/v) sucrose were transferred to the same medium without sucrose, 30 to 45% of the intracellular proteins were degraded in 2 d. An analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that proteins were degraded nonselectively. With the same treatment, protease activity in the cell, which was measured at pH 5.0 using fluorescein thiocarbamoyl-casein as a substrate, increased 3- to 7-fold after 1 d. When the cysteine protease inhibitor (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methyl-butane (10 [mu]M) was present in the starvation medium, both the protein degradation and the increase in the protease activity were effectively inhibited. Light microscopy analysis showed that many small spherical bodies accumulated in the perinuclear region of the cytosol 8 h after the start of the inhibitor treatment. These bodies were shown to be membrane-bound vesicles of 1 to 6 [mu]m in diameter that contained several particles. Quinacrine stained these vesicles and the central vacuole; thus, both organelles are acidic compartments. Cytochemical enzyme analysis using 1-naphthylphosphate and [beta]-glycerophosphate as substrates showed that these vesicles contained an acid phosphatase(s). We suggest that these vesicles contribute to cellular protein degradation stimulated under sucrose starvation conditions.     

Heterogeneous expression of TASK-3 and TRAAK in rat paraganglionic cells

In the present study, we investigated the immunohistochemical localization of the two-pore K⁺ channels, TASK-3 and TRAAK, in paraganglionic cells within the superior cervical ganglion, stellate ganglion, and aortic body in comparison with membrane channels in chief cells of the carotid body. TASK-3 immunoreactivity was observed in the paraganglionic cells in all tissues examined. TRAAK immunoreactivity was observed in the chief cells of the aortic body as well as these of the carotid body, but not in the paraganglionic cells in the sympathetic (superior cervical and stellate) ganglia. Our findings indicate that sympathetic paraganglionic cells and glossopharyngeal/vagal paraganglionic cells were different from each other in the expression patterns of TASK-3 and TRAAK to result in the different chemoreception properties of sympathetic paraganglionic cells from those of chief cells of the aortic and carotid bodies.     

Light and electron microscopic observations on the carotid bodies of rats following adaptation to high altitude.

The carotid bodies of rats were investigated by light and electron microscopy following adaptation to a simulated altitude of 7000 m. Some animals were studied immediately after readaptation to sea-level, other groups up to 41 days later. The animals of the first group show enlarged glomera with dilated capillaries as reported earlier.The type-I-cells are large with a light staining cytoplasm. The lobular configuration of the glomus is lost and intracapillary platelet thrombosis are frequently found. Under these thrombi there is usually a chief cell degeneration with edema and vacuolisation. There is also a noticeable decrease in so-called catecholamine bodies and the remaining ones are usually arranged along the cell borders. Electron microscopically the intercapillary tissue reveals an increase in collagen material partially replanning lost chief cells. Essentially unaltered sustentacular cells frequently enclose with their processes only collagen bundles and nerve fibres. During 41 days following the readaptation there is a narrowing of capillaries and vacuolisation, swelling and thromboses are no longer observed. The amount of collagen, however, remains the same. Dense cored vesicles again increase in number and the chief cells show a prominent interdigitation. Light microscopically the restoration to normal appears to be almost complete. Fine structural analysis, however, shows an appearently permanent distortion in the composition and cellular arrangement of the glomus.     

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.     

Innervation of the parathyroid in the european starling (Sturnus vulgaris)

Anatomical studies were conducted to characterize the source, type, and distribution of parathyroid gland innervation in European starlings. Denervation experiments demonstrated that the parathyroid glands and adjacent carotid bodies are innervated by nerve fibers originating in the nodose ganglion of the vagus nerve. In the parathyroid parenchyma, these fibers terminate adjacent to chief cells or near vascular smooth muscle. Vagal fibers also form synapses with catecholamine-containing glomus cells of the carotid body. Blood that first perfuses the carotid body subsequently perfuses the parathyroid parenchyma. These observations suggest that vagal innervation may influence parathyroid function in starlings either through direct chief cell innervation or through alteration of vascular perfusion. A neurohemal relationship also may exist between the carotid body and parathyroids.     

Light- and electron microscopic observations on parathyroid glands in different age groups of rats

The parathyroid hormone (PTH) acts on bones, intestines, and kidneys to maintain the calcium homeostasis which, in turn, is a main factor in controling the parathyroid (PT) gland activity. In all mammals studied, the chief cells of PT glands changed their size, shape, and cytoplasmic structure due to different functional states which vary the serum calcium levels. The chief cells of the rat PT glands were classified as dark and light. The dark cells may constitute an active form, characterized mainly by the abundant free ribosomes, conspicuous rough endoplasmic reticulum, and GOLGI complexes, greater number of secretory granules (SG) and increased tortuosity of the plasma membranes as compared to the light ones which were considered as a less active type of cells. Due to different calcium requirements in newborn and young rats for the ossification of growing skeleton and in adult and senile rats with consolidate mature bones, the PT glands studied with electron microscope showed various cytological features. The parenchyma of newborn and young PT glands was composed by dark chief cells. The light chief cells were more frequent in adult and senile animals as a less active type of cell. Mature SG were only occasionally observed in dark cells of newborn, young and adult PT glands. They may constitute a reserve supply of PTH but probably not the main way of secretion, according to their little number. Another pool of PTH probably answers the needs for the small basal variations in the steady-state secretion and may be represented by the vesicles observed in the chief cells cytoplasm.     

Peptidergic innervation in the rat carotid body after 2, 4, and 8 weeks of hypocapnic hypoxic exposure

The distribution and abundance of neuropeptide-containing nerve fibers were examined in the carotid bodies of rats exposed to hypocapnic hypoxia (10% O2 in N2) for 2, 4, and 8 weeks. The carotid bodies after 2, 4, and 8 weeks of hypoxic exposure were enlarged by 1.2-1.5 times in the short axis, and 1.3-1.7 times in the long axis in comparison with the normoxic control ones. The enlarged carotid bodies contained a number of expanded blood vessels. Mean density per unit area (10(4) microm2) of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive fibers was transiently high in the carotid bodies after 4 weeks of hypoxic exposure, and decreased significantly to nearly or under 50% after 8 weeks of hypoxic exposure. Density of vasoactive intestinal polypeptide (VIP) immunoreactive fibers increased significantly in all periods of hypoxic exposure observed, and was especially high in the carotid bodies after 4 weeks of hypoxic exposure. Density of neuropeptide Y immunoreactive fibers was unchanged in the carotid bodies during hypoxic exposure. These characteristic changes in the density of SP, CGRP, and VIP fibers in the carotid bodies after 4 weeks of hypoxic exposure suggest that the role of these neuropeptide-containing fibers may be different in the carotid bodies after each of three periods of hypoxic exposure, and that the peptidergic innervation after 8 weeks of hypoxic exposure may show an acclimatizing state.     

Cellular bodies in developing chlemydospores of Thielaviopsis basicola1,2

The development of chlamydospores of Thielaviopsis basicola from hyphal cells involves the thickening and pigmentation of the cell wall. Electron microscope studies showed that membrane-bound cellular bodies appearing in the cytoplasm of differentiating cells developed from dilated cisternae in the endoplasmic reticulum. Within the membrane-bound bodies, vesicles of up to 0.2-micronm diameter were observed which contained electron-dense particles. Vesicles resembling those seen in the cellular body were also present in the cytoplasm close to the plasmalemma. In newly formed chlamydospore cells where wall thickening was complete, the cellular bodies showed loss of internal organization, and most of the vesicles disappeared, leaving a structure resembling a vacuole. The cellular bodies were not present in undifferentiated hyphae or in mature chlamydospores.     

Lysosomal integral membrane glycoproteins are expressed at high levels in the inclusion bodies of I-cell disease fibroblasts

The localization, expression, and transport of two lysosomal integral membrane glycoproteins of human cells, hLAMP-1 and hLAMP-2, have been studied in mucolipidosis II (I-cell disease) fibroblasts. These cells are deficient in N-acetylglucosaminylphosphotransferase, one of the enzymes required for addition of the mannose 6-phosphate recognition signal to newly synthesized lysosomal hydrolases and a prerequisite for the sorting and transport of the hydrolases to lysosomes. I-cells analyzed by immunofluorescence microscopy with monoclonal antibodies against hLAMP-1 and hLAMP-2 showed intense staining of the inclusion bodies covering most of the cytoplasm of the cells. Immunoelectron microscopy confirmed this localization and showed that the hLAMP-positive vesicles commonly contained membrane structures or electron-dense homogeneous material characteristic of secondary lysosomes. Studies of the biosynthesis of hLAMP-2 in I-cells pulse-labeled with [35S]methionine indicated that the molecule is glycosylated in the Golgi system, is transported to vesicles with the high density characteristic of lysosomes, and has chemical properties similar to those of the glycoprotein synthesized in normal cells. The concentration of the hLAMP-2 glycoprotein was three- to fourfold greater than that in normal fibroblasts, in sharp contrast to the reduced levels of lysosomal hydrolases seen in I-cells. These experiments demonstrate that the inclusion bodies in I-cells have properties of secondary lysosomes and that the transport and targeting of the lysosomal membrane glycoproteins to the inclusion bodies of these cells is not coupled to the mannose 6-phosphate system for transporting soluble acid hydrolases.     

Ultrastructural changes in the rat carotid body in severe haemorrhagia

Summary Rat carotid bodies were studied electron microscopically after short-term severe hypovolaemia, which is known to induce a marked chemoreceptor activation in the carotid body. Altogether 84 nerve-endings in the hypovolaemic rats' carotid bodies and 91 nerve-endings in the control carotid bodies were investigated. An increased accumulation of the glomus cell granular vesicles near the synaptic specializations of the nerve-endings was observed after hypovolaemia. Moreover, a statistically significant increase in the contacts between the nerve-ending synaptic specializations and the glomus cell granular vesicles was observed after hypovolaemia. A suggestion was made that the glomus cells might act as modulating, probably inhibitory, interneurones, whose catecholamines are responsible for the inhibition.The authors are greatly indebted to lecturer Pekka Korkala Ph.L. from the Department of Psychology for his skilful statistical analysis of the results.     

Cytochemical studies on the internal polarity of the golgi apparatus and the relationship between this organelle and GERL

Summary Cytochemical studies were performed to clarify the occurrence of an internal polarity of the Golgi apparatus and the relationship between this organelle and GERL in many kinds of cells having different morphologies and functions. The fine structural localizations of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) were examined in anterior pituitary cells, thyroid epithelial cells, gastric chief and parietal cells, duodenal absorptive epithelial cells, hepatocytes, adrenal cortical and medullary cells of mice, and thyroid epithelial cells of domestic fowls. TPPase activity is usually localized in the cisternae of 1–3 stacks and vesicles on the trans-side of the Golgi apparatus of all the cells examined, and in some immature secretory granules of anterior pituitary cells and of gastric chief cells. Rigid lamellae and multivesicular bodies are rarely positive to this reaction, in several kinds of cells. AcPase activity was usually demonstrable in the cisternae of 1–3 stacks and vesicles on the trans-side of the Golgi apparatus, and also in rigid lamellae, coated vesicles, multivesicular bodies and lysosomes in all varieties of cells studied. Some immature secretory granules are positive to the AcPase reaction in anterior pituitary cells and gastric chief cells. The areas positive for both enzyme activities were partially or almost completely overlapping in all the cells examined, though there were minor variations among them. The grades of overlap are classified into three types. Prolonged osmication was performed on thyroid epithelial cells, duodenal absorptive epithelial cells, hepatocytes, adrenal cortical cells, Leydig cells, the epithelial cells of the vas deferens and the theca cells of mice. Cisternae of 1–3 stacks on the cis-side of the Golgi apparatus of all the cells examined were stained with osmium tetroxide. In all these cells we observed that the Golgi apparatus has an internal polarity and that GERL is a part of this organelle in cytochemical respects.This study was supported by grants from the Japan Ministry of Education     

Electron-dense precipitates in glomus cells of rat carotid body after fixation in glutaraldehyde and pyroantimonate-osmium tetroxide mixture as possible indicators of calcium localization

Summary An attempt was made to study the subcellular localization of calcium in carotid body glomus cells of adult rats using fixation with glutaraldehyde followed by treatment with a mixture of pyroantimonate and osmium tetroxide. Precipitates were seen as electron-dense particles (EDP) in the glomus cells, mostly within membrane-bound organelles, such as dense-cored vesicles, mitochondria, small clear vesicles, multivesicular bodies, and especially in lysosomes. However, EDP were also seen in the nuclei and in the free cytoplasm of the glomus cells and even outside them.Preincubation of carotid bodies in media containing calcium and either high potassium or calcium-ionophore A 23187 resulted in a marked increase in the general precipitation pattern, there being an increased amount of EDP both in the glomus cell nuclei and in the cytoplasm. Dense-cored vesicles more often showed precipitates than those in the controls. Some dense-cored vesicles contained multiple precipitates, typically located in the electron-lucent area between core and vesicle membrane.Extensive diffusion of ions probably occurred during fixation before precipitation, making the localization of calcium and other precipitating cations unreliable. However, it is possible that precipitates, which were regularly seen in the dense-cored vesicles, may reflect the content of bound calcium. The possible significance of calcium in glomus cell function is discussed, and the need for more adequate methods is emphasized.The present study has been supported by grants from the Finska Läkaresällskapet and the Sigrid Jusélius Foundation, Helsinki, FinlandWe wish to express our gratitude to Dr. Robert Hamill of Eli Lilly Co. for kindly providing us with the ionophore A 23187. Technical assistance by Mrs. S. Huhtaniitty and Mrs. T. Stjernvall is gratefully acknowledged     

Detection of calcitonin gene expression in human infant and monkey carotid body chief cells by in situ hybridization

Calcitonin mRNA was detected in human and monkey carotid bodies by in situ hybridization histochemistry, using a ³⁵S-labeled oligonucleotide probe for human calcitonin. In both human and monkey carotid body, moderate to high hybridization signal for calcitonin mRNA was observed in all cases. The hybridization signal in the formalin-fixed, paraffin-embedded samples was comparable to that obtained from frozen paraformaldehyde-fixed tissue. Our observations extend the finding of calcitonin-like immunoreactivity in the carotid body chief cells and indicate that calcitonin is produced in the carotid body, probably in the chief cells.     

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.     

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.     

Structure of taste buds in foliate papillae of the rhesus monkey, Macaca mulatta

Taste buds in foliate papillae of the rhesus monkey were examined by electron microscopy. Three distinct cell types were identified. Type I cells were narrow elongated cells containing an oval nucleus, bundles of intermediate filaments, several Golgi bodies, and characteristic apical membrane-bounded dense granules. These cells exhibited morphological variations: some had a moderately dense cytoplasm, perinuclear free ribosomes, and flattened sacs of rough endoplasmic reticulum; others had a more lucent cytoplasm, dilated irregular rough endoplasmic reticulum, lysosome-like dense bodies, and lipid droplets. Type II cells typically contained a spherical, pale nucleus, a prominent nucleolus, supranuclear and infranuclear Golgi bodies, mitochondria with tubular cristae, and one or two centrioles. This cell type, too, showed some variation in the relative amounts of ribosomes and smooth endoplasmic reticulum, which varied inversely with each other. Type III cells were characterized by a clear apical cytoplasm essentially devoid of ribosomes and containing microtubules. In a few type III cells, the peri- and infranuclear regions contained many ribosomes and some rough endoplasmic reticulum. In most Type III cells, there were large numbers of dense and clear vesicles in the peri- and infranuclear regions; some of the vesicles were grouped in synapse-like arrangements with adjacent nerves. The morphological variations exhibited by all three cell types could be accounted for by age differences in each of the cells. This would be consistent with the notion that cell renewal occurs in each of the three cell populations.