An ultrastructural stereological analysis of the aortic body chief cell of adult rabbits

Summary The chief cells of the aortic body (subclavian body) of adult New Zealand white rabbits were examined by ultrastructural stereological analysis. The chief cell nuclei occupy 26.5% of the total volume. Dense-core vesicles account for 16.5% of the cytoplasmic volume, followed by mitochondria (11.6%), endoplasmic reticulum (3.3%), and Golgi apparatus (0.6%). The dense-core vesicles measure approximately 131.6nm in diameter (corrected) and exhibit a heterogeneous size distribution. Both perivascular adrenergic nerve terminals and presumptive afferent terminals presynaptic to the chief cells are observed. The mean synaptic vesicle size of the terminals adjacent to chief cells is 54 nm. The heterogeneous size distribution of the dense-core vesicles of chief cells may indicate the storage of different biogenic amines and/or different secretion or maturation states within the chief cells.Supported by a Grant-in-Aid from the American Heart Association (77630) and with funds contributed in part by the Texas Affiliate. The author wishes to thank Ms. Teri Heitman for her excellent technical assistance     

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.)     

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     

Transduction of chemostimuli by the type I carotid body cell

Summary The postulated mechanisms for hypoxic and acidic chemotransduction by type I cells that we have described here are summarized in the diagrams of Fig. 4. Most if not all of these require more complete evaluation and, as we have described, there are obvious points of contention that need to be resolved. Nevertheless, it is apparent that studies of isolated type I cell preparations carried out over the last six years have provided significant advancements in our understanding of chemotransduction in the type I cell. Only when the functioning of these cells has been fully described can we hope to understand the mechanisms underlying the responses of the intact organ to chemostimuli.Many of the findings reported here, including those to which the authors have contributed, were supported by The Wellcome Trust. We are also grateful to colleagues for allowing us to reproduce parts of their data.     

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.     

X-ray micronalysis of copper and sulphur-containing granules in the fat body cells of homopteran insects

Regions of the fat body of larvae of Chaetophyes compacta and Pectinariophyes sp. (Machaerotidae, Homoptera) which are closely associated with mycetomes have been analysed by electron probe X-ray microanalysis. It is shown that cells in these regions contain electron probe X-ray microanalysis. It is shown that cells in these regions contain electron dense granules which are rich in copper and sulphur. These two elements occur in the atomic ratio of 3:2 respectively. It is conjectured that copper may be bound to a sulphur containing metallothionein and that the granules represent either the end products of copper detoxification or serve as copper stores for synthesis of enzymes and macromolecules by the mycetomal symbionts.     

Calcium pathway through a mineralizing epithelium in the crustacean Orchestia in pre-molt: Ultrastructural cytochemistry and X-ray microanalysis

During the pre-exuvial period of the terrestrial crustacean Orchestia, the calcium of the old cuticle is almost entirely reabsorbed and stored as calcareous concretions in the lumen of the midgut posterior caeca. The elaboration of these concretions is due to transport by the caecal epithelium. With ultrastructural cytochemistry controlled by X-ray microanalysis, it can be demonstrated that the main sites of ionized or ionizable calcium are the apical microvilli and an extracellular (lateral and basal) network of channels. Direct precipitating cytochemical methods, using potassium pyroantimonate or pyrophosphate, potassium oxalate or oxalic acid, sodium fluoride, sodium tungstate, and indirect substitution methods, using lead acetate or nitrate and cobalt nitrate were comparatively used. The results are interpreted in favour of the hypothesis of an extracellular transport pathway for calcium through the lateral smooth septate junctions, in conjunction with a more classical apical transport through the microvilli.     

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.     

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     

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.     

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.     

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.     

Arteriovenous anastomoses in the cat carotid body

Summary In artery surrounding areas in the periphery of the carotid body of the cat we found arteriovenous anastomoses differing in respect to their character. So far, it is not yet to decide the frequency of their occurrence and their functional significance. The anastomoses were demonstrated by light microscopy of serial sections and by scanning electron microscopy with a more developed corrosion casting technique.

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Zusammenfassung In den arteriennahen, peripheren Bereichen des Glomus caroticum der Katze wurden arteriovenöse Verbindungen nachgewiesen, die ihrem Charakter nach unterschiedlich sind. Es ist allerdings noch nicht zu entscheiden, ob derartige Anastomosen regelmäßig vorkommen und wie sie funktionieren. Die beschriebenen Gefäßverbindungen konnten lichtmikroskopisch anhand von histologischen Serienschnitten und mit Hilfe einer weiterentwickelten Korrosionstechnik im Rasterelektronenmikroskop dargestellt werden.

     

Calcium in the synergid cells and other regions of pearl millet ovaries

Summary The synergids and other cells of mature, unpollinated pearl millet ovaries were investigated using: (1) freeze-substitution fixation in conjunction with scanning electron microscope observations and energy-dispersive X-ray microanalysis to localize total calcium (Ca) and other elements, and (2) antimonate precipitation to selectively localize loosely sequestered, exchangeable calcium (Ca⁺⁺). In freeze-fixed ovaries, the synergid cells, ovary wall, nucellus, and other regions of the ovary displayed, respectively and relatively, extremely high, high, moderate, and low levels of Ca. In antimonate-fixed ovaries, Ca-containing antimonate precipitates exhibited similar distribution patterns. In ovaries fixed using the conventional 2% (w/v) antimonate in fixatives, the synergids were disrupted due to precipitate overload. In the ovary wall, precipitates were mainly located in the intercellular spaces. Some precipitates were observed at the micropyle and along the outer ovule integument, associated with diffuse extracellular material, and in the cell walls of nucellar cells proximal to the micropyle. Examination of precipitate distribution inside the synergids was possible in ovaries fixed using 0.5% (w/v) antimonate in the fixatives. Cytoplasmic organelles of all synergids examined exhibited variable states of disintegration. The amount of precipitates associated with the degenerated organelles appeared to be proportional to the degree of their degeneration. Distinct precipitates were localized in contiguous regions of the nucellar cells fused with the embryo sac, the micropylar half of the embryo sac wall, and the filiform apparatus. The results are discussed in relation to the involvement of Ca⁺⁺ in mediating the functions of synergid cells during fertilization in angiosperms.On Specific Cooperative Agreement 58-43YK-8-0026 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Endocytotic uptake of cationized ferritin tracer into glomus cells dissociated from the adult rat carotid body

Summary Glomus cells from carotid bodies of adult rats dissociated by means of collagenase or collagenase + trypsin were used to study by electron microscopy the endocytotic uptake of cationized ferritin (CF) tracer into subcellular compartments. The glomus cells were incubated with the tracer (1) in a basic salt medium (BM), or (2) in the BM into which calcium ionophore A23187 had been added, or (3) in a potassium-rich medium.Incubation of the cells in BM containing CF for 30 min resulted in attachment of the tracer to the cell membrane and uptake of a few solitary tracer particles into small vesicles and multivesicular bodies. No uptake into the cisternae of the Golgi apparatus was observed. Further incubation in BM containing CF for another 30 min resulted in increased uptake of the tracer into small vesicles and multivesicular bodies. A similar pattern of uptake was observed when the dissociated glomus cells were first preincubated in BM with CF for 30 min and then incubated for 1 min or 30 min in the BM solution containing both the ionophore and CF. Upon such incubation, CF particles were seen to penetrate into coated pits and sites of exocytosis at the cell surface. When the 30-min preincubation in BM was followed by incubation in a CF-containing potassium-rich medium for 15–30 min, uptake into vesicles, small lysosomes and occasionally also into profiles of the smooth endoplasmic reticulum was seen. Endocytotic mechanisms of the glomus cells are outlined.     

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.     

Calcium content and concretions of pineal glands of young and old rats

Summary Calcium content and pineal concretions were studied in young (2–3 months) and old (28 months) Wistar rats. Samples, deep-frozen by liquid propane/isopentane and freeze-dried were analysed by means of X-ray microanalysis in a scanning electron microscope. Total semi-quantitative measurements revealed that pineals of old rats showed a marked increase of calcium compared with the pineals of young rats. It is thus suggested that a calcium-rich environment is responsible for the growth of pineal concretions, which only appear in old rats. Pineal calcifications in rats could thus be an indicator of aging and/or of a degenerating state.     

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.     

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.