Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat

Summary The cellular localization of carbonic anhydrase (CAH) in the carotid body of the rat was investigated by means of Hansson's cobalt-precipitation technique in cultures of dissociated cells. In both young (2-day-old) and old (77-day-old) cultures, the parenchymal glomus (type-I) cells were selectively stained by this technique, and in addition expressed tyrosine hydroxylase and neuron-specific enolase as revealed by immunofluorescence. Enzymic reaction product of CAH appeared to be predominantly intracellular since staining was more intense and occurred more rapidly following permeabilization of the cell membranes with Triton X-100; its formation was inhibited by the CAH-inhibitor acetazolamide (1–10 M) or by increasing the pH from 5.8 to 7.5. Cryostat sections of the carotid bifurcation revealed intense CAH-reaction product in cell clusters of the carotid body, in a few cells of the nodose ganglion, and in red blood cells. Neuronal cell bodies of the petrosal ganglion and superior cervical ganglion (SCG) were largely non-reactive. The SCG is known to contain clusters of small intensely fluorescent (SIF) cells, which were also non-reactive when grown in dissociated cell culture. Thus, although glomus and SIF cells are often considered to be similar cell types, functional CAH-activity appears unique to glomus cells, and this may be important for the physiological response of the carotid body to certain chemosensory stimuli.     

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.     

Oxygen and glucose sensing by carotid body glomus cells

Carotid body glomus cells sense hypoxia through the inhibition of plasmalemmal K(+) channels, which leads to Ca(2+) influx and transmitter release. Although the mechanism of O(2) sensing remains enigmatic, it does not seem to depend on cellular redox status or inhibition of mitochondrial electron transport. Hypoxia inducible factors appear to be necessary for the expression of the O(2) sensor and carotid body remodeling in chronic hypoxia, but are not directly involved in acute O(2) sensing. Glomus cells are also rapidly activated by reductions of glucose concentration due to inhibition of K(+) channels. These cells function as combined O(2) and glucose sensors that help to prevent neuronal damage by acute hypoxia and/or hypoglycemia.     

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.     

Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body

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.     

Calcium binding sites in the vesicles of the carotid and aortic body chief cells

Summary Chief cells of the carotid and aortic body chemoreceptors possess numerous cytoplasmic dense-core vesicles which are known to contain primarily dopamine. Following fixation in solutions containing 50 mM CaCl₂, a 20–30 nm electron-dense particle (EDP) is often observed eccentrically located in many of the vesicles. Approximately 44 % of the carotid body and 16 % of the aortic body vesicles contain an EDP. The EDP probably represents the Ca^{+ +} binding site critical to the stimulus-secretion coupling events culminating in exocytosis of these vesicles. The presence of Ca^{+ +} in the cytoplasmic vesicles was verified by electron probe X-ray microanalysis.Supported by a Grant-in-Aid from the American Heart Association (77630) and by funds contributed in part by the Texas Affiliate. The authors wish to thank Ms. Teri Heitman for her excellent technical assistance     

Loss of histochemically demonstrable catecholamines in the glomus cells of the carotid body after alpha-methyl-para-tyrosine treatment.

A statistically significant decrease in the intensity of catecholamine fluorescence of some carotid body glomus cells was observed after inhibition of the enzyme tyrosine hydroxylase by injection of 80 mg/kg alpha-methyl-paratyrosine. The intensity of the formaldehyde-induced fluorescence was measured in individual glomus cells. The maximum decrease in the intensity was observed 4 to 6 hr after the alpha-methyltyrosine injection. This suggests a rapid turnover in the catecholamines of the carotid body.     

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.     

Extraneuronal effects of 6-hydroxydopamine

Summary The effects of various concentrations of 6-hydroxydopamine (6OHDA) on rat adrenocortical cells in tissue culture were studied with phase contrast and electron microscopy. With 40 mg/l of 6-OHDA the first signs of alteration as revealed by microcinematography appeared in isolated cortical cells as early as 15 min after addition of the drug. There was a cessation of movement of cell organelles and an immobilisation of membrane undulations followed by the development of dark inclusion bodies. The cells underwent increasing shrinkage and collapsed by 11/2 h. Chromaffin cells were not affected until 45 min after exposure to the drug and neurons were the most resistant population. However 61/2 h after application of the drug most cells in the culture were dead. 6-OHDA applied in different doses and to adrenal expiants did not alter the sequence of events. Ultrastructurally cortex cells underwent damage along two lines: they either showed lytic changes or developed various types of dense bodies before reaching the lytic stage.Treatment of cortical cells with 40 mg/l 5-or 6-OHDA followed by exposure to buffered 2% glyoxylic acid and heat did not produce a fluorescence within the cells. Microspectrofluorimetry on amine models with noradrenaline, 5- and 6-OHDA revealed that neither 5-nor 6-OHDA are capable to form a fluorophore with glyoxylic acid.Dedicated to Professor Berta Scharrer in honor of her 70th birthdaySupported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3) and a Research Fellowship of the University of Melbourne to K.U., and a Research Fellowship and Grant-in-Aid from the Life Insurance Medical Research Fund of Australia and New Zealand to J.H.C.