Immunohistochemical and biochemical study on the development of the noradrenaline- and adrenaline-storing cells of the adrenal medulla of the rat

Summary The pre- and postnatal development of the adrenal medulla was examined in the rat by immunohistochemistry and by assay of catecholamines. Immunohistochemistry involved the use of antibodies to noradrenaline (NA), adrenaline (A) and the biosynthesizing enzymes dopamine -hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Adrenal glands were obtained from animals from the 16th day of gestation to the 7th postnatal day at daily intervals, and at the 14th postnatal day, and from adult rats. Tissues were fixed in ice-cold, 4% paraformaldehyde, buffered at pH 7.3. Cryostat sections (7 m) were stained with the indirect immunofluorescence technique. Adrenals from the same developmental stages were assayed for the presence of DA (dopamine), NA and A by ion-pair reversed-phase liquid chromatography with electrochemical detection.In adult adrenals the majority of the medullary cells (approximately 80%) were highly immunoreactive to A and moderately immunoreactive to NA. They also showed immunoreactivity to both DBH and PNMT, i.e., they are synthesizing and storing A. The remaining cell clusters were only stained by antibodies to DBH and NA (NA-synthesizing and -storing cells). These findings correlate well with the relative concentrations of A and NA as determined by assay.Three developmental phases could be distinguished. In the first phase, the 16th and 17th prenatal day, medullary cells were only immunoreactive to DBH and NA, and only very small amounts of A as compared to NA were found. During the second period, from the 18th prenatal day to 2 or 3 days after birth, all medullary cells were immunoreactive to DBH, NA, PNMT and A, and during this phase the adrenaline concentration increased daily and became the predominant amine on the 20th day of gestation. Adrenaline represented 75% of total catecholamine on the 1st to 3rd day after birth. The third phase started at the 2nd or 3rd postnatal day and was characterized by the presence of an increasing number of medullary cells solely immunoreactive to DBH and NA, hence synthesizing and storing NA. The remaining cells were immunoreactive to DBH, NA, PNMT and A. Postnatally, the relative concentration of A continued to rise reaching 79% by the 4th postnatal day. These results indicate that initially the adrenal medullary cells are synthesizing and storing almost exclusively NA. Probably, adrenaline synthesis begins at the 16th–17th day of gestation and the cells are then capable of synthesizing and storing both NA and A (mixed cell type) with A synthesis and storage rapidly becoming predominant. Finally, after birth, separate NA-synthesizing and -storing cell types are formed and the so-called A cells stored predominantly (probably >90%) adrenaline with a small proportion of noradrenaline.In the medullary blastema and in the sympathetic ganglia of prenatal animals two cell types, only immunoreactive to DBH and NA, were observed. Presumably, these cells represent developing sympathetic neurons and extra-adrenal chromaffin cells; the latter cell type occasionally invades the adrenal gland. Thus, prospective medullary cells are able to synthesize and store NA before they have made contact with the cortical blastema but A-synthesizing cells are found only within the adrenal gland.Low but significant amounts of DA were found in the adrenal before birth and during the first two postnatal weeks but in the adult animal this accounted for less than 0.1% of total catecholamine.Preliminary reports of this study were made to the American Association of Anatomists (Anat. Rec. 196; 196A, 1980), the Dutch Anatomical Society (Acta Morphol. Neerl. Scand. 19; 330, 1981, and the XIIIth Acta Endocrinologica Congress (Acta Endocrinol. 97: Suppl. 243, 285, 1981)     

Annual cycle of the chromaffin cells of Triturus cristatus

Morphology of the chromaffin cells of Triturus cristatus during a complete annual cycle has been investigated. General ultrastructural characteristics are similar for all chromaffin cells, including numerous small mitochondria, well-developed Golgi apparatus and rough endoplasmic reticulum with short cisternae. The primary difference among cells is the type of the chromaffin granules they posses. These are of two kinds: adrenalin (A) and noradrenalin granules (NA). Both types are simultaneously present in the chromaffin cells but with different ratios during the year. During December–January and May–August, NA granules largely prevail, while in September–November and February–April, A and NA granules are present in about equal quantities. The total quantity of catecholamine granules, however, is relatively constant throughout the year. These findings suggest that T. cristatus has a single type of chromaffin cell, the granule content of which varies according to different functional states. The catecholamines are apparently discharged by exocytosis.     

G-Protein activation mediates prepulse facilitation of Ca2+ channel currents in bovine chromaffin cells

The effects of G-protein activation were investigated on tonic, large depolarization-induced Ca²⁺ channel facilitation in cultured bovine adrenal chromaffin cells. Under whole-cell voltage clamp, activation of G proteins by intracellular dialysis with 200 M GTP-S did not significantly affect prepulse facilitation or whole-cell Ba²⁺ current (I _Ba) density. In contrast, inactivation of G proteins by intracellular GDP-S or pertussis toxin (PTX) pretreatment completely abolished or markedly attenuated facilitation of I _Ba, respectively. GDP-S dialysis resulted in nearly a threefold increase in peak I _Ba density, whereas PTX pretreatment resulted in a 50% increase. Our results indicate that under control recording conditions (200 m intracellular GTP), G proteins are tonically activated and suppress high-voltage-activated (HVA) Ca²⁺ channels in a voltage-dependent and voltage-independent manner. Local superfusion of chromaffin cells with normal bath solution produced a rapid and reversible increase (50%) in I _Ba amplitudes that also abolished prepulse facilitation. Together, these results demonstrate that tonic facilitation of HVA Ca²⁺ channels in bovine chromaffin cells involves the voltage-dependent relief of a G-protein-mediated suppression, imposed by chromaffin cell secretory products that feedback and activate G-protein-coupled autoreceptors.This work was supported by a National Science Foundation grant (DCB-8812562), American Heart Association-Ohio Affiliate grant (SW-91-18), and an American Parkinson's Disease Association grant. C.A.D. was supported by a predoctoral National Research Service Award (National Institutes of Health training grant HL07571-08). The authors thank Kluener's Packing Co. for their generous supply of adrenal glands.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process

Summary Synexin (annexin VII) is a Ca²⁺- and phospholid-binding protein which has been proposed to play a role in Ca²⁺-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 g/mg) and Ca²⁺ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/m²) and in the cytosol (5.3 particles/m²), but mainly around the granule membrane in the granular cell area (11.7 particles/m²). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.     

Morphology and distribution of chromaffin cells in the adrenal gland of cordylidae (Reptilia,Sauria): A comparative study

The distribution of the adrenaline and noradrenaline chromaffin cells in the adrenal glands of 10 members of the family Cordylidae have been examined. In the genus Gerrhosaurus, all the catecholamine cells lie on the surface of the adrenal gland, forming a continuous envelope of one or two layers of cells that mainly contain noradrenaline (NA). In the genus Platysaurus, the chromaffin envelope is intermittent. There are relatively large tracts of interspersed interrenal tissue containing some adrenaline cells (A). Islets of chromaffin cells are scattered between these interrenal tracts. In the genus Pseudocordylus and the genus Cordylus, the superficial chromaffin cells tend to gather into a multilayered dorsal mass, containing mainly NA cells. Inside the interrenal parenchyma, there are always numerous chromaffin islets, containing mainly A cells.     

Survival,morphology, and catecholamine storage of chromaffin cells in serum-free culture: Evidence for a survival and differentiation promoting activity in medium conditioned by purified chromaffin cells

Adult bovine and young rat chromaffin cells cultured in serum-free medium were examined for their survival and differentiation following exposure to various additives, trophic agents and conditioned media. Adrenal chromaffin cells dissociated from 8 day old rats were maintained by dexamethasone, NGF and CNTF or without any additives in an N1-supplemented medium in similar numbers as in serum-containing medium for up to 6 days. Neuritic growth elicited by NGF or CNTF was enhanced in the absence of serum. Medium conditioned by purified bovine chromaffin cells improved cell survival and caused neurite outgrowth in a dose-dependent manner. The activiti(es) was sensitive to heat and trypsin and not blocked by the addition of anti-NGF antibodies. Bovine chromaffin cell survival was reduced by 30% when cells were maintained for one week in the absence as compared to the presence of serum. Addition of insulin, the N1 supplement, dexamethasone or dbcAMP single or in combinations improved the survival to different extents. A combination of insulin (5 g/ml) and dexamethasone (5×10^–6M) proved to be optimal in this respect. However, these supplements failed to restore the cellular catecholamine, noradrenaline and adrenaline contents to levels seen in the presence of serum. This was also true for a chromaffin cell-conditioned medium, which improved survival without elevating the catecholamine contents. Conditioned medium, however, partly restored a more physiological adrenaline-noradrenaline-ratio.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).     

Naphthalenesulfonamide derivatives ML9 and W7 inhibit catecholamine secretion in intact and permeabilized chromaffin cells

The role of protein phosphorylation in catecholamine secretion from bovine adrenomedullary chromaffin cells was studied using different protein kinase inhibitors. Naphthalenesulfonamide derivatives as ML9 and ML7, more specific for the myosin light chain kinase, and the calmodulin antagonist W7 inhibited catecholamine secretion 20 and 40% respectively in digitonin-permeabilized chromaffin cells. ML9 also decreased calcium evoked protein phosphorylation of different proteins including tyrosine hydroxylase in permeabilized cells. These naphthalenesulfonamide derivatives showed also an effect in intact cells, ML9 and W7 produced 50% inhibition in catecholamine secretion and⁴⁵Ca²⁺ uptake, however H8 had no effect. The partial [³H]nitrendipine binding displacement of these drugs to adrenomedullary membranes suggests that these sulfonamide derivatives could interact directly with L-type calcium channels in intact cells. The results obtained in permeabilized cells suggest a possible role of protein phosphorylation in the regulation of catecholamine secretion in chromaffin cells.The abbreviations used are ML9 1-(5-Chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine hydrochloride - ML7 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4 diazepine hydrochloride - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - H8 N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride - W7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride - PKI protein kinase A inhibitor - HEPES N-(2-hydroxyethylpiperazine-N-(2 ethanesulfonic acid) - PIPES piperazine-N, N-bis (2-ethanesulfonic acid) - EGTA [ethylene-bis (oxyethylenenitrilo)] tetraacetic acid - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DMEM Dulbecco's Modified Eagle's medium - MLC myosin light chain - MLCK myosin light chain kinase - TH tyrosine hydroxylase     

Immunohistochemical localization of bioactive peptides and amines associated with the chromaffin tissue of five species of fish

Biogenic peptides and amines associated with the chromaffin tissue in Atlantic cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla), spiny dogfish (Squalus acanthias) and Atlantic hagfish (Myxine glutinosa) were identified utilizing immunohistochemical techniques. Within the posterior cardinal vein (PCV) in cod, trout and eel, a subpopulation of chromaffin cells displayed immunoreactivity to tyrosine hydroxylase (TH) and dopamine--hydroxylase (DH) but not to phenylethanolamine-N-methyltransferase (PNMT). TH-like immunorectivity was observed within cells in hagfish hearts. Nerve fibres displaying vasoactive intestinal peptide (VIP) immunoreactivity and pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity innervated cod, trout and ell chromaffin cells. In eel, neuropeptide Y (NPY)-like and peptide YY (PYY)-like immunoreactivity was located within cells in the PCV, including chromaffin cells. Serotonin-like immunoreactivity was observed within eel and cod chromaffin cells and in hagfish hearts. In the dogfish axillary bodies, nerves displaying TH-like, VIP-like, PACAP-like, substance P-like and galanin-like immunoreactivity were observed. These results are compared with those of other vertebrates, and potential roles for these substances in the control of catecholamine release are suggested.     

Ultrastructural changes in adrenaline- and SGC-cells after morphine coincide with alterations of adrenaline and dopamine levels

Summary The effects of morphine on chromaffin cell ultrastructure and catecholamine contents were studied using the adrenal glands from male mice (ICR strain). After 2 h adrenaline was increased 25% from 8.1 to 11.6 mol/g tissue, followed by a 50% decrease to 5.2 mol/g between 8–24 h and low values persisting at 72 h. Dopamine increased initially, reaching peak values of 0.5 mol/g between 8–24 h, but had returned towards control values of 0.29 mol/g by 72 h. Noradrenaline remained unchanged at 2.5 mol/gram. Naloxone prevented alterations in adrenaline and dopamine levels.Ultrastructural examination revealed several types of catecholamine-storing cells. Of these the adrenaline and small-granule chromaffin (SGC) cells were more affected by morphine than noradrenaline cells. While the initial elevation of adrenaline 2 h after morphine was not accompanied by significant ultrastructural changes, the decrease after 8 and 24 h was paralleled by a significant (p<0.001) loss of adrenaline granules. Signs of active membrane turnover included an increase in the number of vacuoles, and the appearance of numerous coated omega profiles and coated (77.7±0.6 nm) vesicles. Clusters of synaptic-like vesicles (59.8±8.2 nm), slightly larger than neuronal vesicles (45.4±6.4), increased in the SGC-cells. After 72 h, the chromaffin granules in adrenaline cells remained low in numbers and were heterogeneous in electron density. Many synapticlike vesicles were aligned along the SGC-cell membranes where only few chromaffin granules (109.3±20 nm) remained. Thus, continuous morphine exposure for 8–72 h increases the turnover of storage granules in adrenaline and SGC-cells with less effect on the noradrenaline cells which maintain catecholamine levels as indicated by biochemical analysis.     

Characterization of adrenal medullary chromaffin cells by flow cytometry.

BACKGROUND: Adrenomedullary chromaffin cells are neural crest derivatives widely used as a model system to study neurosecretory mechanisms. Morphological, immunohistochemical, and functional data indicate that chromaffin cells are heterogeneous and support the distinction between adrenaline (A)- and noradrenaline (NA)-producing and secreting cells. The aim of this study was to characterize by flow cytometry the two main chromaffin cell subtypes in suspensions of cultured bovine chromaffin cells. METHODS: An indirect immunofluorescence method was used for the specific labeling of two intracellular enzymes, dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT), involved in the synthesis of NA and A, respectively. Flow cytometry analysis of fluorescence labeling was performed in two chromaffin cell fractions differentially enriched in A-containing cells by centrifugation through density gradients. PNMT and DBH-related fluorescence was also correlated with the A and NA content of the cells assayed by HPLC measurements. RESULTS: No significant differences were found in forward-side scatter plots between the two cell fractions (A-enriched cells and mixed cells); however, the degree of labeling of the enzymes and the corresponding PNMT/DBH-related fluorescence ratio was significantly greater in the A-enriched cell fraction. The existence of changes in DBH and PNMT content of chromaffin cells over time (1 week) in culture was also examined. No significant variation in enzyme related fluorescence values was detected in any of the two cell fractions, and this result correlated well with HPLC determinations of the catecholamine content (A and NA) of the cells. CONCLUSIONS: Flow cytometry appears to be a useful technique to characterize chromaffin cell subtypes and to follow their phenotypic changes in response to growth factors.     

Early molecular events during the interaction of enveloped riboviruses with cells

A kinetic model was constructed and partly solved to describe the migration of the fluorescence label 1,6-diphenylhexatriene (DPH) in both directions when enveloped viruses, labelled with DPH in their envelopes are in contact with unlabelled cells or cell labelled in their membranes are in contact with unlabelled enveloped viruses. The central assumption is that two types of receptor sites exist on the cell surface, i.e., physical adsorption sites (P-sites), available to all the viruses studied in these papers and binding sites (B-sites) available only to the viruses which penetrate into the specific cells.The differential equations for the label migration, for different values of the ratio number of viruses number of sites were numerically solved, assuming different fractions of P- and B-sites.The equations also describe, appropriately the mechanism of rapid label migration in the system and substantiate the magnitude time of residence of the nonpenetrating viruses adsorbed on the cell surface. The resulting curves match satisfactorily those for the label release by the viruses and account well for the steady state values of the kinetics of label migration in the virus-cell system.     

Osmotic properties of human red cells

Summary When an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of nonosmotic water which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol. 52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (z_Hb). A number of investigators, particularly Freedman and Hoffman (1979,J. Gen. Physiol. 74:157) have been unable to confirm Gary-Bobo and Solomon's experimental evidence for this concentration dependence of z_Hb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5±0.8% of the total isosmotic cell water (P0.0005,t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.     

Development of adrenal chromaffin cells in Sf1 heterozygous mice

Adrenal medullary chromaffin cells are derivatives of the neural crest and are widely believed to share a common sympathoadrenal (SA) progenitor with sympathetic neurons. For decades, the adrenal cortical environment was assumed to be essential for channelling SA progenitors towards an endocrine chromaffin cell fate. Our recent analysis of steroidogenic factor 1(Sf1) −/− mice, which lack an adrenal cortex, has challenged this view: in Sf1 −/− mice chromaffin cells migrate to the correct “adrenal” location and undergo largely normal differentiation. In contrast to Sf1 homozygous mutants, heterozygous animals have an adrenal cortex, which, however, is smaller than in wildtype littermates. We show here that the Sf1 +/− adrenal cortical anlagen attract normal numbers of chromaffin progenitor cells into their vicinity by embryonic day 13.5 (E13.5). Two days later, however, only a few scattered cells with highly immature features have immigrated into the adrenal cortex, whereas the remainder form a coherent cell assembly ectopically located at the medial surface of the gland. These cells appear more mature than the scattered intracortical chromaffin progenitors and express the adrenaline synthesizing enzyme PNMT with a delay of 1 day in comparison with wildtype littermates. Nevertheless, chromaffin progenitor cells undergo a numerical reduction of approximately 30% by E17.5. Together, our data suggest that normal adrenocortical development is critical for the correct immigration of chromaffin progenitors into the cortical anlagen, for the timing of PNMT expression and for the regulation of chromaffin cell numbers.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 488, TP A6).     

Fate of intraocular chromaffin cell suspensions: role of initial nerve growth factor support

Summary Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin. Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine--hydroxylase (DBH). After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if -nerve growth factor (NGF) was given at grafting. One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber net-works and all cells were DBH-immunoreactive. Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris. Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes. The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls. A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections. In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathically denervated iris. Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.     

Expression of the intercellular adhesion molecule-1 on high endothelial venules and on non-lymphoid antigen handling cells: interdigitating cells,antigen transporting cells and follicular dendritic cells

Intercellular adhesion molecule-1 (ICAM-1)¹ has been implicated in the development of germinal center reactions in vitro, and the present study was undertaken to determine the distribution of ICAM-1 in active germinal centers in vivo and in murine secondary lymphoid tissues in general. Anti-ICAM-1-specific monoclonal antibodies were used in conjunction with immunohistochemistry at both the light and ultrastructural levels of resolution. Examination of cryostat sections of lymph nodes, spleens, and Peyer's patches revealed that anti-ICAM-1 distinctly labeled cells in the light zones of germinal centers, a few cells in the T cell zones (e.g. paracortex of lymph nodes), cells in the sinus floor of the subcapsular sinuses of lymph nodes, and high endothelial venules (HEV). Ultrastructural studies revealed that the cells labeling with anti-ICAM-1 in germinal centers were follicular dendritic cells (FDC) which appeared to have more ICAM-1 than any other cell type. The surfaces of well-developed, intricate, convoluted FDC processes were intensely labeled even under conditions where B cells appeared negative. Interdigitating cells (IDC) were also labeled as were certain endothelial cells in the HEV. The cells in the subcapsular sinus floor labeling with anti-ICAM-1 were the antigen transporting cells (ATC) that carry antigen-antibody complexes into lymph node follicles. We suspect ATC are FDC precursors which mature into FDC in the follicles. Interestingly, FDC, IDC, and ATC are 3 important accessory cells known to handle antigens in specific compartments of lymphoid tissues. The marked localization of this adhesion molecule on these critical antigen handling cells supports the concept that ICAM-1 is important in providing the intercellular adhesion necessary for optimal initiation of immune responses in vivo.Abbreviations ICAM-1 Intercellular adhesion molecule-1 - LFA-1 leukocyte functional antigen-1 - IDC interdigitating cells - ATC antigen transporting cells - FDC follicular dendritic cells - HEV high endothelial venules - DC dendritic cells - PBS phosphate-buffered saline - PLP periodate-lysine-4% paraformaldehyde - GPLP periodate-lysine-0.1% glutaraldehyde-2% paraformaldehyde - EM electron microscopy - HRP horseradish peroxidase - DAB diaminobenzidine tetrahydrochloride - HSA human serum albumin     

Increased numbers of extra-adrenal chromaffin cells in the abdominal paraganglia of senescent F344 rats: A possible role for the glucocorticoid receptor

Summary The increase in numbers of extra-adrenal chromaffin cells of abdominal paraganglia in senescent F344 rats was investigated by 5-bromo-2-deoxyuridine immunocytochemistry. A monoclonal antibody raised against 5-bromo-2-deoxyuridine was used to react with tissue-sections of paraganglia taken from 28-month-old animals given weekly injections of the thymidine analog over a 14-week period. No immunoreactivity was detected in the extra-adrenal chromaffin cells, whereas control sections of intestinal epithelium showed abundant immunoreactivity. Also, the profile for immunoreactivity of the glucocorticoid receptor in relation to age was compared between extra-adrenal and adrenal chromaffin cells, which share cytological characteristics, but not the increase associated with senescence. In the extra-adrenal chromaffin cells, the intensity of receptor immunostaining was unchanged, while in the adrenal chromaffin cells it decreased with age. These results indicate that hypertrophy of the paraganglia in aged F344 rats is not due to the proliferation of extra-adrenal chromaffin cells. Instead, they suggest that the chromaffin cell phenotype may be induced in pre-existing cells and that the expression of the glucocorticoid receptor has an intrinsic role in this change.     

Nicotonic and muscarinic components in acetylcholine stimulation of porcine adrenal medullary cells

Adrenal medullary chromaffin cells secrete catecholamines (CA) in response to cholinergic receptor activation by acetylcholine (ACh) released from splacnic nerve terminals. In cultured bovine chromaffin cells nicotinic receptors play a preponderant (> 90%) role in the control of CA release. By contrast, we found and report here that up to 40% of the ACh-evoked CA secretion from cultured porcine chromaffin cells can be associated with muscarinic receptor activation. The following results support our belief that in porcine adrenal medullary cells ACh (100 M) evoked CA secretion is mediated by both nicotinic and muscarinic cholinergic receptors. 1) Hexamethonium (100 M), a nicotinic receptor antagonist, inhibited ACh-induced CA secretion to ca. 40% of the control release and atropine (1 M), a muscarinic receptor antagonist, inhibited to ca. 60% of the control value. 2) We also found that ACh (100 M) evoked intracellular Ca2+ concentration ([Ca2+]i) rise was inhibited by these receptor antagonists to a different extent, and reversibly reduced by lowering the concentration of Ca2+ in the external medium ([Ca2+]o). This last maneuver ([Ca2+]o < 0.1 M) per se caused a marked reduction in the peak phase of the [Ca2+]i rise evoked by ACh (40% of the control response). Switching the external medium back to physiologic [Ca2+]o in the continued presence of ACh caused a partial recovery of the elevated [Ca2+]i. This [Ca2+]o-dependent [Ca2+]i rise was blocked by hexamethonium (100 M) but not by atropine (1 M). Conversely, the ACh-evoked [Ca2+]i rise in low external [Ca2+]o was blocked by atropine but not by hexamethonium. From these data we conclude that in porcine adrenal medullary cells an important fraction (ca. 0.4) of both ACh-induced CA secretion and peak [Ca2+]i rise is due to muscarinic receptor activation.     

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca²⁺/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-³²P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca²⁺ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca²⁺ in digitonin-treated cells. In the presence of 1 M Ca²⁺, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca²⁺. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl₂ - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution     

The distribution and ultrastructure of sensory elements in the baroreceptor region of the truncus arteriosus of the lizard Trachydosaums rugosus

Summary The proximal truncus arteriosus of the lizard Trachydosaurus rugosus was studied with light-, fluorescence and electron-microscopical techniques. Three vessels comprised the truncus: the pulmonary, left aortic, and caroticoaortic arteries. Right and left truncal nerves, each derived from the ipsilateral vagus nerve, innervated the truncus, particularly its proximal 3 mm.Ultrastructurally, the nerves had a variety of appearances: some were clearly adrenergic, c-type or p-type. A number of profiles contained large numbers of mitochondria and were classified as sensory. Some profiles defied exact classification, having characteristics common to two different types of profile.Within the outer medial layers, profiles up to 7 m in diameter were found. These contained large numbers of mitochondria, myelin bodies and structures intermediate between the two. In addition, the profiles contained large amounts of glycogen and small numbers of vesicles. These nerve fibres were classified as baroreceptors, since they closely resemble carotid sinus and aortic arch baroreceptors in mammals.Large numbers of chromaffin cells were found, particularly in the common wall of the pulmonary and left aortic arteries. Many of these cells emitted a long tapering process, which sometimes entered a nearby nerve bundle. Sensory, p-type and c-type profiles, but not adrenergic profiles, made extensive close contacts with chromaffin cells.