An antibody to agmatine localizes the amine in bovine adrenal chromaffin cells.

Agmatine, a newly identified amine in mammalian brain, is an endogenous ligand for imidazoline and alpha 2-adrenergic receptors. We sought to develop a polyclonal antibody to agmatine suitable for immunocytochemistry. Agmatine was conjugated to keyhole limpet hemocyanin and injected into rabbits. The polyclonal antiserum so generated dose-dependently recognized the agmatine conjugate but not carrier protein by dot blot. Its reaction with the conjugate was selectively antagonized by agmatine but not related compounds. The antiserum, but not pre-immune or pre-adsorbed antiserum, selectively stained cultured adrenal chromaffin cells. Our results indicate that agmatine immunoreactivity is contained in a sub-population of adrenal chromaffin cells and, thus, these antibodies are useful for immunocytochemical localization of the amine in mammalian tissues.

Kinetics of adrenal medullary cells.

The adrenal medulla of mammals has a heterogeneous population of cells. In adults most are epithelial cells containing a particular type of cytoplasmic granule. Based on a variety of cytochemical and ultrastructural studies it is now accepted that 2 different adrenal medullary chromaffin cell types can be distinguished, i.e. noradrenaline (NA) and adrenaline (A) synthesising and storing cells. Other cell types present in the adrenal medulla include neuronal elements comprising either cell bodies or nerve fibres entering from outside the gland (extrinsic innervation). It is assumed that adrenal medullary cells have a limited life span, i.e. they are replaced after a certain period. Data on this replacement process are scarce. Recently, we initiated an investigation into this question using cytochemical procedures that enable the detection of DNA duplication to measure mitotic activity in individual cells. Female Sprague-Dawley rats aged 22-36 wk received a single i.p. injection of BrdU or BrdU was administered continuously via an implanted mini-osmotic pump. Cell nuclei that had incorporated BrdU were demonstrated using an indirect immunoperoxidase staining technique. At 1 h after a single injection, 0.46 +/- 0.07% of the adrenal medullary (chromaffin) cells were labelled. This increased to 0.77 +/- 0.08% after 12 h with no further increase during the next 7-8 d. With continuous infusion of BrdU the fraction of labelled cells increased gradually to about 40% after 73 d (the longest period studied). These results show that in adult rats adrenal medullary cells are able to divide, although at a slow rate (renewal rate of about 1%/day).

Perinatal development of galanin-like immunoreactivity in chromaffin tissues of the rabbit.

We have analyzed the perinatal development of galanin-like immunoreactivity (GAL-LI) and catecholamines (CA) in the paraaortal paraganglia (PGGL) and adrenal glands. In the PGGL, the tissue content of GAL-LI was highest on the day of birth and decreased postnatally. The fetal levels were lower than at birth. In contrast, the content of CA in the PGGL increased with age. In the adrenal glands, the contents of both GAL-LI and CA also increased with age. During the first postnatal week the contents of both GAL-LI and CA in the PGGL were markedly higher than in the adrenal glands. Chromatographic analysis of GAL-LI in extracts of fetal and postnatal rabbit PGGL, respectively, indicated that most of the GAL-LI from both age groups co-eluted with synthetic porcine GAL. An additional, apparently more polar, component was also detected at both ages, which may represent a differently processed form of the peptide. The high content of GAL-LI in the PGGL at birth may reflect an enhanced synthesis associated with birth.

Immune cells mediate epinephrine secretion from bovine chromaffin cells in vitro.

Potential immunological influences on peripheral catecholamine secretion were investigated by measuring epinephrine secretion from chromaffin cells in vitro in response to cell-free conditioned media from mononuclear cells. Chromaffin cells were isolated from bovine adrenals whereas mononuclear cells were isolated from bovine spleen tissue or whole bovine blood. In secretion experiments epinephrine release and epinephrine remaining in cells was determined such that secretion was expressed as % of total cell content. After 90 minutes exposure to conditioned media, 22.8 +/- 1.1% of content was released compared to 1.7 +/- 0.2% with RPMI media. Secretion after filtration (< 3,000 MW cutoff) was 21.6 +/- 0.9% whereas after boiling and boiling in acid, secretion was 10.2 +/- 0.2 and 4.3 +/- 0.1% respectively. Dialysis (< 3,000 MW cutoff) reduced the 90 min conditioned media-stimulated epinephrine secretion from 22.5 +/- 3.8% to 2.3 +/- 0.3%. Neither atropine nor hexamethonium blockade altered the conditioned media-stimulated epinephrine secretion. These results suggest that mononuclear cells produce a low molecular weight substance--most likely a peptide--that contributes to the stimulation of epinephrine secretion.

Lambert-Eaton syndrome: antigen-antibody interaction and calcium current inhibition in chromaffin cells.

Plasma and IgG obtained from 10 Lambert-Eaton myasthenic syndrome (LES) patients (5 with carcinoma, 5 without associated cancer), 6 healthy subjects, and 1 patient with small-cell lung cancer (SCLC) were examined in their ability to recognize chromaffin cell antigens on Western blots. The pattern of antigen recognition was compared with the magnitude of inhibition of voltage-dependent calcium and sodium currents recorded with the patch-clamp technique from chromaffin cells. Eight of the 11 patients with LES and/or SCLC recognized plasma membrane proteins and 9 of the patients' IgG interacted with cytoplasmic antigens with no apparent pattern of antigen recognition between patients. Also, there was no obvious band pattern distinguishing patients with LES from those with LES and concurrent SCLC. Eighty percent of the LES patients' antibodies were capable of reducing the calcium current (ICa) in chromaffin cells. One of the novel findings of this study is that 30% of the patients had produced antibodies which were able to inhibit both calcium and sodium currents (INa). The heterogeneous response of the IgG on the Western blots does not appear to correlate with the efficacy of reducing the inward currents.

The generation of monoclonal antibodies that bind preferentially to adrenal chromaffin cells and the cells of embryonic sympathetic ganglia.

Adrenal chromaffin cells, sympathetic neurons, and small intensely fluorescent (SIF) cells are each derived from the neural crest, produce catecholamines, and share certain morphological features. These cell types are also partially interconvertible in cell culture (Doupe et al., 1985a,b; Anderson and Axel, 1986). Thus, these cells are said to be members of the sympathoadrenal (SA) lineage and could share a common progenitor. To investigate the origins of this lineage further, we used the cyclophosphamide immuno-suppression method (Matthew and Patterson, 1983) to generate five monoclonal antibodies (SA1-5) that bind strongly to chromaffin cells, with little or no labeling of sympathetic neurons or SIF cells in frozen sections from adult rats. Competition experiments indicate that these antibodies bind to at least three distinct epitopes in tissue sections. The SA antibodies also label most of the cells of embryonic sympathetic ganglia and adrenal primordia. Labeling of sympathetic ganglia appears as the cells initially coalesce and express high levels of tyrosine hydroxylase (TH). Not all TH+ cells in the embryo are SA 1-5+, however; carotid body SIF cells, nodose ganglion TH+ cells, and the transiently TH+ cells in the dorsal root ganglia do not display detectable SA 1-5 labeling. Thus, the expression of these markers for the SA 1-5 lineage is selective. SA antigen expression is hormonally controlled; removal of glucocorticoid and addition of NGF to cultured adrenal chromaffin cells result in the loss of SA 1-5 labeling. These results suggest that the presumed precursors for sympathetic neurons and SIF cells initially express chromaffin cell markers.

Histogenesis of the human adrenal medulla. An evaluation of the ontogeny of chromaffin and nonchromaffin lineages.

The authors previously evaluated the expression of a panel of chromaffin-related genes during histogenesis of the human adrenal medulla. In these studies, chromaffin and nonchromaffin adrenal neuroblasts were identified. To better characterize these nonchromaffin neuroblasts, the authors evaluated two additional markers: HNK-1, an antibody recognizing the migratory neural crest cell; and S-100, a protein expressed by sustentacular cells of the adrenal medulla. HNK-1 immunoreactivity was found in both chromaffin and nonchromaffin cell types at different times during development, marking the nonchromaffin lineage during the second trimester of gestation as well as the chromaffin lineage in the neonatal period. In addition, S-100 expression was noted in some nonchromaffin neuroblasts, and sustentacular cells were first identified at approximately 28 weeks of gestational age. These data suggest a model of human adrenal medullary histogenesis that incorporates the chromaffin, ganglionic, and sustentacular lineages known to constitute the adult adrenal medulla.

[Cellular and humoral immunity in duodenal peptic ulcer with gastrin cell hyperplasia]. / Kletochnyi i gumoral'nyi immunitet pri iazvennoi bolezni dvenadtsatiperstnoi kishki s giperplaziei gastrinovykh kletok.

A total of 61 patients with frequently relapsing duodenal ulcer were examined. Of these, 18 patients were in the acute phase and 43 experienced remission. Using 1 mm2 of the mucosa, measurements were made of the counts of duodenal and pyloric G-cells (by immunomorphologic assay), of the absolute and relative counts of T and B lymphocytes, the content of IgA, IgM and IgG, histamine and serotonin (by fluorometry) in the blood, and of the concentration of uropepsin in the urine. In the stages of exacerbation and remission, the patients suffering from duodenal ulcer with hyperplasia of G-cells manifested, as compared with the analogous patients without hyperplasia, a decrease of the absolute and relative counts of T cells, especially of those of B cells, combined with a rise of the content of IgM and IgG during exacerbation, followed by its returning to normal in the phase of remission. Over one year part of the duodenal ulcer patients with hyperplasia of G-cells received preventive treatment with ranitidine, which resulted in a tendency towards the lowering of the count of pyloric G-cells and the rise of the absolute and relative counts of T cells.

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens.

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.

Preparation and characterization of anti-human chromogranin A and chromogranin B (secretogranin I) monoclonal antibodies.

Chromogranin A, chromogranin B/secretogranin I and chromogranin C/secretogranin II are acidic sulphated and phosphorylated secretory proteins present in a large number of endocrine and neuronal tissues. It has been suggested that these proteins may be useful immunohistochemical markers for human tumours of endocrine origin and their measurement in plasma has been proposed as a diagnostic tool in patients with these tumours. In order to obtain anti-human chromogranins/secretogranins antibodies for clinical applications, we immunized mice with whole chromaffin granules isolated from human pheochromocytoma. The immune sera analysed by two-dimensional immunoblotting were found to recognize chromogranins/secretogranins and other unidentified proteins and to react in immunocytochemistry with pheochromocytoma as well as with a number of endocrine cells of different types. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analysed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach we were able to identify several monoclonal antibodies against human chromaffin granule components. In particular, we have characterized one anti-human chromogranin A and one anti-human chromogranin B/secretogranin I monoclonal antibody which showed a very specific pattern both in immunocytochemistry and in two-dimensional immunoblotting.

Monoclonal antibodies to chromaffin cells can distinguish proteins specific to or specifically excluded from chromaffin granules.

I have prepared a number of monoclonal antibodies to chromaffin cell membranes. One of these antibodies recognizes a number of antigenically related proteins that are present in all tissues examined. In the adrenal, these proteins are completely excluded from chromaffin granules but are present in other subcellular membrane fractions. This non-granule membrane-specific antibody has been designated NG3. A second antibody, CG7, binds to a single protein which segregates specifically into chromaffin granules. The protein recognized by CG7 is cytochrome b561, or chromomembrin B, one of the major protein components of chromaffin granule membranes. CG7 also labels a protein (the identical cytochrome b561) in bovine posterior pituitary neurosecretory vesicle membranes indicating that it functions in both peptidergic and catecholaminergic secretory granules. These two monoclonal antibodies provide useful probes of both granule and extra-granule membrane proteins for studies of membrane trafficking in chromaffin cells.

Exposure of an antigen of chromaffin granules on cell surface during exocytosis.

The synthesis rate of the membrane proteins of the catecholamine-storing vesicles (chromaffin granules) of the adrenal medulla is lower than that of the secretory proteins of the contents. Based on these results we proposed that after exocytosis the membranes of chromaffin granules are retrieved and are re-used for several secretion cycles (see also ref. 4). This concept of re-use of granule membranes has been further strengthened by the finding that exogenous markers which are taken up by secretory cells during stimulation can be traced to the Golgi region and to immature secretory organelles. However, one basic question remains: are the membranes of secretory organelles specifically and completely removed from the plasma membrane and if so, how fast is this process? By using an antiserum against a membrane glycoprotein of chromaffin granules we have now obtained quantitative data which demonstrate that during exocytosis this antigen becomes exposed on the cell surface and disappears again to a large degree within 30 min.

Amines of the mucosal mast cell of the gut in normal and nematode infected rats.

Infection with the nematode N. brasiliensis is accompanied by a marked increase of the number of mucosal mast cells (MMC) and the mucosal content of histamine and 5-hydroxytryptamine (5-HT). We compared amine levels, determined by ion exchange and high performance liquid chromatography (HPLC) with numbers of MMC and enterochromaffin cells (ECC). Furthermore, we measured 5-HT cytofluorometrically in individual MMC and ECC. The cellular distribution of 5-HT was studied immunohistochemically. Our results corroborate previous findings that histamine is stored in MMC. Quotients between histamine content and numbers of MMC decreased throughout the period of worm expulsion, followed by a recovery, suggesting a histamine release during this defense reaction. The HPLC analysis gave no evidence for a storage of dopamine in MMC. ECC and MMC of normal and infected rats showed a formaldehyde induced fluorescence and 5-HT immunoreactivity. The formaldehyde induced fluorescence of MMC from normal rats was about 10% that of ECC, but MMC exceeded ECC three times by numbers. These findings suggest that a considerable proportion of the intestinal 5-HT in the normal rat is stored in MMC. ECC numbers did not change during the infection and their content of 5-HT was unchanged, as judged by cytofluorometry. The cytofluorometric measurements showed that the intensity of the monoamine fluorescence from the MMC of infected animals was about three times as high as that of controls. It was concluded that the increased tissue levels of 5-HT was due to both an increase in MMC numbers and an increase in the 5-HT content of individual MMC. The results suggest a different role for histamine and 5-HT in the defense reaction towards the nematode infection.

Immunochemical evidence for exocytosis in isolated chromaffin cells after stimulation with depolarizing agents.

After chemical stimulation with depolarizing agents (Ba2+ or Ca2+/carbachol) isolated living chromaffin cells display a drastically increased binding capacity for anti-DBH, distributed spotwise on or near the outer cell membrane. This effect is inhibited by noradrenaline; it is not evoked by the non-exocytotically releasing agents tyramine and reserpine. the effect of apparent externalization of DBH is paralleled by the observation of a DBH-dependent binding of 125I-labelled protein A upon the same depolarizing stimuli. These observations are discussed as possible evidence for exocytotic activities.