Cytofluorometric quantitation of 5-hydroxytryptamine and heparin in individual mast cell granules.

Cytofluorometric quantitation of 5-hydroxytryptamine (5-HT) and heparin in individual mast cell granules is described. The technique is based on micromanipulation of intact mast cells reacted with formaldehyde or stained with Berberine sulfate and the use of a cytofluorometer equipped with a sensitive peak detecting device. The quantities of 5-HT and heparin contained in mast cell granules which are of the order of 10(-16) and 10(-13) g, respectively were expressed as relative fluorescence guanta. The results of measurements on representative samples of mast cell granules indicate that all granules contain heparin as well as 5-HT, and that there are large variations in both 5-HT and heparin content within the granule populations of individual cells. A dose dependent increase in 5-HT content in both cells and individual mast cell granules occurred 24 hr after the injection of 10--50 mg L-5-hydroxytryptophan/kg intraperitoneally. There was no evidence for an increase in the heparin content of granules or cells, indicating that a new synthesis of granular macromolecules is not required for the 5-HT uptake. The results further suggest that 5-HT may be stored initially in a cytoplasmic extragranular pool and then taken up in the mast cell granules.     

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.     

Indole reactions of mast cells and enterochromaffin cells related to fixations with and without aldehydes

Summary Survey of a considerable number of rat, mouse and hog tissues which presented large numbers of mast cells in preparations stained with toluidine blue and other metachromatic or basic dyes at low pH levels, revealed numbers of oval bodies of about the same size as mast cells which reacted weakly or even moderately to the postcoupled benzylidene indole reaction. The numbers of these were always less than that of mast cells in toluidine blue sections of the same blocks. They never occurred in clusters of perhaps 15–20 in a single high power field, as mast cells often do. Smooth and especially striated muscle which often formed the background tissue where most mast cells are found with metachromatic stains, regularly present indole reactions due to protein tryptophan. This is usually equal to or stronger than that in the supposed mast cells.Indole reactive bodies whose morphology suggests mast cells are also present in similar numbers in formaldehyde and glutaraldehyde fixed tissue as well as with aldehyde free fixations. Glutaraldehyde and formaldehyde are known to inhibit the benzylidene reaction of 5-HT in vitro (30 min for glutaraldehyde, 3 h for formaldehyde) (Lillie, 1977). This action was avoided in mercury and lead heavy metal fixations and in acetone, Carnoy, chloroform methanol and similar fixations.The mast cell-like bodies are best explained as tangential or oblique sections of individual muscle fibers. We have described the same phenomenon with the ferric ferricyanide (Golodetz-Unna, 1909) reaction (Lillie et al., 1978a), and the PCB reaction is that of tryptophan in these muscle cell sections.In contrast to the DMAB type reaction failure acid diazosafranin successfully demonstrated mast cells with both aldehyde and aldehyde free fixations. This reaction has been shown to occur with 5-HT and 5-HTP (Lillie et al., 1973).     

Visualisation of messenger RNA directing peptide synthesis by in situ hybridisation using a novel single-stranded cDNA probe. Potential for the investigation of gene expression and endocrine cell activity

Recently (Pecci Saavedra et al. 1982; Brusco et al. 1982, 1983) we have showed that the actual specificity of the rabbit anti-5-HT antibodies, is for the beta-carboline derivatives of 5-HT as a result of cyclization of the lateral chain. We explained this as resulting from the use of formaldehyde which acted both as a fixative in the preparation of the tissues, and as the coupling agent in the preparation of the immunogen. Following this line we have fixed several brain stem specimens with 0.5% p-benzoquinone; 3% glutaraldehyde; 4% paraformaldehyde plus 0.25% glutaraldehyde and compare the results with tissues fixed in 4% paraformaldehyde. Glutaraldehyde and p-benzoquinone do not produce cyclization of 5-HT but immobilize monoamines in situ. As expected, the antibodies applied according to the PAP technique did not stain the neuronal bodies of the raphe system, known to contain 5-HT when 3-4% glutaraldehyde or 0.5% p-benzoquinone were used. Good staining was obtained with 4% paraformaldehyde alone or with 4% paraformaldehyde plus 0.25% glutaraldehyde. A quantitative assay of the spot test of Larsson (1981) was devised for measuring in vitro the inhibitory effects of 5-HT, of the 5-HT-BSA complex and of the cyclic derivative, 6-OH-1,2,3,4-tetrahydro-beta-carboline. The results confirmed that the avidity of the antiserum is much greater for the cyclic derivatives contained in the 5-HT-BSA complex and for 6-OH-1,2,3,4-tetrahydro-beta-carboline than for 5-HT. It is concluded that the formation of a new ring by the lateral chain of 5-HT is responsible of the in-vitro and in the tissue immunoreactivity of the anti-5-HT-antibodies.     

Protein content, dry mass and chemical composition of individual mast cells related to body growth.

Recently developed quantitative microscopical techniques were used to study relations between body growth and protein content as well as dry mass of individual mast cells. Since previous studies had shown an age-related increase of mast cell content of 5-hydroxytryptamine (5-HT) and heparin, these mast cell components were also included in the present study. The cells were obtained from the peritoneal cavity of rats aged 44--269 days (body weights 189--610 g). All studied mast cell parameters showed an increase that was related to the growth of the animals. The dry mass increased 60%, protein 50%, heparin 50% but 5-HT increased as much as 260% during the studied growth period. There was a mutual and linear correlation between all studied mast cell parameters. Population studies, based on large scale measurements of individual mast cells from young and adult rats, were made. These studies showed that histograms of 5-HT content, protein content and dry mass of individual mast cells were skewed with a tail towards higher values and approximately lognormal. On the other hand, the frequency distribution of heparin content of individual mast cells was approximately normal.     

Protein content,dry mass and chemical composition of individual mast cells related to body growth

Summary Recently developed quantitative microscopical techniques were used to study relations between body growth and protein content as well as dry mass of individual mast cells. Since previous studies had shown an age-related increase of mast cell content of 5-hydroxytryptamine (5-HT) and heparin, these mast cell components were also included in the present study. The cells were obtained from the peritoneal cavity of rats aged 44–269 days (body weights 189–610 g). All studied mast cell parameters showed an increase that was related to the growth of the animals. The dry mass increased 60%, protein 50%, heparin 50% but 5-HT increased as much as 260% during the studied growth period. There was a mutual and linear correlation between all studied mast cell parameters. Population studies, based on large scale measurements of individual mast cells from young and adult rats, were made. These studies showed that histograms of 5-HT content, protein content and dry mass of individual mast cells were skewed with a tail towards higher values and approximately lognormal. On the other hand, the frequency distribution of heparin content of individual mast cells was approximately normal.Supported by grants from the Swedish Medical Research Council, Project no 2235     

Cytofluorometric demonstration of a catecholamine in rat mast cells after the administration of DOPA

Synopsis A catecholamine, probably dopamine, was identified in rat peritoneal mast cells after subcutaneous injection of DOPA. Its identity was established by cytofluorometry of cells treated with hot formaldehyde vapour according to the Falck-Hillarp technique. Injections of 50–200 mg/kgl-DOPA were followed by a dose-dependent increase in fluorescence intensity, measured at the emission maximum for catecholamines. The increase in fluorescence intensity was accompanied by a change in the emission spectrum with displacement of the fluorescence maximum towards a shorter wavelength characteristic for a catecholamine. Recordings of rates of photodecomposition showed a rapid exponential fading of the fluorescence in mast cells of control rats comparable to that of 5-hydroxytryptamine-containing protein droplets, whereas the mast cells of DOPA-treated rats showed a slower fading rate, intermediate between that of dopamine- and 5-hydroxytryptamine-containing models.     

Uptake of 5-hydroxytryptamine by mast cells in vivo: a cytofluorometric study of mast cells and individual mast cell granules.

Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.     

A modification of the histochemical fluorescence method for the improved localization of 5-hydroxytryptamine

Summary By using a modified formaldehyde gas treatment, it has been possible to improve the histochemical fluorescence method for the demonstration of 5-hydroxytryptamine. A certain improvement of the localization of catecholamines has also been obtained.The modified histochemical procedure involves a primary treatment with a formaldehyde gas of a humidity which does not cause any noticeable diffusion. After this it is possible to retreat the tissue specimen with a formaldehyde gas of higher humidity resulting in increased yields of the final fluorescent compounds. In spite of this improvement of the formaldehyde gas technique for the demonstration of 5-hydroxytryptamine, it cannot be considered as sensitive as that for catecholamines. The reasons for this are discussed.     

Uptake of 5-Hydroxytryptamine by mast cells in vivo: A cytofluorometric study of mast cells and individual mast cell granules

Summary Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.Supported by grants from the Swedish Medical Research Council, Project no 2235     

活细胞内5-羟色胺可见荧光的多光子激发成像

应用多光子激发激光扫描显微镜对5-羟色胺（5-hydroxytryptamine, 5-HT）孵育的大鼠粘膜型肥大细胞进行自发荧光成像,首次观察到了活细胞内5-HT相关的可见荧光,并对其产生机理进行了初步探讨.实现了对活细胞内5-HT空间分布的高分辨成像,为研究活组织或细胞内5-HT的空间分布和含量与细胞功能状态的关系提供了新的实验方法.     

Hormones in the nucleus. Immunologically demonstrable biogenic amines (serotonin, histamine) in the nucleus of rat peritoneal mast cells

Using 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide (EDAC) fixation and immunocytochemical confocal microscopic study, bright serotonin and histamine fluorescence appeared in the nucleus of rat peritoneal mast cells. In case of paraformaldehyde fixation, this was not observed. The phenomenon can be explained by the cross-linking effect of EDAC, which did not allow the efflux of biogenic amines from the nucleus. This means that biogenic amines are present in the nucleus of mast cells, and this is supported by the flow cytometric measurement data of the whole cell. Other hormones studied (triiodothyronine, insulin, and endorphin) were not present in the nucleus. Four pharmaca with biogenic amine-influencing character in the central nervous system were used for studying the relation between the external (surrounding and cytoplasmic) and nuclear biogenic amine content of mast cells. Fluoxetine, a serotonin reuptake inhibitor depleted nuclear as well as cytoplasmic serotonin content. Clorgyline, a MAO-A inhibitor, decreased cytoplasmic serotonin content and weakened nuclear serotonin fluorescence. The tryptophan hydroxylase inhibitor, para-chlorophenylalanine (PCPA), and the mast cell degranulator, Compound 48/80, reduced cytoplasmic serotonin content without influencing nuclear content. Histamine fluorescence was influenced solely by fluoxetine. The results show that nuclear 5-HT content is dependent firstly of serotonin uptake and reuptake. To our knowledge, this is the first exact report on the presence of non-steroid-type-receptor-transported hormones inside the nucleus of a cell.     

The histologic and functional characterization of enzymatically dispersed intestinal mast cells of nonhuman primates: effects of secretagogues and anti-allergic drugs on histamine secretion

Despite the apparent involvement of gastrointestinal mast cells in hypersensitivity reactions in the mucosa, remarkably little information is available concerning the characteristics of these cells from man and higher animals. To study the characteristics of gastrointestinal mast cells from nonhuman primates, a previously described technique which uses a combination of mechanical and enzymatic methods to obtain mast cells from the tissues of rodents required modification to permit the successful dispersion of normal gastrointestinal tissues of higher animals. This modified procedure, as described in this report, appears to be relatively selective for mast cells located in the mucosal site, and typically yields ca 9 X 10(5) mast cells per gram of tissue. The mucosal mast cells obtained comprised ca 2% of the total nucleated cells, contained approximately 1 pg of histamine per cell, and stained metachromatically with toluidine blue only at low pH. The cells exhibited a dose-dependent release of histamine on challenge with goat anti-human IgE or the ionophores A23187 and Br-x537A but were refractory to the action of compound 48/80. IgE-mediated histamine release from monkey intestinal mast cells differed from that observed from rat intestinal mast cells in that release was inhibited not only by quercetin but also by theophylline. Disodium cromoglycate gave variable results. The data indicate that viable nonhuman primate mucosal mast cells can be obtained for study, and that these cells, although sharing some characteristics of mucosal mast cells from lower species, have distinct and unique properties. The availability of this nonhuman primate model for the study of mast cell function in higher animals should contribute to the understanding of mast cell-mediated diseases in man.     

Morphologic and cytochemical characteristics of amine-containing globule leukocytes in rat tracheal epithelium

Amine-containing cells in the tracheal epithelium are typically of the small-granule type (diameter approximately 100 nm). However, in the rat, another amine-containing cell type has been identified that possesses the amine-handling features of the APUD-series of cells (amine precursor uptake and decarboxylation) but not the ultrastructural characteristics. It has been postulated that these cells may be related to cutaneous melanocytes. In this study, fluorescent cells were present in the laryngeal and tracheal epithelial lining of adult Sprague-Dawley rats following freeze-drying and exposure to formaldehyde vapor (FIF or formaldehyde-induced fluorescence). Microspectrofluorimetry revealed an emission maximum at 493 nm. The excitation maximum could not be calculated but appeared to be around or below 350 nm (to record spectra below requires the use of quartz optics). Yellow fluorescence also emanated from serotonin-containing mast cells (excitation and emission maxima: 401/515 nm). Tracheal segments processed according to the aqueous formaldehyde ( AFIF ) technique, for the demonstration of 5- hydroxytryptophan (5-HTP) or serotonin (5-HT), failed to identify fluorescent cells in the epithelial lining even though connective-tissue mast cells were evident. Subsequent treatment of AFIF -fixed sections with formaldehyde and HCl vapors ( AFIF -HCl) resulted in the formation of a fluorogenic compound within numerous cells in the tracheal lining (455/537 nm). This spectral shift and increase in intensity of fluorescence following acidification are characteristic for standards and/or cells that contain tryptamine, tryptophan, or peptides with NH2-terminal tryptophan and are markedly different from microspectrofluorimetric data reported for the phenylethylamines or serotonin. It is therefore postulated that these cells contain a closely related beta-(3-indolyl) ethylamine-like compound, serotonin excluded. The morphology of the fluorescent cells was similar when prepared according to the FIF or AFIF -HCl techniques. Conjunctive staining, the examination of a single section first by fluorescence microscopy and subsequently by other histochemical and cytochemical methods, demonstrated that the fluorescent granules were also methylene blue, alcian blue, periodic-acid Schiff, and ferric- fericyanide positive. Subsequent correlative electron microscopic examination of Epon-embedded AFIF -HCl-treated tracheal sections demonstrated that these amine-containing cells were globule leukocytes.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

Production of Clostridium pasteurianum in a Defined Medium

A method for the growth of Clostridium pasteurianum in a 140-liter (total capacity) stainless-steel vessel is described. By preventing the pH value from falling below 5.6, the growth of cultures was prolonged. Larger amounts of the carbon source (sucrose) and the nitrogen source (ammonium ion) were supplied and consumed, and cell yields of up to 5.56 g (dry weight) per liter were obtained. The highest cell yield previously reported was 1.7 g (dry weight) per liter obtained under nitrogen-fixing conditions in 500-ml cultures. The ferredoxin content of the cells was comparable with that obtained by earlier workers.     

A RAPID AND SIMPLE METHOD FOR THE DETERMINATION OF PICOGRAM LEVELS OF SEROTONIN IN BRAIN TISSUE USING LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

A rapid and simple technique using solvent extraction and high pressure liquid chromatography with electrochemical detection has been developed for the determination of serotonin in small brain tissue samples (1-20 mg). The method has a reasonably good specificity and a very low experimental error (less than 3%s.e ., calculated from six samples processed and analysed from the same brain homogenate). The recovery of authentic 5-HT added is 80-90%. The 5-HT levels of rat whole brain was found with the present technique to be 690 ± 17.5 ng/g and of mouse neocortex 304 ± 16 ng/g. Monoamine oxidase inhibition with pargyline (2 h) increased 5-HT levels in mouse neocortex to 194 ± 15% (N = 5) of control, while reserpine depleted 5-HT to 13 ± 4% of control. The method has a sensitivity level of about 20 pg (0.1 pmol) per brain sample.     

A quantitative cytochemical study of the growth of individual mast cells

Summary For individual mast cells, relationships between their dry mass and their content of heparin and 5-hydroxytryptamine (5-HT) were studied. This was achieved by measuring these parameters successively on identical cells, by means of quantitative cytochemical techniques. The peritoneal mast cells have a very long life span and a slow turnover of granule components. Increase of the dry weight of the cells may therefore be taken as an expression of cellular growth. Mast cell populations from younger and older animals were analysed in an attempt to evaluate the influence of cell-aging and animal-aging on the growth of the mast cells. The analysis was based on allometric (log-log) plots and linear regressions. Within the cell populations there were strong mutual correlations between the cell parameters studied, without any obvious deviations from linearity. However, the slopes of the allometric lines indicated a somewhat different mode of growth for mast cells from younger and older animals. The capacity of the mast cells to accumulate 5-HT after a single injection of its precursor, 5-hydroxytryptophan, was used as a functional test. In relation to the cell weight, the induced increase of 5-HT was greater for lighter than for heavier mast cells. This difference between light and heavy mast cells was greater for cells from younger than from older animals. These differences in growth and functional properties between mast cells from younger and older animals were interpreted as an effect of aging of the animals rather than of aging of the cells.Supported by grants from the Swedish Medical Research Council, Project no 2235I wish to express my sincere thanks to Professor Lennart Enerbäck for valuable and constructive criticism of this paper. I also wish to thank Lecturer Erik Leander for valuable statistical advice     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Subcellular localization of serotonin immunoreactivity in rat enterochromaffin cells

Serotonin immunoreactive material was localized to rat enterochromaffin cells (EC cells) at the subcellular level using antibodies to serotonin (5-HT) raised in rabbits. Ultrathin sections from paraformaldehyde fixed plastic embedded tissues were directly labelled with the 5-HT antiserum, using the protein A-gold technique to visualize the immunoreaction. The 5-HT immunoreactivity (5-HT-IR) in the rat gastrointestinal mucosa was exclusively localized to epithelial EC cells with a low background over other epithelial non-enterochromaffin cells. Quantitative evaluation of the immunoreaction revealed that most of the 5-HT-IR in the cytoplasm of EC cells (60%) was located over the dense cores of the secretory granules. However, a significant part of the cytoplasmic 5-HT-IR (40%) was located outside the dense cores of the secretory granules which suggests that different forms of 5-HT storage may exist.