Biomedical subjects

L Enerbäck

Publications and source records attributed to L Enerbäck.

At least 73 records · Page 4Linked to original sources

PubMed1985

Allometric growth of the rat peritoneal mast cell mass and of the granular constituents: heparin, histamine and 5-hydroxytryptamine.

This paper reports an attempt to measure the normal growth of connective tissue mast cells in rats aged 6 to 24 weeks. We used peritoneal mast cells as a model, and calculated the total mast cell mass and the mass of its components from total peritoneal mast cell numbers and their content of protein, heparin, histamine and 5-hydroxytryptamine (5-HT). The growth process was analysed with the aid of allometric, log-log plots of mast cell quantities versus body weight and linear regression, in order to facilitate comparisons with other systems, notably the lymphoid apparatus. We found that the growth of peritoneal mast cells conformed to the allometric principle (r = 0.91 to 0.93). There were no deviations from linearity or changes in the slope (growth rate constant, k) of the allometric lines within the studied growth interval. K ranged from 1.3 to 1.7, indicating that the mast cell mass and its different components grew at a faster rate than the body as a whole, typical of a late maturing cell system. The mode of growth of the peritoneal mast cells is thus distinctly different from that of the lymphoid system, and neither thymus involution nor sexual maturation appears to influence the growth of these cells.

Aging↗

PubMed1984

A method for the preparation of imprints from the nasal mucosa.

Important immunological reactions take place on the surface of mucosal membranes. Improved methods for the sampling and quantitative study of the cells taking part in these reactions are therefore desirable. We here describe a new technique for the preparation of imprints from the nasal mucosa. The method utilizes a plastic film coated with a thin layer of an albumin-glycerol mixture to improve cell adherence to the surface. The membrane is gently pressed onto a defined portion of the mucous membrane. Fixation and staining procedures are performed on the plastic film, which is then mounted on a slide and covered by a coverslip. The preparations have excellent optical properties and specific cell types can be easily studied, quantified and related to the specific area of the mucosa from which the imprint was taken.

Cell Adhesion↗

PubMed1984

Turnover of histamine in mucosal and connective tissue mast cells of the rat.

Mast cells constitute a heterogenous cell system. The specific type of mucosal mast cell (MMC) of the gut differs with respect to a number of properties from the classical connective tissue mast cell ( CTMC ) found in, e.g. skin, tongue and the peritoneal cavity. This report summarizes recent findings concerning turnover rates of amines and heparin in the two cell types. The elimination rate of radioactively prelabelled histamine, 5-hydroxytryptamine (5-HT) and heparin from peritoneal CTMC was compared with the elimination of radiolabelled histamine from tongue, where histamine is stored in CTMC and duodenum where it is stored in MMC. The elimination of histamine from peritoneal CTMC was slow (t 1/2 = 23 days) and did not differ significantly from that of 5-HT (t 1/2 = 25 days) and heparin (t 1/2 = 35 days) suggesting a low degree of secretory activity in the normal rat. The elimination rate of histamine from the tongue was also very slow. The specific radioactivity of histamine in duodenum was decreasing more rapidly. This was explained by a dilution of the radioactivity since the histamine content increased during the experimental period, and also by MMC death. The results are compatible with the assumption that CTMC and MMC are secretory cells, but with low activity until recruited by adequate, immunological or other stimuli.

Animals↗

PubMed1984

The dynamics of mast cell secretion studied by vital berberine staining.

The fluorescent cationic dye berberine in combination with histamine release studies have been used to explore the different steps of the mast cell secretory process. We have previously shown that quantitation of heparin release by the binding of berberine to fixed mast cells can be used as a direct measure of release of granules. This report summarizes recent work using berberine as a vital stain demonstrating the secretory activity of mast cells. After membrane stabilization with polyethylene glycol (PEG) normal mast cells exclude the dye while mast cells stimulated to secretion with polymyxin B show a strongly fluorescent dye binding to individual cytoplasmic granules. The mean fluorescence intensity of the cell populations after vital berberine staining was compared both to heparin and histamine release. The results strongly suggest that berberine, under the vital staining conditions used, is a marker of intracellular granules that have released histamine. The vital staining method was also used to study membrane events following a polymyxin B-induced secretion. The mean fluorescence intensity decreased by 75% during the first hour after the termination of a polymyxin B stimulation while the mast cell content of histamine and heparin remained constant. The findings support the idea that the membranes are rapidly restored after mast cell secretion, permitting a selective amine release without accompanying release of heparin or other matrix components of the granules.

Animals↗

PubMed1984

Mast cell secretion. Rapid sealing of exocytotic cavities demonstrated by cytofluorometry.

Mast cell secretion is closely associated with extrusion of granules from the cell, and results in rapid release of both amines and heparin. Amines may also be released from intracellular granules communicating with the extracellular environment through channels formed by membrane fusions. We have recently reported the use of the fluorescent, cationic dye berberine as a vital stain marking amine-releasing granules located within the domains of the cell. The present report concerns early membrane events and the fate of such granules after termination of the secretory process. Purified mast cells were stimulated to a moderate secretory response by a low concentration of polymyxin B. The cells were then kept in a medium without the secretagogue. The fluorescence intensity of individual cells after vital berberine staining (reflecting the number of intracellular granules in contact with the extracellular environment) decreased rapidly after terminated mast cell secretion. The heparin content (reflecting the total number of intracellular granules) and the histamine content of the cells, both reduced by the secretory process, remained constant over a period of 1 h after terminated mast cell secretion. These findings suggest that mast cell membranes are rapidly restored after secretion, permitting selective amine release without accompanying release of heparin or other matrix components.

Animals↗

PubMed1983

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.

Animals↗

PubMed1983

Mucosal mast cells of the rat intestine: a re-evaluation of fixation and staining properties, with special reference to protein blocking and solubility of the granular glycosaminoglycan.

Mucosal mast cells of the gastrointestinal tract constitute a separate cell line within the mast cell system of the rat, differing in several respects from the classical connective tissue mast cells and, unlike the latter, requiring special fixation techniques for their demonstration. We have examined some histochemical properties of mucosal mast cells of the duodenum and compared them with connective tissue mast cells of the tongue or skin. The results indicate that the structural integrity of the granules of both types of mast cell is partly dependent on ionic linkages between glycosaminoglycan and protein. The so far unidentified glycosaminoglycan of mucosal mast cells appears to be more soluble than the heparin of connective tissue mast cells. The strongly fluorescent binding of Berberine to the granules of connective tissue mast cells and, depending on their content, of heparin is absent from mucosal mast cells, confirming previous findings which suggested that they contain a glycosaminoglycan with a lower degree of sulphation. Aldehyde fixation by routine procedures reversibly blocks the cationic dye binding of mucosal mast cell granules. The dye binding groups may be unmasked by trypsination or by long staining times of the order of several days. The results suggest that the blocking of staining by aldehydes is caused by a diffusion barrier of a protein nature. Mucosal and connective tissue mast cells thus differ with respect to the spatial arrangement of glycosaminoglycan and protein in their granules. As a result of the study a modified method for the demonstration of mucosal mast cells in tissue sections is described, based on normal formaldehyde fixation and staining in Toluidine Blue for a long time. It has some advantages over previous methods and preserves the structure of mucosal and connective tissue mast cells equally well.

Acids↗

PubMed1983

Suggestive evidence for a direct innervation of mucosal mast cells.

Mast cells are often observed near nerves and functional evidence suggests an innervation of these cells. In the present ultrastructural study, nerve terminals containing many small clear vesicles and a few large vesicles with dense matrix were observed in direct contact with the plasma membrane of mucosal mast cells in the rat ileum, strongly suggestive of a direct innervation.

Animals↗

PubMed1983

Raised 5-hydroxytryptamine concentrations in enterochromaffin cells in adult coeliac disease.

We measured cytofluorometrically the concentration of 5-hydroxytryptamine (5-HT, serotonin) of individual enterochromaffin (EC) cells in adult coeliac and non-coeliac small intestinal mucosa. The distributions of 5-HT concentration within populations of EC cells in control and coeliac mucosae were log normal and thus contained one single population of EC cells. The median 5-HT concentration per EC cell, and the number of EC cells both increased in coeliac disease, but showed signs of normalisation when gluten was withdrawn from the diet. The results indicate that, besides inducing EC cell hyperplasia, gluten is capable of producing reversible changes in functions of EC cells in adult coeliac disease.

Adult↗

PubMed1983

Storage and turnover of histamine, 5-hydroxytryptamine and heparin in rat peritoneal mast cells in vivo.

The biogenic amines and heparin of rat peritoneal mast cells were labelled in vivo by the injection of amine precursors (3H-histidine and 3H-5-hydroxytryptophan) and 35S-sodium sulfate. Uptake of label was rapid, probably reflecting the synthesis of new granule material, but the elimination was slow. Half-lives of radiolabelled histamine (23 days) and 5-hydroxytryptamine (5-HT; 25 days) did not differ statistically from that of heparin (35 days). The slow elimination rates suggest that mast cell secretion is of little biological significance under normal conditions but are well compatible with the idea that mast cell function is related to secretion evoked by appropriate immunological stimuli. It further permitted an analysis of the amine storage by repeated injections of unlabelled 5-HT. A 15-fold increase in 5-HT content was obtained while the total amine content remained constant. The uptake of 5-HT was balanced by a reduction of histamine in a molar 1:1 ratio. A displacement of histamine by 5-HT was further indicated by increased elimination rate of radiolabelled histamine in response to 5-HT injections. The results support previous binding studies in vitro and indicate that histamine and 5-HT are bound to identical storage sites in the mast cell granules.

Animals↗

PubMed1983

Fluorescent berberine binding as a marker of secretory activity in mast cells.

We have previously shown that the weakly fluorescent cationic dye, berberine, forms a strongly fluorescent complex with the heparin of fixed mast cells and can be used for cytofluorometric measurement of heparin in both mast cells and individual mast cell granules. Now, we report on the use of berberine as a vital stain, demonstrating the secretory activity of mast cells. At a dye concentration of 0.025%, and after membrane stabilization with polyethylene glycol, normal mast cells exclude the dye while mast cells stimulated to secretion with polymyxin B show a strongly fluorescent dye binding to individual cytoplasmic granules. The mean fluorescence intensity (reflecting the number of stained granules) of the cell populations increased with increasing polymyxin B concentrations up to 2.0 micrograms/ml, thereafter remaining constant up to 10 micrograms/ml. Fluorescence intensity after vital staining with berberine was compared both to release of heparin measured by berberine binding to fixed cells (reflecting exocytosis of mast cell granules) and also to histamine release. The results strongly suggest that berberine specifically stains secreting granules that are located within the domains of the cells but may have released histamine. Vital berberine staining combined with histamine assays and cytofluorometric measurements of heparin is therefore of great potential interest for analyzing the dynamics of mast cell secretion.

Animals↗

PubMed1982

Uptake and turnover of dopamine in rat mast cells studied by cytofluorometry and high performance liquid chromatography.

Uptake and turnover of dopamine (DA) in rat peritoneal mast cells were studied by a cytofluorometric technique. The main advantage of the method is that it permits the study of the distribution of amine content within populations of cells. Catecholamines and indolamines can be differentiated, but subtler structural differences in this group of compounds cannot be distinguished. We, therefore, combined the cytofluorometric measurements with a liquid chromatographic method based on reversed-phase chromatography followed by amperometric detection in a thin layer flow cell. Intraperitoneally injected L-DOPA was rapidly decarboxylated to DA, which was accumulated in mast cell granules. The elimination of DA from the mast cells was much faster than previously published 5-hydroxytryptamine and histamine elimination rates. No evidence of intracellular conversion of DA before its elimination was found and simultaneous heparin quantitations gave no evidence of an elimination pathway due to exocytosis of granules. Electron microscopy disclosed no structural changes that could be related to exocytosis during the elimination phase of DA. The rapid elimination together with absence of inhibition of DA-uptake after storage of exogenous 5-hydroxytryptamine suggest that the mechanism of DA storage differs from the mechanism of storage of endogenous mast cell amines.

Animals↗

PubMed1982

Effect of polyethyleneglycol (PEG) on spontaneous and polymyxin B-induced histamine release from rat peritoneal mast cells.

Mast cells were isolated from the peritoneal cavity of rat and purified by centrifugation in a gradient of Percoll. The spontaneous and polymyxin B-induced release of histamine was studied after preincubation of the cells with polyethyleneglycols (PEGs) of different molecular weight (200-20,000 dalton) and with fatty acid derivatives of PEG 6000. It was found that very low concentrations (less than 0.1%) of PEG 6000 reduced the spontaneous and polymyxin B-induced release of histamine to a greater extent than the same concentration of bovine serum albumin. The inhibition increased with the size of the PEGs, but was little affected by the presence of fatty acid ligand bound to PEG.

Animals↗

PubMed1981

The effect of polymyxin B and some mast-cell constituents on mucosal mast cells in the duodenum of the rat.

Mucosal mast cells in the rat duodenum show no morphological signs of exocytosis of granules and do not release histamine after treatment with polymyxin B in doses large enough to cause almost complete degranulation of connective-tissue mast cells of tongue, skin, and mesentery with concomitant release of approximately 60% of the tissue histamine. Administration of polymyxin B in gradually increasing doses over a period of 5 ds resulted in a statistically significant increase in mucosal mast cells and a comparable increase in duodenal histamine content, whereas the connective-tissue mast cells in the other tissues examined became fewer in number, the remaining cells showing profound morphological changes, and tissue histamine levels, were reduced to approximately 40% of the controls. A similar increase in mucosal mast cells has been observed after treatment with another mast-cell secretagogue, compound 48/80. This suggests that the increase in mucosal mast cells may be an indirect effect of these compounds, related to their activation of other mast cells and mediated by material(s) secreted by the connective-tissue mast cells. Possible mediators such as heparin, histamine, and 5-hydroxytryptamine injected for 5 ds in doses large enough to account for the amount released from the degranulated mast cells had no effect on the morphology or numbers of mast cells in any of the tissues examined.

Animals↗

PubMed1981

DNA distribution of mast cell populations in growing rats.

The proliferation of rat peritoneal mast cells was examined under normal conditions in vivo. DNA content of individual mast cells was measured by cytofluorometry after staining with the bibenzimidazole dye Hoechst 33258. Diploid non mast cells from each rat were used as a biological standard, which resulted in small long-term variations in the method. The proportion of mast cells in the S + G2 region of the DNA distribution was about 4% for young rats (24 days old, body-weights about 60 g). It decreased in relation to body-weight, and was less than 1% for 105-day-old rats weighing 400 g. During the same growth period the total number of mast cells in the peritoneal cavity increased about 8-fold. The total number of proliferating cells, about 30,000, remained constant throughout the observation period. No evidence of polyploidization or accumulation in G2 of mast cell nuclei was found. It is concluded that peritoneal mast cells increase in number by mitotic proliferation of differentiated cells.

Animals↗

PubMed1980

Cytophotometric quantification of retrograde axonal transport of a fluorescent tracer (primuline) in mouse facial neurons.

A method for cytophotometric quantifications of retrograde axonal transport of a fluorescent tracer in tissue sections is described. As a fluorescent tracer the anionic vital stain primuline proved to be suitable since it resulted in a strong yellow-green fluorescence, which faded very slowly permitting localization of cells during illumination with UV-light. Primuline injected into the muscles of the vibrissae in mice was transported to the corresponding nerve cell bodies in the facial nucleus, where it appeared as fluorescent granules 9 h after the injection. The fluorescence intensity increased with increasing exposure times and concentrations of the injected tracers. The motor endplates showed no ultrastructural changes after the tracer injections. The motor endplates showed no ultrastructural changes after the tracer injections. With this method a substantial increase in tracer accumulation in facial neurons could be revealed during nerve regeneration 11 days after crushing the facial nerve. Small alterations in neuronal tracer accumulation could be measured after intoxication of the mice with botulinum and tetanus toxins. Since these toxins should cause a decrease or increase in the degree of synaptic activity the amount of retrograde axonal transport may to a certain extent be dependent on the synaptic function. The findings with this new technique therefore indicate that quantitative changes occur in axonal transport in materials from the periphery during different pathological and physiological conditions, which may be important for an understanding of how a nerve cell body is dependent on its peripheral field of innervation.

Animals↗

PubMed1980

Histamine content of peritoneal and tissue mast cells of growing rats.

The histamine and 5-hydroxytryptamine (5-HT) content of mast cells was measured in rat peritoneal mast cells (isolated by density-gradient centrifugation or in crude peritoneal cell suspensions) and in some solid, mast-cell-rich tissues (tongue, skin, and duodenum). The duodenum contains large numbers of mast cells belonging to the specific type of mucosal mast cell. The peritoneal cavity, tongue, and skin contains the classical, mature connective-tissue-type of mast cell. The approximate amine content in mast cells of solid tissues was calculated by combining the biochemical assays with cell counting. The amine content was related to the age and body weight of the rats, studied during a period of rapid body growth (25-233 days). In the connective-tissue-mast cells both amines showed an increase that was strongly correlated to age and body weight. The increment of histamine was not as large as that of 5-HT. In peritoneal mast cells the histamine content per cell was doubled during the growth period studied, whereas there was a sixfold increase of 5-HT. The estimated 5-HT content per mast cell of tongue and skin also increased in relation to body weight. The histamine/5-HT quotients in these tissues were similar, and decreased with increasing age as did the same quotients for peritoneal cells. Parallel cell counts and histamine assays indicated that the mucosal mast cells contained much less histamine than the connective-tissue mast cells, and this findings was supported by histochemical observations. The observations did not suggest that histamine is stored else-where than in mast cells. In the mucosal mast cells, too, the histamine content appeared to increase as a function of age and body weight. Duodenal 5-HT, which is to a large extent contained in enterochromaffin cells, did not increase in relation to body growth.

Aging↗

PubMed1980

Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll.

Rat mast cells from peritoneal washings were purified by centrifugation on a medium containing silica particles coated with PVP (Percoll). By isopycnic centrifugation on continuous Percoll gradients, the buoyant density of mast cells ranged from 1.09 to 1.17 and was consistently lower in mast cells from younger rats than in mast cells from older rats. In suspensions uncontaminated with blood, the mast cells could be isolated by a simple centrifugation procedure based on density differences (step I). Contaminating red blood cells were removed by a second short centrifugation of velocity gradient type, using a linear Percoll gradient of low density. The mast cell purity was 91-98% and the recovery 63-100%. Purified mast cells reacted normally in a viability test employing fluorescein diacetate, retained amines and heparin, and reacted normally with amine release following incubation with Polymyxin B or antigen (immunised rats). Electron microscopy revealed excellent ultrastructural preservation of the mast cells and infrequent uptake of Percoll particles by endocytosis.

Animals↗