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 quantitation of 5-hydroxytryptamine in mast cells: an improved technique for the formaldehyde condensation reaction

A simple technique for the condensation of cellular 5-hydroxytryptamine (5-HT) with formaldehyde gas is described. The technique, which is especially suited for quantitative cytofluormetric studies, involves the generation of formaldehyde gas from dry paraformaldehyde in a closed reaction vessel with the addition of a measured quantity of water. The fluorescence yield of 5-HT was tested at various humidities. Optimal results were obtained with the addition of 100 mg water to a 1000 ml reaction vessel containing 6 g of dry paraformaldehyde. A major advantage of the method if the fact that the humidity during the reaction can be precisely controlled. The fluorescence yield of 5-HT, tested over a 50 day period showed excellent reproducibility. The stoichiometry of the reaction was tested by comparison of cytofluormetic data with that obtained by analysing the 5-HT content of pooled mast cells with an independent biochemical method. A highly satisfactory correlation (r = 0.96) was obtained within the range of 0.1 to 4 pg of 5-HT per cell. The limit of sensitivity of the cytofluorometric method was found to be of the order of 10(-13) g, and was determined by the fluorescence blank of the mast cells. This contributes to between 10 and 30 per cent of the total fluorescence emission from mast cells containing about 0.2 pg of 5-HT.     

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.     

Aging differentially modifies arterial sensitivity to endothelin-1 and 5-hydroxytryptamine: Studies in dog coronary arteries and rat arterial mesenteric bed

The influence of age on vascular reactivity to endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) was studied in coronary artery rings from dogs of 9 years of age or younger, and dogs older than 9 years. ET-1 caused concentration-dependent contractions that developed about 100% of the 70 mM KCl-induced tension in the younger dogs; those from older dogs did not generate more than 20%. In contrast, 5-HT developed only about 20% of the KCl-induced tension in rings from young dogs, whereas in the older animals, it developed up to 120% of the KCl tension. No significant difference in the tension developed by 70 mM KCl was noted between both groups of dogs. Mechanical denudation of the endothelial cell layer caused a modest, yet significant, leftward shift of the ET-1 and 5-HT concentration-response curves only in the younger dogs. Nω-Nitro-l-arginine (15 μM) shifted the ET-1 concentration-response curves to the left in rings from both groups of dogs. Rings precontracted with 20 mM KCl relaxed in a concentration-dependent fashion with acetylcholine; its sensitivity was about threefold less in the older group of dogs. To validate the changes in vascular reactivity with age, a parallel study was performed perfusing the arterial mesenteric bed of rats of 3, 7, and 30 weeks of age. In this experimental model, the efficacy of ET-1 significantly decreased with age and that of 5-HT was significantly increased. The vasomotor reactivity of noradrenaline was modestly affected by aging, whereas the acetylcholine-induced vasorelaxation was significantly reduced with age.     

Potentiation of antigen-induced mast cell activation by 1-34 bovine parathyroid hormone

Peptides such as parathyroid hormone (PTH), somatostatin, and gastrin have been reported to stimulate mast cell mediator release. Preincubation of rat serosal mast cells with synthetic 1-34 bovine parathyroid hormone (1-34bPTH) significantly enhanced antigen-induced 5-hydroxytryptamine (5-HT) release. Enhancement of 5-HT release by 1-34bPTH was dose dependent between 5 and 2000 nM. In the absence of antigen, mean net 5-HT release was less than 1% when naive or passively sensitized mast cells were incubated with 1000 nM 1-34bPTH for time intervals up to 90 min. These findings indicate that 1-34bPTH, at relatively low concentration, potentiates antigen-induced 5-HT release from mast cells.     

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.     

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     

Biochemical and immunohistochemical determination of 5-hydroxytryptamine located in mast cells in the trigeminal ganglion of the rat and guinea pig

The presence of 5-hydroxytryptamine (5-HT), as well as its precursor (5-HTP) and metabolite (5-HIAA), were biochemically determinated in the trigeminal ganglion of the guinea pig and rat. The distribution of 5-HT in the ganglion and in its posterior root was studied using both indirect immunofluorescence and the peroxidase-antiperoxidase method. In order to increase the possible 5-HT content of primary sensory neurons for subsequent immunohistochemical visualization, animals were first treated with nialamide, an inhibitor of monoamine oxidase, and then loaded with L-tryptophan. Another group of animals received colchicine to inhibit intra-axonal transport of transmitter substances. However, even combined use of loading and colchicine treatment did not reveal 5-HT immunoreactivity in ganglion cells. The only source of 5-HT immunoreactivity in the trigeminal ganglion and its posterior root was mast cells. These cells were located around the ganglion in adjacent leptomeningeal and connective tissues, as well as between the ganglion cells and nerve fibers. Only occasionally were mast cells found in the posterior root of the ganglion.     

Changes in numbers and heparin content of peritoneal fluid mast cells of growing rats measured by flow cytofluorometry

A cytofluorometric method, based on berberine staining of mast cell heparin, was used for flow cytofluorometric counting and heparin quantitation of mast cells in crude peritoneal suspensions of growing rats. The automatic flow cytofluorometric counting of mast cells correlated well with hemocytometer cell counts. The mean mast cell heparin content obtained by flow cytofluorometry showed good agreement with such obtained by cytofluorometry of microscopically identified mast cells. The number of peritoneal mast cells and the mean mast cell heparin content was found to increase as the animals grew older. The results of the microscope fluorometric measurements suggested that the heparin content was normally distributed within mast cell populations of both young and old rats. However, the heparin distributions obtained by flow cytofluorometry were often positively skewed but did not fulfill the condition of the log-normal distribution.