Perinuclear localization of biogenic amines (serotonin and histamine) in rat immune cells

EDAC fixation, which polymerizes biogenic amines, and clorgyline, which blocks monoamine oxydase, were used to demonstrate the perinuclear and nuclear localization of histamine and serotonin (5-HT) of immune cells in rat peritoneal fluid and thymus. For detection, an immunocytochemical method combined with confocal microscopy was used. In peritoneal cells (lymphocytes) 5-HT formed spots around the nucleus, while histamine was localized diffusely. In thymocytes, 5-HT was distributed in patches, while histamine formed a garland around the nuclear envelope. Clorgyline enhanced the fluorescence and increased the number of fluorescent positive cells only for histamine, and then histamine-positive nuclei were also present. The results show that: (1) EDAC fixation demonstrates the presence of biogenic amines in immune cells, (2) using this fixation, the perinuclear localization of biogenic amines can be demonstrated, (3) the localization of the two amines is different and (4) the nuclear localization of histamine in thymocytes can be surmised and in mast cells (described previously) is proven again. The potential importance of the nuclear and near-nuclear localization of biogenic amines is discussed.     

Influence of paraformaldehyde and EDAC fixation on the demonstrability of hormones (histamine, endorphin, triiodothyronine) in rat immune cells: an immunocytochemical comparative analysis

The amount and localization of three hormones (histamine, endorphin and triiodothyronine [T(3)]) was measured in male and female rat peritoneal cells (lymphocytes, mast cells, monocyte-macrophage-granulocyte group [mo-gran]) using flow cytometry as well as confocal microscopy after paraformaldehyde (PFA) or EDAC fixation. In the EDAC fixed lymphocytes and mo-gran of female animals two-magnitude higher levels of histamine were measured after EDAC fixation and one magitude higher in mast cells. The amount of T(3) was almost four-fold in lymphocytes and 2.5-4-fold in mast cells and mo-gran. Endorphin content was not altered by the type of fixation. In each cell type in males one magnitude higher levels of histamine and T(3) were measured after EDAC fixation and a small, but significant, elevation of endorphin. Confocal microscopy supports the quantitative data. The results show that (1) the fixation with the crosslinking molecule, EDAC, is more suitable for immunocytochemical studies of amino-acid type hormones in immune cells, (2) more histamine and T(3) are present in the immune cells than it was supposed previously when studying PFA-fixed preparations, (3) the estimation of the amount of peptide hormones seems to be accurate after PFA fixation, (4) there is a quantitative difference comparing the results of PFA and EDAC fixation between males and females.     

Secretin-cells of the mammalian intestine contain serotonin

Various endocrine cells contain biogenic amines in addition to their peptide hormones. In the digestive tract, one of these amines is serotonin that is regularly present in enterochromaffin (EC-) cells. Previously, it has been assumed that other entero-endocrine cell types also contain this amine. Moreover, it was presumed that chromogranin A, an acidic glycoprotein, is involved in storage mechanisms for biogenic amines in endocrine cells. Using immunohistochemical techniques, we now exemplarily investigated cholecystokinin (CCK-) and secretin (S-) cells of five adult mammalian species for their content of serotonin and of chromogranin A. In all mammalian species, CCK-cells were devoid of serotonin but contained chromogranin A immunoreactivity of varying densities. In contrast, S-cells of all mammals were immunoreactive for serotonin; however, immunoreactivities for this biogenic monoamine were heterogeneous and varied from dense to faint or lacking immunostainings. Likewise, immunoreactivities for chromogranin A in S-cells showed inter-species and inter-cellular heterogeneities. S-cells containing serotonin were simultaneously immunoreactive for chromogranin A and the density of immunoreactivities for both were correlated in given S-cells. Based on mutual relationships of chromogranin A and serotonin immunoreactivities, we assume that chromograinin A is virtually a prerequisite for the S-cells' content of serotonin and that this protein participates in storage mechanisms for biogenic amines in endocrine cells. S-cells have now to be added to the family of amine-storing endocrine cells. Basically, serotonin-storing endocrine cells in the digestive tract cannot be simply regarded as enterochromaffin (EC-) cells any longer; the current nomenclature and classification of entero-endocrine cells should be reviewed in this respect.     

Secretin-cells of the mammalian intestine contain serotonin

Summary Various endocrine cells contain biogenic amines in addition to their peptide hormones. In the digestive tract, one of these amines is serotonin that is regularly present in enterochromaffin (EC-) cells. Previously, it has been assumed that other entero-endocrine cell types also contain this amine. Moreover, it was presumed that chromogranin A, an acidic glycoprotein, is involved in storage mechanisms for biogenic amines in endocrine cells. Using immunohistochemical techniques, we now exemplarily investigated cholecystokinin (CCK-) and secretin (S-) cells of five adult mammalian species for their content of serotonin and of chromogranin A. In all mammalian species, CCK-cells were devoid of serotonin but contained chromogranin A immunoreactivity of varying densities. In contrast, S-cells of all mammals were immunoreactive for serotonin; however, immunoreactivities for this biogenic monoamine were heterogeneous and varied from dense to faint or lacking immunostainings. Likewise, immunoreactivities for chromogranin A in S-cells showed inter-species and inter-cellular heterogeneities. S-cells containing serotonin were simultaneously immunoreactive for chromogranin A and the density of immunoreactivities for both were correlated in given S-cells. Based on mutual relationships of chromogranin A and serotonin immunoreactivities, we assume that chromogranin A is virtually a prerequisite for the S-cells' content of serotonin and that this protein participates in storage mechanisms for biogenic amines in endocrine cells.S-cells have now to be added to the family of amine-storing endocrine cells. Basically, serotonin-storing endocrine cells in the digestive tract cannot be simply regarded as enterochromaffin (EC-) cells any longer; the current nomenclature and classification of entero-endocrine cells should be reviewed in this respect.This work was supported by grants of the Deutsche Forschungs-gemeinschaft (EN 65/15-2)     

Effect of serotonin-acting agents on the serotonin content of immune cells. A peculiar observation

The effect of the tryptophan hydroxylase inhibitor, PCPA methylester, the serotonin reuptake inhibitor fluoxetine and MAO-A inhibitor clorgyline on the serotonin content of rat immune cells was studied, using labelled antibodies and flow cytometry. Each molecule significantly increased in males the serotonin concentration of peritoneal lymphocytes and the monocyte-macrophage-granulocyte group (mo-gran), however the agents were ineffective towards mast cells. In females fluoxetine and clorgyline increased the serotonin concentration in peritoneal lymphocytes and mo-gran. Fluoxetine also increased the serotonin level in mast cells. Thymus was absolutely resistant to the drugs in both genders. The results call attention (1) to the reverse effect of serotonin-acting agents on immune cells, (2) to the influence of the milieu where the cell is located and (3) the effect of gender.     

In vitro effect of biogenic amines on the hormone content of immune cells of the peritoneal fluid and thymus. Is there a hormonal network inside the immune system?

Immune cells contain different hormones and hormone-like molecules, such as insulin, endorphin, triiodothyronine (T3) histamine, serotonin. In earlier in vitro experiments insulin down-regulated histamine, serotonin and T3 content of thymus cells. Now we studied the effect of biogenic amines on the endorphin, T3, serotonin and histamine content of rat peritoneal and thymic cells. Cells were obtained from male rats of 100g body weight. 100 ng/ml serotonin or 300 ng/ml histamine was added for 30 min. After that the cells were prepared for flow cytometric analysis with antibodies to endorphin, T3, histamine and serotonin as primary antibodies and anti-rabbit IgG as secondary antibody. Finishing the measurements the cells were also studied by confocal microscopy. T3 concentration (binding of anti-T3 antibody) increased in peritoneal mast cells after serotonin treatment and in the monocyte-macrophage-granulocyte group after histamine treatment. Thymocytes' T3 content radically decreased after both treatments. Serotonin and histamine treatment also radically reduced the amine content of each other. Endorphin level was resistant to hormonal treatments. The results call attention to a possible hormonal network inside the immune system in which hormones produced by the immune cells themselves can influence each other.     

Localization of serotonin and dopamine in the brown adipose tissue of the rat and their variations during cold exposure

The variations of several biogenic amines in brown adipose tissue (BAT) during cold exposure were studied and their localization investigated with histological methods. The study of serotonin and its metabolite 5-HIAA suggests that BAT serotonin is mobilized during acute and chronic cold exposure. This amine was found to be principally stored, together with histamine, in mast cells. The mast cell number in BAT was doubled during cold adaptation, as was the histamine content of the tissue. Using radio-enzymatic assay and high pressure liquid chromatography, only small amounts of dopamine were found in BAT. Since no specific dopamine-storing structure was detected (for example SIF cells), this low amount of dopamine is probably the precursor pool for noradrenaline synthesis and is most likely stored in the noradrenergic innervation of the tissue. BAT is known to be sensitive to both exogenous serotonin and exogenous dopamine; according to our results serotonin could play a role in BAT regulation while the role of dopamine remains hypothetical.     

Biogenic amines modulate pulse rate in the dorsal blood vessel of Lumbriculus variegatus

The biogenic amines are widespread regulators of physiological processes, and play an important role in regulating heart rate in diverse organisms. Here, we present the first pharmacological evidence for a role of the biogenic amines in the regulation of dorsal blood vessel pulse rate in an aquatic oligochaete, Lumbriculus variegatus (Müller, 1774). Bath application of octopamine to intact worms resulted in an acceleration of pulse rate, but not when co-applied with the adenylyl cyclase inhibitor MDL-12,330a. The phosphodiesterase inhibitor theophylline mimicked the effects of OA, but the polar adenosine receptor antagonist 8(p-sulphophenyl)theophylline was significantly less potent than theophylline. Pharmacologically blocking synaptic reuptake of the biogenic amines using the selective 5-HT reuptake blocker fluoxetine or various tricyclic antidepressants also accelerated heart rate. Depletion of the biogenic amines by treatment with the monoamine vesicular transporter blocker reserpine dramatically depressed pulse rate. Pulse rate was partially restored in amine-depleted worms after treatment with octopamine or dopamine, but fully restored following treatment with serotonin. This effect of 5-HT was weakly mimicked by 5-methoxytryptamine, but not by α-methylserotonin; it was completely blocked by clozapine and partially blocked by cyproheptadine. Because they are known to orchestrate a variety of adaptive behaviors in invertebrates, the biogenic amines may coordinate blood flow with behavioral state in L. variegatus.     

Protein phosphorylation during mast cell secretion in radioprophylaxis

A study was made of the role of protein phosphorylation of mast cells and their cytoskeleton upon secretion induced by biogenic amines (histamine and serotonin) and bradykinin, a possible mediator of the effect of MEA, a sulfur-containing radioprotector. The data obtained indicate that the incorporation of phosphate in some proteins of mast cells is an important stage in the process of exocytosis during radioprophylaxis. Cytoskeletal proteins were shown to be involved in mast cell secretion.     

EDAC fixation increases the demonstrability of biogenic amines in the unicellular Tetrahymena: a flow cytometric and confocal microscopic comparative analysis

Earlier experiments demonstrated the presence of hormones of the higher ranked animals in Tetrahymena. In the present experiments two fixatives, paraformaldehyde, which is commonly used and a carbodiimide, EDAC that was recommended by Panula et al. for the immunocytochemistry of histamine, are compared in Tetrahymena for the demonstration of histamine and serotonin by using flow cytometry and confocal microscopy. Both hormone levels were significantly higher after EDAC fixation; serotonin almost doubled and histamine was more than fivefold. The confocal microscopic pictures were clearer and the hormones' localization was easier. The results support earlier observations on the presence of these hormones in Tetrahymena and points to the advantage of EDAC fixation for demonstrating these hormones immunocytochemically.     

Gender differences in the histamine and serotonin content of blood,peritoneal and thymic cells: a comparison with mast cells

The serotonin and histamine content of mast cells and white blood cells in adult male and female rats was compared, using a flow cytometric immunological method. Serotonin was significantly higher in female peritoneal mast cells, peritoneal monocyte-ganulocyte-macrophage cells, blood lymphocytes and blood thymocytes. Histamine was significantly higher in female peritoneal monocyte-granulocyte-macrophage cells, and blood lymphocytes, monocytes and granulocytes, but was significantly less in thymocytes. Peritoneal lymphocytes and the monocyte-granulocyte-macrophage group contained significantly more histamine than mast cells. These experiments call attention to gender differences in the levels of biogenic amines in cells participating in defence reactions, and to the possible non-unique role of mast cells in serotonin and histamine supply.     

Mast cells of the lungs in experimental hyperergic pleurisy

Reaction of mast cells, the content of free and cell histamine and serotonin in the lung tissue at early stage of inflammation were studied on the model of hyperergic pleurisy in albino rats. Intrapleural antigen injection to sensitized rats was followed by progressive degranulation of mast cells of pleural and subpleural lung tissue with release of histamine and serotonin. The maximal increase in the content of free amines was found after 15 min. The level of free amines did not differ significantly from the initial one by the first hour. The early activation of amines synthesis and their storage recovery were observed as well as reactions of the late phase of immunological activation in the mast cells as a leukocytic tissue infiltration by subsequently polymorphonuclear leukocytes and macrophages.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Differential staining of catecholamines, 5-hydroxytryptamine and related compounds in aldehyde-fixed tissues

Summary Factors influencing the fixation of biogenic amines by solutions of glutaraldehyde, formaldehyde, acrolein and glyoxal as well as the influences of these aldehydes on the histochemical reactivity of biogenic amines, have been extensively investigated. Selective stainings of 5-hydroxytryptamine, noradrenaline, dopamine and related substances in many aldehyde-fixed tissues have been achieved by systematically applying some phenol methods such as argentaffin, ferric-ferricyanide, diazonium and thioindoxyl reactions, and some indole methods such as xanthydrol, dimethylaminobenzaldehyde, vanilline, salicilaldehyde and benzaldehyde tests.The list of the structures which react like 5-hydroxytryptamine in in vitro tests includes enterochromaffin cells, carcinoid tumours, rodent mast cells, thyroid C cells, a kind of cell found in the human carotid body and chemodectomas, and some cells secreting protein hormones. NA cells of the adrenal medulla, another kind of cell in the carotid body and chemodectomas, as well as ruminants' mast cells, reacted like catecholamines in in vitro tests.Supported by grant No. 115.1139.04715 from the Italian Consiglio Nazionale delle Ricerche.     

Kinetics of NAD reduction in the nucleus and the cytoplasm

Summary The kinetics of the nuclear and cytoplasmic fluorescence response to glycolytic substrate were studied in ascites cells in culture (EL2 cells) and radiation giants (EL2G) maintained under a variety of conditions, using a beam-splitter supplemented microfluorimeter which allows fluorescence recording simultaneously with microelectrophoretic addition of substrate. A sequence of fluorescence pulses which resemble closely the curve of formation and disappearance of the enzyme-substrate complex were obtained upon repetitive additions of substrate. The pulses were analyzed in terms of peak fluorescence response (PFR), duration of steady state, halftime of fluorescence rise and decay (tin1/2off), number of consecutive cycles elicited, etc. There is a considerable parallelism in the kinetics of the nuclear and cytoplasmic fluorescence after addition of glycolytic substrate, over the whole time course of consecutive pulses. However in untreated, Amytal- or Rotenone-perfused cells the peak magnitudes of the cytoplasmic fluorescence are significantly lower and the cytoplasmic pulses are damped earlier than the nuclear upon repeated additions of substrate. In Amytal-grown EL2 cells there is a drop of PFR and a prolongation of t _1/2off in the nucleus and cytoplasm, which persists when the cells are transferred to an Amytal-free medium. However, if the cells are maintained for longer periods in Amytal, the nuclear fluorescence tends to return to control level, while the cytoplasmic pulses remain small and are easily damped. In T₃- or Amytal + T₃ -grown EL2 cells the cytoplasmic fluorescence instead of dropping like in the controls, follows the nuclear level over the whole time course of repeated pulses, and can even exceed the nuclear. Comparable phenomena are observed in T₃ -and Insulin-grown radiation giants When the amount of substrate is varied, starting from levels which can barely elicit a response the magnitude of the fluorescence response (integrated fluorescence pulse) in the cytoplasm and nucleus follows a sigmoid curve which can be interpreted as a function of allosteric enzymes.List of Abbreviations EL2 mouse Ehrlich ascites cells in tissue culture - EL2G giant tissue culture mouse Ehrlich ascites cells obtained by X-irradiation - EL2T giant tissue culture EL2 cells obtained by treatment with Trenimon (2.3.5-Tris aethyleniminobenzochinon-1.4) - G6P glucose-6-phosphate - FDP fructose-1.6-diphosphate - 6 PG 6-phospho-gluconate - UDPG uridine-5-diphosphoglucose - AMP adenosine-5-monophosphate - ADP adenosine-5-diphosphate - G1P D-glucose-1-phosphate - PFR peak fluorescence response - t _1/2off halftime of fluorescence rise and decay - T3 triidothyronine     

Biogenic amines in the guinea pig endocrine pancreas.

Pancreata of guinea-pigs were investigated for the presence and cellular distribution of biogenic amines. Out of the established endocrine cell types only insulin (B-) cells contained immunoreactivity for serotonin and noradrenaline. However, the B-cells' content of both amines was quite variable. Serotonin was also confined to enterochromaffin (EC-) cells. No immunoreactivity for dopamine or histamine was present in any islet cell. Treatment of guinea-pigs with Ro-4-4602 led to a marked decrease of serotonin and noradrenaline in pancreatic endocrine cells. The present findings suggest that serotonin and noradrenaline are involved in the function of the endocrine pancreas, particularly of islet B-cells.     

The occurrence of the cell type containing a specific monoamine in the taste bud of the rabbit's foliate papila.

The fluorescence histochemical examination on biogenic amines of the rabbit's foliate papilla revealed that a specific monoamine exhibiting an yellow fluorescence was present in a certain cell type of taste buds. The fluorescence had the emission maximum at 520 mmu and faded rapidly under the influence of the UV-irradiation. The green fluorescence of adrenergic nerve had the emission maximum at 480 mmu and was fairly stable upon the UV-irradiation. The yellow fluorescence disappeared completely following reserpine treatment, while it was markedly enhanced by nialamide treatment. From the observations, it is suggested that certain taste bud cells of the foliate papilla contain a biogenic monoamine, probably 5-hydroxytryptamine (serotonin).     

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptus anser

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca2+ was studied and it was found that calcium cations at concentrations of 0.5-10 microM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 microM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca2+. Calmodulin antagonists--chlorpromazine, W-7, and W-5--caused a dose-dependent decrease of the enzyme activity stimulated by 5 microM Ca2+ with IC50 values of 35, 137, and 174 microM, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 microM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 microM calcium ions, but were markedly inhibited by 100 microM Ca2+. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca2+, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or were restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity does not D. depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

From the nucleus to the plasma membrane: translocation of the nuclear proteins histone H3 and lamin B1 in apoptotic microglia

Nuclear autoantibodies have been found in patients with autoimmune diseases. One possible source for nuclear antigens are apoptotic cells. However, the mechanism of how apoptotic cells make nuclear factors accessible to the immune system is still elusive. In the present study, we investigated the redistribution of nuclear components after UV irradiation in the microglial cell line BV-2 and in primary mouse microglia at the ultrastructural level. We used transmission electron microscopy-coupled electron energy loss spectroscopy (EELS) to measure phosphorus as an indicator for nucleic acids and immunogold labeling to detect histone H3 and lamin B1 in apoptotic cells. EELS revealed elevated concentrations of phosphorus in nuclear and cytoplasmic condensed chromatin compared to the remaining cytoplasm. Furthermore, immunolabeling of lamin B1 and histone H3 was detected in apoptotic microglia not only in the nucleus, but also in the cytoplasm, and even at the plasma membrane. Confocal images of apoptotic microglia, which were not previously permeabilized, showed patches of histone H3 and lamin B1 labeling at the cell surface. The pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) prevented the occurrence of cytoplasmic condensed chromatin in apoptotic microglia. Our findings indicate that nuclear components leak from the nucleus into the cytoplasm in apoptotic microglia. At least histone H3 and lamin B1 reach the cell surface, this may promote autoreactive processes.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.