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.     

Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation

Summary Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1–0.01 m) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized ³H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.     

Maturation of adult rat peritoneal and mesenteric mast cells

Summary Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.     

Electron microscopic study of the regeneration in vitro of rat peritoneal mast cells after histamine secretion

Summary Regeneration of rat mast cells was studied by TEM from 10 s to 48 h after secretion of histamine induced by compound 48/80. During the first 2 h, small intracellular cavities, formed during compound exocytosis and containing non-membrane-bound remnants of the granules, tended to coalesce, and after 2 h of incubation regeneration started. After 6 h, all the cavities had fused into one large central cavity which contained the remnants of the granules and remained open to the exterior during the entire period. The plasma membrane microfolds which disappeared just after secretion were reformed during regeneration. They were apparently involved in endocytotic-like activity and coated vesicles also appeared beneath the plasmalemma (membrane recycling?). The fate of the granule remnants in the cavity is unknown, as regeneration was not completed after 48 h which is the longest survival time obtained so far in ultrastructural studies of mast cell regeneration in vitro.     

Induction of histamine release from rat mast cells by bradykinin analogues

Independently of their agonistic or antagonistic activity on different isolated tissue preparations, the kinin analogues investigated induce histamine release on rat peritoneal mast cells. The effectivity of most compounds is 10 to 100 times higher than that of bradykinin. Beside the positively charged amino acids, the elongation at the N-terminus with hydrophobic amino acids and the replacement of amino acids in the bradykinin sequence (especially at position 7) with aromatic residues is important for a high histamine-releasing activity.     

The effect of calmodulin on histamine release in the rat peritoneal mast cell

To investigate the role of the Ca²⁺-binding protein calmodulin on histamine release in the rat peritoneal mast cell, we exposed cells to exogenous calmodulin in the presence of a variety of histamine secretagogues. Histamine release stimulated by compound , polymyxin B and ionophore A23187 was inhibited while concanavalin A-stimulated release was not affected. Calmodulin in the presence of the secretagogues did not affect cell viability and calmodulin alone had no effect on histamine release. No direct interaction between calmodulin and the secretagogues was observed. Exogenous calmodulin does not appear to be incorporated into the cell. The inhibition of histamine release by calmodulin can be explained as a labile interaction between the protein and the cell that requires externally-bound Ca²⁺. These experiments demonstrate the use of exogenous calmodulin as a probe in the study of the mechanism of histamine release.     

Further studies on the structural requirements for mast cell degranulating (MCD) peptide-mediated histamine release

Mast cell degranulating (MCD) peptide was modified in its two disulfide bridges and in the two arginine residues in order to measure the ability of these analogs to induce histamine release from mast cells in vitro. Analogs prepared were [Ala^3,15]MCD, [Ala^5,19]MCD, [Orn¹⁶]MCD, and [Orn^7,16]MCD. Their histamine-releasing activity was determined spectrofluorometrically with peritoneal mast cells. The monocyclic analogs in which the cysteine residues were replaced pairwise with alanine residues showed three-to ten-fold diminished histamine-releasing activity respectively, compared with the parent MCD peptide. Substantial increases in activity were observed where arginine residues were replaced by ornithines. The ornithine-mono substituted analog showed an almost six-fold increase and the ornithine-doubly substituted analog three-fold increase in histamine-releasing activity compared with the parent MCD peptide. The structural changes associated with these activities were followed by circular dichroism (CD) spectroscopy. Changes in the shape and ellipticity of the CD spectra reflected a role for the disulfide bonds and the two arginine residues in the overall conformation and biological activity of the molecule.     

Presence of hormones (triiodothyronine, serotonin and histamine) in the immune cells of newborn rats

Five hormones in lymphatic cells from the spleen and thymus of newborn rats were measured using flow cytometry and confocal microscopic immunofluorescence. Immunoreactive histamine, serotonin and triiodothyronine (T(3)) were present in the cells. However, growth hormone and insulin were not observed, except for low levels of insulin in a few spleen cells. Serotonin content was modest compared to the histamine and T(3) levels. The localization of each hormone in the cells was different. The importance of these observations in the light of functional data is discussed.     

Surface morphology of rat peritoneal mast cells during in vitro regeneration after histamine secretion

Summary Cell-surface morphology of regenerating mast cells was followed over a period of 48 h after histamine release. Control cells (not stimulated to secrete) were characterized by anastomosing folds of membrane of equal depth and width. During exocytosis these folds disappeared and were replaced by deep cup-shaped flaps of membrane evident in cells incubated for 10 min. During the first hours of regeneration these flaps fused mutually or with the plasma membrane. This activity suggests membrane retrieval, maybe specifically recycling the granule-type patches of membrane. Membrane-fusion activity was observed to some degree also after extended incubation. After 48 h of incubation the regeneration process was still not completed, as indicated by the fact that holes leading to intracellular cavities could still be found.     

Effects of egg removal on maternal aggression,biogenic amines,and stress indicators in ovigerous lobsters (Homarus americanus)

In many disparate taxa, including crayfish, a freshwater decapod crustacean, the presence of one’s offspring has been shown to be an important variable in the level of maternal aggression. Ovigerous American lobsters (Homarus americanus) show a territorial advantage against nonmaternal females, even though no posthatch care is provided. The eggs are attached to the pleopods (swimmerets) throughout embryogenesis. We evaluated the effect of stripping ovigerous H. americanus females of their eggs on maternal territoriality. In one treatment group, maternal females were stripped of all eggs. Twelve days later, including being individually isolated for an additional 48?h in a test tank, each resident was serially intruded upon by 4 non-maternal conspecific females. An identically treated control group of ovigerous female residents was left intact prior to the serial intrusions. Various biochemical parameters of the stripped and unstripped animals were measured before and after the experimental treatment (or control). The behavioral modulators measured were the biogenic amines, octopamine and serotonin, and the stress indicators were a heat-shock protein (HSP70), methyl farnesoate, and crustacean hyperglycemic hormone. The intact ovigerous residents showed a significant territorial advantage over the intruders, whereas the contest outcomes of stripped residents were reduced to chance. The presence of eggs in maternal H. americanus is therefore necessary for the maintenance of the shelter-related territorial advantage. However, no significant differences in any of the biochemical parameters were observed between or within treatment conditions. It appears that these amines are not prominently involved in the mechanism of maternal aggression, and that these changes in territorial defense are not simply due to changes in general stress as a result of stripping the females of their eggs.     

Effects of egg removal on maternal aggression, biogenic amines, and stress indicators in ovigerous lobsters (Homarus americanus)

In many disparate taxa, including crayfish, a freshwater decapod crustacean, the presence of one's offspring has been shown to be an important variable in the level of maternal aggression. Ovigerous American lobsters (Homarus americanus) show a territorial advantage against nonmaternal females, even though no posthatch care is provided. The eggs are attached to the pleopods (swimmerets) throughout embryogenesis. We evaluated the effect of stripping ovigerous H. americanus females of their eggs on maternal territoriality. In one treatment group, maternal females were stripped of all eggs. Twelve days later, including being individually isolated for an additional 48 h in a test tank, each resident was serially intruded upon by 4 non-maternal conspecific females. An identically treated control group of ovigerous female residents was left intact prior to the serial intrusions. Various biochemical parameters of the stripped and unstripped animals were measured before and after the experimental treatment (or control). The behavioral modulators measured were the biogenic amines, octopamine and serotonin, and the stress indicators were a heat-shock protein (HSP70), methyl farnesoate, and crustacean hyperglycemic hormone. The intact ovigerous residents showed a significant territorial advantage over the intruders, whereas the contest outcomes of stripped residents were reduced to chance. The presence of eggs in maternal H. americanus is therefore necessary for the maintenance of the shelter-related territorial advantage. However, no significant differences in any of the biochemical parameters were observed between or within treatment conditions. It appears that these amines are not prominently involved in the mechanism of maternal aggression, and that these changes in territorial defense are not simply due to changes in general stress as a result of stripping the females of their eggs.     

Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo

Summary The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulumlysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors.     

Role of striatal serotonin2A and serotonin2C receptor subtypes in the control of in vivo dopamine outflow in the rat striatum

This study investigated, using in vivo microdialysis in the striatum of freely moving rats, the role of striatal serotonin2A (5-HT2A) and 5-HT2C receptor subtypes in the modulation of dopamine (DA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) outflow, both in basal conditions and under activation induced by subcutaneous administration of 0.01 mg/kg haloperidol. The different 5-HT2 agents used were applied intrastriatally at a 1 microM concentration through the microdialysis probe. Basal DA efflux was enhanced (27%) by the 5-HT2A/2B/2C agonist 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI) and reduced (-30%) by the 5-HT2B/2C antagonist SB 206553. It was unaffected by infusion of the 5-HT2A antagonist SR 46349B. The effect of DOI was abolished by SB 206553 but not modified by SR 46349B. Haloperidol-stimulated DA efflux (65-70%) was reduced by both SR 46349B (-32%) and the 5-HT2A/2B/2C antagonist ritanserin (-30%) but not affected by SB 206553. Conversely, the effect of haloperidol was potentiated (22%) when DOI was coperfused with SB 206553. Also, haloperidol-stimulated DOPAC outflow (40-45%) was reduced (-20%) by SR 46349B and potentiated (25%) by the combination of SB 206553 with DOI. These results indicate that striatal 5-HT2A receptors, probably through activation of DA synthesis, positively modulate DA outflow only under activated conditions. In contrast, striatal 5-HT2C receptors exert a facilitatory control on basal DA efflux, which appears to be both tonic and phasic.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

Influence of in vitro and in vivo insulin treatment on the hormone (histamine, serotonin, endorphin and triiodothyronine) content of thymus and spleen cells

Thymic and spleen cells were treated in vitro or in vivo with insulin. The in vitro treatments were done with 10(-6), 10(-9), 10(-12) and 10(-15) M concentrations for 30 min and after that histamine, serotonin, endorphin and triiodothyronine (T3) content of the cells were detected by using antibodies to the hormones and flow cytometry as well as confocal microscopy. For in vivo treatment 1 IU/kg insulin was given for adult rats and 1 h after that the target hormone contents were determined by the same manner. Histamine and T3 content radically decreased in the thymus after in vitro treatment independent on the insulin concentrations administered. In vivo treatment halved histamine and T3 content. Serotonin content also decreased after in vitro treatment with the two higher concentrations, however the in vivo treatment did not cause a change. Histamine content was elevated after in vitro treatment in the spleen, independent on the insulin concentration. Endorphin level was not influenced at all. The experiments demonstrate that insulin is a factor which regulates the content (production, storage, secretion?) of some immunologically important molecules of the immune cells. Since each hormone molecule studied has important immunomodulatory role, the experiment points to the indirect immunomodulatory role of insulin.     

5-HT(1B) receptor-mediated regulation of serotonin clearance in rat hippocampus in vivo

The 5-hydroxytryptamine (5-HT; serotonin) transporter (5-HTT) is important in terminating serotonergic neurotransmission and is a primary target for many psychotherapeutic drugs. Study of the regulation of 5-HTT activity is therefore important in understanding the control of serotonergic neurotransmission. Using high-speed chronoamperometry, we have demonstrated that local application of 5-HT(1B) antagonists into the CA3 region of the hippocampus prolongs the clearance of 5-HT from extracellular fluid (ECF). In the present study, we demonstrate that the 5-HT(1B) antagonist cyanopindolol does not produce this effect by increasing release of endogenous 5-HT or by directly binding to the 5-HTT. Dose-response studies showed that the potency of cyanopindolol to inhibit clearance of 5-HT was equivalent to that of the selective 5-HT reuptake inhibitor fluvoxamine. Local application of the 5-HT(1A) antagonist WAY 100635 did not alter 5-HT clearance, suggesting that the effect of cyanopindolol to prolong clearance is not via a mechanism involving 5-HT(1A) receptors. Finally, the effect of low doses of cyanopindolol and fluvoxamine to inhibit clearance of 5-HT from ECF was additive. These data are consistent with the hypothesis that activation of terminal 5-HT(1B) autoreceptors increases 5-HTT activity.     

The effect of 2450 MHz microwave radiation on histamine secretion by rat peritoneal mast cells

Isolated rat peritoneal mast cells actively secrete histamine in response to reaginic or chemical stimulation. Mast cells were irradiated in a waveguide microwave exposure chamber at 2450 MHz with power absorptions of 8.2 and 41.0 mW/g for periods up to 3 h. These levels of microwave absorption caused no change in the morphological characteristics or viability of the cells. Irradiated mast cells were stimulated with compound 48/80, a potent, noncytotoxic histamine releasing agent. The dose response curves showed that neither prior nor simultaneous irradiation of mast cells at 37°C affected 48/80-induced secretion. However, microwave power absorptions of 41.0 mW/g inhibited secretion at 44.0°C. Precise measurements of the effect of heat on secretion indicated that this level of inhibition could have been produced by a radiation induced increase in cell temperature between 0.4 and 0.9°C above ambient levels. Alternatively, the heat stress produced at 44°C may have sensitized the cells to the electromagnetic effects of the microwave radiation. Rat peritoneal mast cells can therefore be useful as a model for the study of functioning secretory cells during microwave irradiation and can also be used to monitor the synergistic effects of cell heating during in vitro exposure.     

Activation of extracellular signal-regulated kinase (ERK) and Akt by human serotonin 5-HT(1B) receptors in transfected BE(2)-C neuroblastoma cells is inhibited by RGS4

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G proteins. One of the best-studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all G(i) and G(q) alpha subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)-C human neuroblastoma cells expressing human 5-HT(1B) receptors were used to demonstrate that RGS4 can inhibit the coupling of 5-HT(1B) receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal-regulated kinase. This activation was attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. As RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways.