Effect of femtomolar concentrations of hormones on insulin binding by Tetrahymena, as a function of time

The unicellular ciliate Tetrahymena, contains and binds hormones, characteristic of vertebrates. Earlier experiments demonstrated the effect of extremely low concentrations of hormones. In the present experiments, the effect of various hormones (endorphin, serotonin, histamine, insulin and epidermal growth factor [EGF]) in 10(-15) M, or oxytocin, gonadotropin at 0.001 IU concentrations) on the binding of FITC-insulin was studied by using flow cytometry and confocal microscopy, after 1, 5, 15, 30 and 60 min. Six of the seven hormones promptly decreased the cells' hormone binding capacity, the exception being EGF, and in four cases (endorphin, serotonin, insulin and oxytocin) the reduction was enormous. The decreased binding was durable. However, in the case of endorphin and oxytocin after 30 min, and in the case of serotonin after 60 min the binding returned to the control level. In the case of oxytocin after 60 min, binding significantly surpassed the control level. Histamine returned to the control level after 15 min, but after that the binding became even lower. EGF provoked special behaviour: it increased hormone binding after 30 and 60 min. The results call attention to the extreme sensitivity of Tetrahymena receptors to hormonal inductions and to its quick response ability.     

Comparison of the effect of hormones on the hormone synthesis of Tetrahymena in medium or salt solution

Tetrahymena pyriformis was maintained in TYM (tryptone‐yeast medium) as well as in Losina salt solution. One hour treatment of 10^?15 M histamine, serotonin or insulin was given before the histamine, serotonin, triiodothyronine and adrenocorticotropin contents of the cells were measured by flow cytometry after immunocytochemical staining. Maintenance in salt solution increased the hormone level in the cells, and use of the treatment hormone treatments further increased the endogenous hormone content relative to that in medium. The cells in salt mimic better the natural conditions, which means that the effects of hormones under more natural conditions are expressed to a greater extent than the exogenously given hormones in TYM typically used under laboratory conditions. Intercellular hormonal communication between the cells of a Tetrahymena population might assist in the survival of the individual cells.     

Hormonal interactions in Tetrahymena: effect of hormones on levels of epidermal growth factor (EGF)

Tetrahymena pyriformiswas treated with insulin, histamine or serotonin for 30 min and epidermal growth factor (EGF) level was studied inside the cells using specific antibodies and flow cytometry as well as confocal microscopy. The EGF concentration was highly significantly elevated after hormone treatment, regardless of the hormone used. EGF was localized mainly in the cortical region (mucocysts) and in vesicles and this localization did not differ in untreated and treated cells. The results call attention to the possibility of interactions between hormones at unicellular level and points to the presence of a hormonal system in Tetrahymena that includes receptors, hormones and signal transduction pathways as well as hormonal interactions. This could be the basis of further evolution to the hormonal system of multicellulars.     

Hormonal effects on Tetrahymena: change in case of combined treatment

In order to approach their natural conditions, populations of Tetrahymena were kept in Losina-Losinky's salt solution for 1 h, than in the tryptone+yeast medium. During this time they were treated with histamine, serotonin or insulin, or with the combinations of these hormones. Effect of the combined treatments on the production of serotonin (5HT), or adrenocorticotropic hormone (ACTH) or triiodothyronine (T?) by the cells was compared to the effect of single-hormone treatments. Significant differences were seen between the results obtained following the single or combined treatments. There was no summation of the effects, however an elevation or diminution of the hormone production was observed after the combined treatment, as compared with the untreated controls or with the use of one of the hormones in the samples. The experiments demonstrate that there is a hormonal regulation between the Tetrahymena cells and the hormones influence each other's effect.     

Effect of the inhibition of triiodothyronine (T3) production by thiamazole on the T3 and serotonin content of immune cells

Triiodothyronine (T3) and serotonin are present in the cells of immune system (blood, peritoneal and thymic lymphocytes, monocytes and granulocytes of the blood and peritoneal fluid, mast cells). In the present experiments the effect of thiamazole, an antithyroid drug was studied on the content of these two hormones in immune cells after one and two weeks of continuous treatment (by drinking water, containing 30 mg/100 ml thiamazole, ad libitum) in adult male rats, using flow cytometric and confocal microscopic analysis. In thymic lymphocytes both hormone contents significantly increased in both time points. A significant decrease of T3 was observed in peritoneal monocytes and granulocytes also in both time points, in peritoneal mast cells after one week and in blood lymphocytes after two weeks. Serotonin content in addition to the elevated thymic values (in both measurements) was significantly reduced in blood lymphocytes after two weeks. Confocal microscopy demonstrated heterogeneous cell populations with disparate hormone content and mostly diffuse localization The experiments call attention to the presence of T3 in the immune cells and to its variable concentration under the effect of a thyrostatic drug as well, as to the T3-serotonin relationship in the cells of the immune system.     

Steroid hormone (hydrocortisone, oestradiol and testosterone) uptake, storage or induced synthesis in tetrahymena

After cyclodextrin-coated 10(-6) m steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72 h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48 h, suggesting Tetrahymena has a functional cytochrome P(450)aromatase.     

Increased hormone levels in Tetrahymena after long-lasting starvation

Tetrahymena contains vertebrate hormone-like materials. The level of one of these, insulin increased during starvation in a previous experiment. We hypothesized that other hormones are also influenced by starvation. To prove the hypothesis Tetrahymena pyriformis cultures were (1) starved for 24h; (2) starved for 24h and re-fed for 30min or (3) starved for 30min. Amount and localization of vertebrate-like hormones, produced by Tetrahymena, beta-endorphin, adrenocorticotropin (ACTH), serotonin, histamine, insulin and triiodothyronine (T(3)) were studied by immunocytochemical methods using flow cytometry and confocal microscopy. Long starvation elevated with 50% the hormone levels, while short starvation moderately elevated only the serotonin level in the cells. After short re-feeding endorphin and histamine returned to the basal level, ACTH and serotonin approached the basal level, however, remained significantly higher, while insulin and T(3) stood at the starvation level. The results show that such a stress as long starvation provokes the enhanced production of hormones which likely needed for tolerating the life-threatening effect of stress.     

Effect of concanavalin A (Con-A) on the hormone production of the unicellular Tetrahymena and the immune cells of the rat. A comparative study

Tetrahymena populations were treated with 10(-15) g ml(-1) or 10(-6) g ml(-1) concanavalin-A (Con-A) in tryptone-yeast medium for 1 h. Rat peritoneal immune cells (mast cells, lymphocytes, monocyte-granulocyte group) were also treated with 10(-6) g ml(-1) Con-A, for 1 h. The cells' hormone (ACTH, histamine, serotonin, endorphin, triiodothyronine (T(3))) content was measured by using immunocytochemistry and flow cytometry. The extremely low dose of Con-A universally and significantly elevated the hormone contents, while the result of higher dose was uncertain. In the immune cells, Con-A significantly decreased the ACTH level in each cell type and histamine level in mast cells. The results demonstrate the very high sensitivity of Tetrahymena receptors for a non-hormone (lectin) molecule, which can bind to the insulin receptors and mimics the effect of insulin. The results also show that Tetrahymena receptors are more sensitive to lower concentrations of molecules than to higher ones. The universal hormone-production stimulating effect of Con-A-which is observed in Tetrahymena-is specified in rat.     

Chemical reception mechanisms at a low level of phylogeny. Influence of polypeptide hormones and non-hormone polypeptides on the growth of Tetrahymena

The polypeptide hormones insulin, glucagon, thyrotropin (TSH), pregnant mare serum gonadotropin (PMSG) and adrenocorticotropin (ACTH) stimulated the growth of the Tetrahymena, and the non-hormone polypeptides (bovine serum albumin (BSA), protamine) had a similar effect. Re-exposure after 24 h accounted for a greater growth stimulation than pre-exposure alone in cultures treated with TSH and PMSG, and re-exposure after 7 days had such effect in all polypeptide-treated cultures. It follows that the non-hormone polypeptides had a similar imprinting potential to the polypeptide hormone. The non-hormone polypeptides were also able to cross-imprint for one another, i.e. pre-exposure to one enhanced the binding capacity of the cells for the other on re-exposure, and vice versa. A single treatment with a polypeptide hormone or a non-hormone polypeptide did in itself stimulate the growth of the Tetrahymena for as long as 1 week.     

Hormone receptor studies on frog macrophage cells by means of histamine, serotonin and indoleacetic acid.

The phagocytotic activity of frog macrophage cells could be increased by 50% with histamine and by 24% with serotonin. These hormones have a similar effect at the various stages of phylogenetic development, to judge from the respective responses of the unicellular Tetrahymena which showed roughly the same percentual increases of phagocytosis as frog macrophages at roughly the same hormone concentrations. It is concluded that cytoplasmic membrane receptor patterns for a given function do not change in the course of phylogenetic development and the receptors have a capacity for selection, preferring histamine to serotonin, and the latter to the chemically closely related plant hormone indoleacetic acid.     

Taurine depletion increases phosphorylation of a specific protein in the rat retina

Summary Partial depletion of the taurine content in the rat retina was accomplished for up to 22 weeks by introduction of 1.5% guanidinoethanesulfonate (GES) in the drinking water. Taurine levels decreased by 50% after 1 week of GES treatment and by 80% at 16 weeks. Replacement of GES by taurine to the GES-treated rats from week 16 to 22 returned their taurine content to the control value. Whereas addition of taurine (1.5%) to the drinking water of control rats from week 16 to 22 elevated the retinal taurine content to 118% of the control value, the administration of untreated water to GES-treated animals for the 16 to 22 week time period increased the retinal taurine content to only 76% of the control value.The amplitude of the electroretinogram (ERG) b-wave was decreased by 60% after GES-treatment for 16 weeks and maintained this reduced level for up to 22 weeks. Administration of taurine in the drinking water from week 16 to 22 returned the b-wave amplitude to a range not statistically different from the control values whereas the administration of untreated water produced less improvement.After 6 weeks of GES treatment when the retinal taurine content was reduced by 70% and the amplitude of the b-wave was reduced by 50% (extrapolated from Figure 1), phosphorylation of a specific protein with an approximate molecular weight of 20K was increased by 94%. The increased phosphorylation of the ~20K protein observed after GES treatment was reversed when the animals were treated with taurine (1 1/2%) in the drinking water for an additional 6 weeks. There was no change in the phosphorylation of the ~20K protein when animals were treated with taurine for 6 weeks. The data obtained support the theory that taurine may have a regulatory effect on retinal protein phosphorylation.     

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.     

Is there a hormonal network in Tetrahymena? A systematic investigation of hormonal effects on the hormone content

The effect of six hormones (histamine, serotonin, insulin, epidermal growth factor (EGF), oxytocin and gonadotropin) was studied on the hormone (histamine, serotonin, adrenocorticotropic hormone [ACTH], endorphin and triiodothyronine [T(3)]) content of Tetrahymena. The hormones were given in 10(-9) or 10(-12) M concentrations or as 0.1 and 0.001 I.U. ml(-1) (in the case of oxytocin and gonadotropin) for 1 h. The hormones in picomolar concentration, i.e. at levels which can be present also in natural conditions, influence the amount of other hormones inside the cell. Their effect is not a general one: it is individual, the level of one of the hormones was elevated, while that of the others diminished under the effect of the same hormonal stimulus. Insulin was the only hormone, which influenced the concentration of other hormones in one direction, elevating them. This effect could have a role in the life-saving property of this hormone in Tetrahymena, but the hormones were not studied from this point of view. Usually there is no difference between the effect of the two concentrations used, but there are situations when the effect of the two concentrations is opposite. This means that there is a possible concentration dependence and this could influence differently the cells which are far from or near to the secretor cell. Considering earlier observations, the duration of the treatment can also influence the result. The results give new data to the hormonal regulation at unicellular level (which can be the base of regulation at higher evolutionary levels) and point to the possibility of a hormonal network.     

Prolonged impact of pubertal serotonin treatment (hormonal imprinting) on the later serotonin content of white blood cells

The first encounter between the developing receptor and its target hormone establishes the hormonal imprinting which is needed for the normal function of the cell. In the presence of foreign-however able to bind-molecules, faulty imprinting develops with lifelong consequences. Hormonal imprinting influences not only the receptors, but also the later hormone production of cells. The critical time of hormonal imprinting is the perinatal period, however it can be executed sometimes (in continuously differentiating cells) also at puberty. As in earlier experiments single neonatal serotonin treatment caused a life-long alteration of white blood serotonin content in female rats, the early (10-19 day) and late (8 weeks) effect of single pubertal serotonin treatment was studied presently, by using flow cytometry. In contrast to the earlier (neonatal) results, pubertal treatment caused a radical reduction of serotonin content in male's lymphocytes, monocytes, granulocytes and mast cells, independent on the time of study. The effect in females was rather increasing, however uncertain. The experiments call attention to the possible different effects of neonatal and pubertal hormonal imprinting and to the imprintability of blood cells in adolescence.     

Studies on doses of methimazole (MMI) and its administration regimen on broiler metabolism

We designed three experiments to determine both the optimal dose of and time on experiment for methimazole (MMI; 1-methyl-2-mercaptimidazole). Our goals were to determine if chicken growth was related to thyroid hormone levels and if intermediary metabolism changed along with changes in thyroid hormone levels. Initiating MMI at one week of age decreased (P<0.01) plasma thyroid levels and growth in four-week old birds. In contrast, initiating MMI at two and three weeks of age decreased (P<0.05) hormone levels without affecting growth as severely. Although initiating MMI at two weeks of age depressed (P<0.05) plasma thyroid hormones at four weeks, there was little change in vitro lipogenesis at four weeks. Again, initiating MMI at one week of age decreased body weight, plasma thyroid hormones and in vitro lipogenesis at four weeks of age. In addition, this treatment also decreased (P<0.05) malic enzyme activity at this same age period. The second experiment showed that MMI, initiated at 14 days, had no significant effect on 28-day body weight and again decreased both plasma T(3) and T(4) but T(3) replacement increased plasma T(3) in both 14-28-day treatment groups. All body weights were similar at 30 days, however. Lastly, diets containing graded levels of MMI decreased thyroid hormones and body weight (0>0.25>0.5>1 g MMI/kg). In contrast, only the two higher levels (0.5 and 1 g MMI/kg) decreased in vitro lipogenesis. Growth depression, caused by MMI feeding, can occur without changes in lipid metabolism. The length of MMI administration may be as important as dose level in obtaining effects (growth, thyroid hormone depression and inhibition of lipogenesis).     

Acute and delayed effect of (-) deprenyl and (-) 1-phenyl-2-propylaminopentane (PPAP) on the serotonin content of peritoneal cells (white blood cells and mast cells)

Acute and delayed (hormonal imprinting) effect of (-) deprenyl and its derivative without MAO-B inhibitory activity (-) PPAP, were studied on cells of the peritoneal fluid (lymphocytes, monocytes, granulocytes and mast cells) by flow cytometric and confocal microscopic analysis. Thirty minutes after treatment of 6-week-old female animals, deprenyl was ineffective while PPAP significantly increased the serotonin level of these cells. Three weeks after treatment at weaning, deprenyl drastically decreased the serotonin level of each cell type, while PPAP moderately but significantly increased the serotonin level of monocytes, granulocytes and mast cells. This means that the two related molecules have different effects on the immune cells, which seem to be independent of MAO-B inhibition. The experiments emphasize the necessity of studying the prolonged effects of biologically active molecules, even if they are without acute effects. As serotonin is a modulator of the immune system, the influence on immune cells of the molecules studied can contribute to their enhancing effect.     

Effect of stress and stress hormones on the hormone (insulin) binding of Tetrahymena

The unicellular Tetrahymena pyriformis GL produce, store and secrete vertebrate‐like hormones. In earlier experiments the effect of different stressors on the hormone levels of Tetrahymena was studied and an elevation of these was found. In the present experiments the hormone binding was investigated, using flow cytometric method. FITC‐insulin binding was elevated after concentrated (5, 10, or 20 mg ml^?1) NaCl or 0.01%, 0.1%, or 0.05% formaldehyde treatment, or after thermal stress (37°C). Serotonin given together with NaCl increased and together with formaldehyde decreased the binding. Histamine always decreased the binding and insulin was indifferent. Four hours after osmotic stress, hormone binding significantly decreased and this was not influenced by hormones. However, 4 h after formaldehyde stress the binding elevated and this was further increased by repeated hormone treatments. The results show that the stress in Tetrahymena provokes an activation of its hormonal system (hormone production and binding), which is differently influenced by exogeneously given hormones. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of hormonal imprinting on hormone level. Permanent receptor damaging effect of pituitary hormones administered to newly-hatched cockerels

The overlapping effect of TSH and FSH on the gonad and on the thyroid gland can be demonstrated in cockerels even at the age of five weeks. These hormones influence the secretion of testosterone in a similar way and to a similar extent, while on the thyroxine level the influence of the specific hormone is more pronounced. Neonatal FSH and TSH treatment considerably decreased the basal testosterone level measured at the age of five weeks. Neonatal FSH treatment increased the basal T4 level while TSH treatment decreased it. The effect of TSH treatment administered at the age of five weeks in increasing the testosterone level was weakened after neonatal pretreatment with any iodine hormone. The effect of TSH treatment could only be inhibited by neonatal FSH pretreatment. Neonatal pretreatment with any of the trophormones caused a diminution of the T4 level augmenting of FSH and TSH administered at the age of five weeks.     

Investigations on the triiodothyronine (T3)-specificity of thyrotropic (TSH) and gonadotropic (HCG) hormone in the unicellular Tetrahymena

In a previous experiment thyrotropin (TSH) increased the triiodothyronine (T3) production of Tetrahymena and chorionic gonadotropin (HCG) moderately overlapped the effect. At present the production of three amino acid type (histamine, serotonin, epinephrine) and one peptide (endorphin) hormones were studied under the effect of TSH or HCG, in tryptone-yeast (TY) or salt (Losina-Losinsky) medium. The duration of the effect was 10 min. TSH significantly (with almost 20%) decreased epinephrine production in TY medium and HCG similarly decreased epinephrine and increased histamine level. In salt solution TSH as well as HCG decreased the level of serotonin. The results show that at this low level of phylogeny TSH effect is not completely thyroxine-specific, however it is not general. HCG overlaps TSH effect on epinephrine and serotonin production, however its effect is broader. The experiments also demonstrate that the effect of pituitary trop-hormones can be bidirectional in Tetrahymena, as histamine level was increased and epinephrine level was decreased by HCG, in the same cells.     

The effect of corticosteroids upon the number and organ distribution of "background" immunoglobulin-secreting cells in mice

The influence of the synthetic corticosteroid dexamethasone sodium phosphate (DEXA) upon the immunoglobulin (Ig)-secreting cells was studied in not intentionally immunized BALB/c mice. This was done for IgM-, IgG-, and IgA-secreting cells in spleen, mesenteric lymph nodes (MLN), and bone marrow (BM). A single injection of DEXA (16 to 144 mg/kg body wt) markedly reduced the number of Ig-secreting cells in spleen and MLN within 1 day, but hardly affected their number in the BM. The decrease was immediately followed by a recovery and, at the highest doses and especially in MLN, by an overshoot. Two weeks after the initial decrease a second decrease was found. When mice were subjected to daily treatment with DEXA during 1 week, initially a recovery pattern was found in spleen and MLN similar to that found after a single injection of a high dose. In this case, however, the effects were less dose dependent, and the overshoot reaction was followed by a period of subnormal numbers of Ig-secreting cells which lasted at least 1 week. This late effect of DEXA not only occurred in spleen and MLN, but also in the BM. The most prominent effect of daily treatment with DEXA was the long-lasting decrease of the number of IgG-secreting cells starting 1 week after withdrawal of treatment. This decrease was associated with a severely decreased serum IgG level.