How does the unicellular <Emphasis Type="Italic">Tetrahymena</Emphasis> utilise the hormones that it produces? Paying a visit to the realm of atto-and zeptomolar concentrations

Changes in the hormone content of Tetrahymena pyriformis GL were investigated during histamine, serotonin or insulin treatment at concentrations of 10⁻⁶M to 10⁻²¹M for 30 min. The immunologically demonstrable hormone content was studied by using specific antibodies, flow cytometry and confocal microscopy. Histamine at the higher ranges elevated the serotonin content of Tetrahymena, whereas serotonin at the lower ranges (down to 10⁻²¹M) decreased its histamine levels. Insulin did not affect its serotonin content, whereas serotonin increased its insulin content at each concentration studied (down to 10⁻²¹M). Insulin between 10⁻⁶M and 10⁻²¹M increased the hista-mine levels of Tetrahymena, although histamine influenced its insulin level only at 10⁻⁶M. Our results call attention to the presence of hormonal interactions even at “low” levels of phylogeny and to the extreme sensitivity of the hormone receptors of Tetrahymena. These data might explain (1) the requirement of Tetrahymena for (vertebrate) hormone production and hormone receptors and (2) the way that it uses these hormones under natural conditions.This work was supported by the National Research Fund (OTKA-T-037303), Hungary.     

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.     

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.     

Effects of extremely low concentrations of hormones on the insulin binding of Tetrahymena

FITC-insulin binding to previously hormone-treated Tetrahymena was studied by flow cytometry and confocal microscopy. Hormones produced by Tetrahymena were chosen for study and the hormone concentrations were administered between 10(-6) and 10(-21)M for 30 min. Endorphin, serotonin and insulin significantly reduced the hormone binding however histamine did not influence it at all. Endorphin, serotonin and insulin were significantly effective down to 10(-18)M and the effect of insulin and endorphin suggest a similar mechanism. The results call attention to the efficacy of very low hormone concentrations, which can influence the hormone content (earlier experiments) and receptor binding capacity (present study) of a unicellular organism. This seems to be very important, as in wild (natural) conditions the dilution of signaling materials secreted by a water-living protozoan is very high. In addition, the results point to the selectivity of response, as not all of the hormones that deeply influence other physiological indices (e.g. histamine) have an effect on insulin content or insulin receptors.     

How applicable is the general adaptation syndrome to the unicellular Tetrahymena?

Hormone receptors, hormones and signal transduction pathways characteristic of higher vertebrates can be observed also in the unicellular Tetrahymena. Previous work showed that stress conditions (starvation, high temperature, high salt concentration, formaldehyde or alcohol treatment) elevated the intracellular level of four hormones (ACTH, endorphin, serotonin and T₃). Here, the effect of other stressors (CuSO4 poisoning, tryptophan hydroxylase inhibitor parachlorphenylalanine (PCPA) treatment) on the same and other hormones (epinephrine, insulin, histamine) was studied, using immunocytochemistry and flow cytometric analysis. It was found, that each effect increased the intracellular hormone contents, but some hormones (histamine, T₃) were less reactive. Insulin—which is a life‐saving factor for Tetrahymena—itself provoked elevation of hormone amounts in association with a stressor, further increased the level of hormones. It was concluded that the ancestor of Selye's General Adaptation Syndrome (GAS) can be found already at unicellular level, and this possibly has a life saving function. Copyright © 2008 John Wiley & Sons, Ltd.     

The hormonal system of the unicellular Tetrahymena: a review with evolutionary aspects

The unicellular ciliate, Tetrahymena has receptors for hormones of the higher ranked animals, these hormones (e.g. insulin, triiodothyronine, ACTH, histamine, etc.) are also produced by it and it has signal pathways and second messengers for signal transmission. These components are chemically and functionally very similar to that of mammalian ones. The exogenously given hormones regulate different functions, as movement, phagocytosis, chemotaxis, cell growth, secretion, excretion and the cells' own hormone production. The receptors are extremely sensitive, certain hormones are sensed (and response is provoked) at 10-21 M concentration, which makes likely that the function could work by the effect of hormones produced by the Tetrahymena itself. The signal reception is selective, it can differentiate between closely related hormones. The review is listing the hormones produced by the Tetrahymena, the receptors which can receive signals and the signal pathways and second messengers as well, as the known effects of mammalian hormones to the life functions of Tetrahymena. The possible and justified role of hormonal system in the Tetrahymena as a single cell and inside the Tetrahymena population, as a community is discussed. The unicellular hormonal system and mammalian endocrine system are compared and evolutionary conclusions are drawn.     

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.     

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.     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

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.     

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.     

TIME- AND CONCENTRATION-DEPENDENCE OF THE GROWTH-PROMOTING ACTIVITY OF INSULIN AND HISTAMINE IN TETRAHYMENA. APPLICATION OF THE MTT-METHOD FOR THE DETERMINATION OF CELL PROLIFERATION IN A PROTOZOAN MODEL

The unicellular ciliateTetrahymena pyriformiswas treated with different concentrations of insulin or histamine and at different time points the cell density was measured, using a tetrazolium-based semiautomated colorimetric assay (MTT). The assay was suitable to determine the rate of cell proliferation of Tetrahymena. Insulin in each concentration significantly elevated the cell count up to 3h. After that, it was neutral or its effect was insignificant. Histamine at 10^?5m concentration diminished cell count at 3, 5, 7 and 24h. At 10^?6m concentration there was no difference and at 10^?7m concentration it enhanced cell division up to 5h, after that there being no difference. The two hormones have cell division promoting activity for cells of higher animals and the experiments demonstrate this effect already at a unicellular level.     

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.     

Ibuprofen and warfarin modulate allosterically ferrous human serum heme-albumin nitrosylation

Ferrous human serum heme–albumin (HSA–heme–Fe(II)) displays globin-like properties. Here, the effect of ibuprofen and warfarin on kinetics of HSA–heme–Fe(II) nitrosylation is reported. Values of the second-order rate constant for HSA–heme–Fe(II) nitrosylation (k_on) decrease from 6.3 × 10⁶ M⁻¹ s⁻¹ in the absence of drugs, to 4.1 × 10⁵ M⁻¹ s⁻¹ and 4.8 × 10⁵ M⁻¹ s⁻¹, in the presence of saturating amounts of ibuprofen and warfarin, respectively, at pH 7.0 and 20.0 °C. From the dependence of k_on on the drug concentration, values of the dissociation equilibrium constant for ibuprofen and warfarin binding to HSA–heme–Fe(II) (i.e., K = 3.2 × 10⁻³ M and 2.6 × 10⁻⁴ M, respectively) were determined. The observed allosteric effects could indeed reflect ibuprofen and warfarin binding to the regulatory fatty acid binding site FA2, which brings about an alteration of heme coordination, slowing down HSA–heme–Fe(II) nitrosylation. Present data highlight the allosteric modulation of HSA–heme–Fe(II) reactivity by heterotropic effectors.     

Tryptamine as an endogenous modulator of neuronal sensitivity to serotonin

Research was performed on sensory ganglia isolated from adult rats using intracellular techniques for recording membrane potential and by measuring resistance at the membrane of individual units. It was found that tryptamine at high concentrations manifests serotoninlike activity, but, at concentrations not affecting potential and resistance at the neuronal membrane, either reinforces (at a concentration of 10^–7 M) or attenuates (at 10^–5 M) serotonin (5-HT) effects mediated by type 1A (but not type 2) 5-HT receptors. 5-HT-modulated effects were produced by tryptamine-induced changes in 5-HT sensitivity at the neuronal membrane and remained unchanged by maximum level of this transmitter. Harmane acts similarly to tryptamine, although harmane derivatives (C-412 and C-506 respectively) produce either potentiation or inhibition of 5-HT_1A over the entire concentration range used (of 10^–7 M-10^–5 M). The allosteric nature of 5-HT-modulation by tryptamine and harmane is discussed.M. Gor'kii Medical Institute, Donetsk, Ukrainian SSR. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 352–357, May–June, 1989.     

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.     

Effects of L-alanine and L-alanine peptides on the chemotaxis of tetrahymena: Evolutionary conclusions

L-alanine and its peptides (L-Ala-2–6) do not attract or repulse Tetrahymena in a 10^–8M concentration. In 10^–10M concentration there is a consistent repellent effect. Twenty four hours after L-alanine or L-alanine-peptides' pretreatment (imprinting) the progeny generation of the cells react differently to the same materials. L-Alanine, L-alanine penta- and hexapeptide in both concentrations are chemoattractant, while L-alanine tetrapeptide is repellent. L-Alanine dipeptide is inert in 10^–10M and repellent at 10^–8M concentrations, while L-alanine tripeptide is strongly repellent at 10^–10M and attractant at 10^–8M concentrations. This means, that the first encounter (imprinting) with an exogeneous amino acid or peptide is decisive to the later reaction of the protozoan cell. The chain length is important in the imprinting, however the reaction is not consistent. The experiments call the attention to the significance of imprinting in the receptor and hormone evolution.     

Au-nanoclusters incorporated 3-amino-5-mercapto-1,2,4-triazole film modified electrode for the simultaneous determination of ascorbic acid, dopamine, uric acid and nitrite

A novel biosensor has been constructed by the electrodeposition of Au-nanoclusters (nano-Au) on poly(3-amino-5-mercapto-1,2,4-triazole) (p-TA) film modified glassy carbon electrode (GCE) and employed for the simultaneous determination of dopamine (DA), ascorbic acid (AA), uric acid (UA) and nitrite (NO₂⁻). NH₂ and SH groups exposed to the p-TA layer are helpful for the electrodeposition of nano-Au. The combination of nano-Au and p-TA endow the biosensor with large surface area, good biological compatibility, electricity and stability, high selectivity and sensitivity and flexible and controllable electrodeposition process. In the fourfold co-existence system, the linear calibration plots for AA, DA, UA and NO₂⁻ were obtained over the range of 2.1–50.1 μM, 0.6–340.0 μM, 1.6–110.0 μM and 15.9–277.0 μM with detection limits of 1.1 × 10⁻⁶ M, 5.0 × 10⁻⁸ M, 8.0 × 10⁻⁸ M and 8.9 × 10⁻⁷ M, respectively. In addition, the modified biosensor was applied to the determination of AA, DA, UA and NO₂⁻ in urine and serum samples by using standard adding method with satisfactory results.     

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 °C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 °C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 °C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 °C than at 22 °C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell⁻¹) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x^−0.858. Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29–46%), followed by SPX-D (21–28%), 13-desMeC (21–23%), and 13-desMeD (17–21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 × 10⁴ cells mL⁻¹. As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x^−0.8986. In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60–87%) and lower proportions of SPX-D (3–12%) and 13-desMeD (1.6–10%) as compared to batch culture.     

Endogenous ouabain-like factor (OLF) secretion is modulated by nicotinic mechanisms in rat adrenocortical cells

This study tested the hypothesis that rat adrenocortical secretion of endogenous ouabain-like factor (OLF) is regulated by nicotinic mechanisms. OLF secreted by dispersed cell suspensions of zona glomerulosa (ZG) and fasciculata/reticularis (ZFR) cells was found to co-elute with authentic ouabain by reverse phase HPLC; OLF concentrations in cell supernatants were measured by radioimmunoassay. Nicotine (10⁻⁶ − 10⁻³ M) stimulated significant OLF secretion in rat adrenocortical cells. Acetylcholine (10⁻⁷ − 10⁻⁴ M) and eserine (10⁻⁷ − 10⁻³ M) stimulated OLF secretion in ZG cells at lower concentrations and stimulated at higher concentrations. Acetylcholine had no effect on ZFR secretion of OLF, but eserine stimulated OLF secretion. ACTH (10⁻⁸ M) strongly potentiated the OLF stimulatory effect of nicotine in ZG cells; however significant interactions between nicotine and ACTH or angiotensin II on OLF secretion in ZFR cells were not apparent. The ganglionic blockers hexamethonium and mecamylamine further potentiated the effect of nicotine, implicating nicotinic acetylcholine receptors (nAChRs) in regulation of OLF secretion. The α7-receptor antagonist methyllycaconitine (MLA) dose-dependently inhibited the effect of nicotine in the ZG cells, and in ZFR cells MLA potentiated nicotine-induced OLF secretion. These data suggest that nicotinic regulation may underlie OLF secretion by rat adrenocortical cells, and strongly suggest presence of functional nicotinic acetylcholine receptors on these cells.