|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Comparison of the effect of hormones on the hormone synthesis of Tetrahymena in medium or salt solution
G Csaba*1, Eszter Lajkó† and Éva Pállinger†
*Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary, and †Research Group for Inflammation Biology and Immunogenomics of Hungarian Academy of Sciences, Budapest, Hungary
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.
Key words: hormonal effect, hormone production, medium, milieu, protozoan
Abbreviations: ACTH, anti-adrenocorticotropic hormone, TYM, tryptone-yeast medium
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Part of a series marking the 70th birthday of the Cell Biology International Editor-in-Chief Denys Wheatley.
The unicellular Tetrahymena react to hormones of higher vertebrates (Csaba and Lantos, 1973; 1975a; 1975b; Shemarova et al. 2002; 2004; Kőhidai et al., 2003; Csaba, 1980, 1984, 1985, 1993, 2000, 2008) having receptors for them (Zipser et al., 1988; O'Neill et al., 1988; Christopher and Sundermann, 1995; Leick et al., 2001; Christensen et al., 2003). These hormones are also present inside the Tetrahymena and produced by it (LeRoith et al., 1980; 1982; 1983; Hegyesi et al., 1998; Csaba and Kovács, 1999; Kőhidai et al., 2002; 2003; Csaba et al., 2004), and they are identical with that of the hormones of mammals (Csaba et al., 1999; Rodriguez et al., 2004). The hormones in the Tetrahymena could serve as a signal function in this low level of phylogeny and show some specificity, as insulin influences its sugar metabolism (Csaba and Lantos, 1975a), histamine and serotonin regulate its phagocytosis (Csaba and Lantos, 1973, 1975b) and thyroxin as well as its precursors stimulate its cell division (Csaba et al., 1979; Csaba and Németh, 1980; Csaba, 1980). The hormones are effective at a very low femtomolar or zeptomolar concentrations (Csaba et al., 2007a), as the Tetrahymena hormone receptors are very sensitive. This is very important from a functional point of view, as the grade of dilution is very high.
In natural conditions Tetrahymena is living in a watery condition; however, in laboratory conditions it is maintained in a medium which contains nourishments needed for life. Nevertheless, it can be maintained—for a short period—in a physiological salt solution which mimics the natural conditions, when the cell is starving. In the present experiments, the effect of three hormones (produced also by the Tetrahymena; Pállinger et al., 2005; Csaba et al., 2009) on Tetrahymena (Quinones-Maldonado and Renaud, 1987; Castrodad et al., 1988; Csaba 2000, 2008) is studied (and compared) on the production of four hormones under nourishment-rich and starved conditions.
2. Materials and methods
2.1. Cells and treatments
Tetrahymena pyriformis GL strain was used in the logarithmic phase of growth. The cells were cultured at 28°C in tryptone medium (Sigma, St. Louis, MO, U.S.A.) containing 0.1% yeast extract, for 24 h. The density of Tetrahymena cultures studied was 104 cell/ml. For the experiments, cells were placed in fresh medium or in Losina salt solution (10 ml of 1% NaCl, 10 ml of 0.1% MgCl, 10 ml of 0.1% CaCl, 10 ml of 0.1% KCl, +950 ml of boiled distilled water + 10 ml of 0.2% NaHCO3; Losina-Losinsky, 1931), which is physiological for Tetrahymena. There were samples without treatment and other samples treated with serotonin (5-HT; Sigma), histamine (Sigma) or insulin (Actrapid, Novo, Denmark) at 10−15 M concentration, for 1 h.
2.2. Flow cytometric analysis
After the experimental procedure, cells were fixed with 4% paraformaldehyde solution (dissolved in pH 7.2 PBS) for 5 min, and then washed twice in wash buffer (0.1% BSA; 20 mM Tris/HCl; 0.9% NaCl; 0.05% Nonidet NP-40; pH 8.2).
To block nonspecific binding of antibodies, the cells were treated with blocking buffer (1% BSA in PBS) for 30 min at room temperature. Aliquots from cell suspensions (50 μl) were transferred into tubes, and 50 μl of primary antibody (anti-serotonin, anti-histamine, anti-triiodothyronine (T3) or ACTH [anti-adrenocorticotropic hormone], purchased from Sigma; diluted 1:200 in antibody buffer [1% BSA in wash buffer]) were added for 30 min at room temperature. Negative controls were carried out with 50 μl of PBS containing 10 mg/ml BSA instead of primary antibody. After washing four times with wash buffer to remove excess primary antibody, the cells were incubated with FITC-labelled secondary antibody (anti-rabbit IgG; Sigma; dilution 1:50 with antibody buffer) for 30 min at room temperature.
To control the specificity, the autofluorescence of the cells and unspecificity of the secondary antibodies were checked. This meant that the fluorescence of cells treated only with the secondary antibody (without the specific first antibody) was also measured in the antibody-treated series. The measurement was done in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, U.S.A.), using 5000 cells for each measurement. In the cell populations, the hormone content (concentration) was compared. Dead and living cells were separated during analysis. As dead cells lose their membrane integrity, forward and side scatter (FS/SS dot plot) was used to exclude debris and dead cells. For measurement and analysis, the CellQuest Pro software was used. The numerical comparison of detected values was done by comparison of percentage changes in geometric mean channel values (Geo-mean) relative to the appropriate control groups by using the Origin software and Student's t test. The experiments were done twice with similar results, and Tables 1–3 present the results of one of these experiments.
3. Results and discussion
In the experiments the effect of 1 h treatment with three hormones (histamine, serotonin or insulin) on the hormone production of Tetrahymena was compared. The cells were maintained in the medium or Losina salt solution. The medium was rich in nourishment and growth factors, which are needed for cell proliferation (Wheatley et al., 1993), while the salt solution does not contain nutrients and growth factors at all, except those produced by Tetrahymena itself (Christensen et al., 1995). This means that the cells starved in the latter condition. Tables 1–3 show the results.
Histamine in medium significantly increased the cells' serotonin content (Table 1). In the case of histamine treatment in salt, there is a significant increase related to the treatment in medium and to the control in salt. T3 was significantly elevated after histamine treatment, and this high value remained under the salty condition. However, histamine treatment in salt resulted in a higher level of T3 related to the control, in medium. Histamine caused a marked increase in ACTH content both in the medium and salt solution. However, salt alone provoked an important increase, and histamine level in salt produced the highest value.
Table 1 Effect of 1 h histamine (10−15 M) treatment on the hormone concentration of cells
In salt solution, the histamine content was significantly higher than in the medium, and the stimulating effect of serotonin was observed only in salt (Table 2). Similarly, the T3 production stimulating effect of serotonin was observed only in salt or in a more expressed form in the case of serotonin in salt. The very strong stimulating effect to ACTH formation was observed only in the salt solution.
Table 2 Effect of 1 h serotonin (10−15 M) treatment on the hormone concentration of cells
Under the effect of insulin treatment (Table 3) in the medium, there was no difference when compared with the control. However, in the case of T3, insulin reduced the hormone content in salt, while in the case of histamine and ACTH it was neutral. Here, the control in salt always contained significantly more hormones than in the medium.
Table 3 Effect of 1 h insulin (10−15 M) treatment on the hormone concentration of cells
The study of hormone concentrations in the cells is an index, with which the effect of an exogenously given hormone can be measured. At the same time, the change in hormone level inside the cells points to the effect of Tetrahymena-produced hormones. The femtomolar concentration of hormones with which the treatment was done could be equivalent to the hormone concentration around the Tetrahymena produced by itself (Csaba et al., 2006, 2007a). The conditions in which the Tetrahymena is found in the salt, better approach the natural conditions than the experiments done in the medium. The results of the experiments show that the milieu in which the treatment takes place influences the effect of hormones.
In earlier experiments, it was demonstrated that stress increases the hormone content of Tetrahymena (Csaba and Pállinger, 2008), and starvation is also a form of stress (Csaba et al., 2007b, 2007c, 2008). One hour (the period of starvation) is not a long time, however, considering the generation time of Tetrahymena (Phillips and Lloyd, 1978; Scherbaum, 1957), it is almost the half of it, although it varies individually inside a population (Prescott, 1959). Although the effect of hormones (on other hormones) was more expressed in salt milieu, it is known that this milieu is more ‘natural’ than the nourishment-rich tryptone medium. However, in natural watery conditions there are available sources of food for Tetrahymena. This means that although results in salt milieu approaches that of the natural one, it is not identical with it.
If Tetrahymena senses signals in such a high dilution of hormones, it means that there is a possibility of information transmission by cells over relatively long distances. This could help the survival of the population. The intercellular communication between the cells of Tetrahymena can be accepted as an ancient form of communication, pointing to signalling systems in mammals (Christensen et al., 1998; Rasmussen et al., 1996; Wheatley et al., 1994; Csaba, 2008).
Serotonin and histamine are biogenic amines, small amino-acid-type hormones. Insulin is a polypeptide. Treatment with the two biogenic amines in salt solution increased the hormone content of the cells, while insulin in salt was neutral (histamine, ACTH) or decreased (T3) the hormone level. This could explain why insulin in the medium was indifferent to the intracellular hormone levels, whereas in earlier experiments (Csaba et al., 2007c), at higher concentrations insulin increases it. This was also observed in previous experiments (Csaba and Pállinger, 2008). It must be considered that insulin, as a life-saving factor in Tetrahymena (Christensen, 1993; Wheatley and Christensen, 1999), is inclined to produce a biphasic reaction (Christensen et al., 1996a; 1996b), and this could be manifested in different effects of different concentrations. However, this phenomenon warrants further investigation.
Gyorgy Csaba planned the experiments, evaluated the results and wrote the paper. Eszter Lajkó maintained and treated the Tetrahymena, and was involved in control of the draft. Éva Pállinger was involved in measurement of the samples and mathematical evaluation of them, and control of the draft.
The authors thank Ms. Katy Kallay and Ms. Angela Kozák for their expert technical assistance.
Christensen, ST, Wheatley, DN, Rasmussen, MI and Rasmussen, L (1995) Mechanisms controlling death, survival and proliferation in a model unicellular eukaryote Tetrahymena thermophila. Cell Death Differ 2, 301-308
Christensen, ST, Kemp, K, Quie, H and Rasmussen, L (1996a) Cell death, survival and proliferation in Tetrahymena thermophila. Effects of insulin, sodium nitroprusside, 8-bromo-cyclic GMP, NG-methyl-l-arginine and methylene blue. Cell Biol Int 20, 653-6
Christensen, ST, Quie, H, Kemp, K and Rasmussen, L (1996b) Insulin produces a biphasic response in Tetrahymena thermophila by stimulating cell survival and activating proliferation in two separate concentration intervals. Cell Biol Int 20, 437-44
Christopher, GK and Sundermann, CH (1995) Isolation and partial characterisation of the insulin binding site of Tetrahymena pyriformis. Biochem Biophys Res Commun 212, 515-23
Csaba, G, Gaál, A, Kovács, P, Simon, G and Kőhidai, L (1999) Prolonged elevation of insulin content in the unicellular Tetrahymena after insulin treatment: induction of insulin production or storage. Cell Biochem Funct 17, 165-73
Csaba, G and Kovács, P (1999) Localization of beta-endorphin in Tetrahymena by confocal microscopy. Induction of the prolonged production of the hormone by hormonal imprinting. Cell Biol Int ;23, 695-702
Csaba, G, Kovács, P and Pállinger, É (2004) Presence and localization of epidermal growth factor (EGF)- and EGF-receptor-like immunoreactivity in Tetrahymena. Cell Biol Int 28, 491-6
Csaba, G, Kovács, P, Tóthfalusi, L and Pállinger, É (2006) Effects of extremely low concentrations of hormones on the insulin binding of Tetrahymena. Cell Biol Int 30, 957-62
Csaba, G, Kovács, P and Pállinger, É (2007a) How does the unicellular Tetrahymena utilise the hormones that it produces? Paying a visit to the realm of atto- and zeptomolar concentrations. Cell Tissue Res 327, 199-203
Csaba, G, Kovács, P and Pállinger, É (2008) Comparison of the insulin binding, uptake and endogenous insulin content in long- and short-term starvation in Tetrahymena. Cell Biochem Funct 26, 64-9
Csaba, G and Lantos, T (1973) Effect of hormones on Protozoa. Studies on the phagocytotic effect of histamine, 5-hydroxytryptamine and indoleacetic acid in Tetrahymena pyriformis. Cytobiologie 7, 361-5
Csaba, G and Németh, G (1980) Effect of hormones and their precursors on protozoa—the selective responsiveness of Tetrahymena. Comp Biochem Physiol 65B, 387-90
Csaba, G and Pállinger, É (2008) Is there a hormonal network in Tetrahymena? A systematic investigation of hormonal effects on the hormone content. Cell Biochem Funct 26, 303-8
Hegyesi, H, Kovács, P, Falus, A and Csaba, G (1998) Presence and localization of histidine decarboxylase enzyme (HDC) and histamine in Tetrahymena pyriformis. Cell Biol Int 22, 493-7
Kőhidai, L, Vakkuri, O, Keresztesi, M, Leppaluoto, J and Csaba, G (2002) Melatonin in the unicellular Tetrahymena pyriformis: effects of different lighting conditions. Cell Biochem Funct 20, 269-72
Kőhidai, L, Vakkuri, O, Keresztesi, M, Leppaluoto, J and Csaba, G (2003) Induction of melatonin synthesis in Tetrahymena by hormonal imprinting—a unicellular “factory” of the indoleamine. Cell Mol Biol 49, 521-4
LeRoith, D, Schiloach, J, Roth, J and Lesniak, MA (1980) Evolutionary origins of vertebrate hormones: substances similar to mammalian insulin are native to unicellular eukaryotes. Proc Natl Acad Sci USA 77, 6184-6
LeRoith, D, Schiloach, J, Berelowitz, M, Frohman, LA and Roth, J (1983) Are messenger molecules in microbes the ancestors of the vertebrate hormones and tissue factors? Fed Proc 42, 2602-7
LeRoith, D, Liotta, AS, Roth, J, Shiloach, J, Lewis, ME, Pert, CB and Krieger, DT (1882) Corticotropin and beta-endorphin-like materials are native to unicellular organisms. Proc Natl Acad Sci USA 79, 2086-90
Losina-Losinsky, LK and Zur Ernährungsphysiologie der Infusorien, (1931) Unterschungen über die Nahrungsmittel und Vermehrung bei Paramecium caudatum. Arch Protistenk 74, 18-20
O'Neill, JB, Pert, CB, Ruff, MR, Smith, CC, Higgins, WJ and Zipser, B (1988) Identification and characterization of the opiate receptor in the ciliated protozoan, Tetrahymena. Brain Res 450, 303-15
Phillips, CA and Lloyd, D (1978) Coninuous-flow size selection of Tetrahymena pyriformis ST: changes in volume, DNA, RNA and protein during synchronous growth. J Gen Microbiol 105, 95-103
Rasmussen, L, Christensen, ST, Shousbue, P and Wheatley, DN (1996) Cell survival and multiplication, the overriding need for signals:from unicellular to multicellular systems. FEMS Microbiol Lett 137, 123-8
Shemarova, IV, Selivanova, GV and Vlasova, TD (2002) The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates. Tsitologia 44, 1097-103
Shemarova, IV, Selivanova, GV and Vlasova, TD (2004) A cytophotometric study of the influence exerted by epidermal growth factor on RNA and protein synthesis in the ciliata Tetrahymena pyriformis. Tsitologia 46, 993-5
Wheatley, DN, Rasmussen, L and Tiedtke, A (1994) Tetrahymena: a model for growth, cell cycle and nutritional studies, with biotechnological potential. Bioessays 16, 367-72
Wheatley, DN, Christensen, ST, Schousbue, P and Rasmussen, L (1993) Signalling in cell growth and death: adequate nutrition alone may not be sufficient for ciliates. Cell Biol Int 17, 817-23
Zipser, B, Ruff, MR, O'Neill, JB, Smith, CC, Higgins, WJ and Pert, CB (1988) The opiate receptor: a single 110 kDa recognition molecule appears to be conserved in Tetrahymena, leech and rat. Brain Res 463, 296-304
Received 12 July 2010; accepted 17 August 2010
Published online 28 September 2010, doi:10.1042/CBI20100513
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