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Increased hormone levels in Tetrahymena after long-lasting starvation
Cell Biology International 31 (2007) 924e928 www.elsevier.com/locate/cellbi
Increased hormone levels in Tetrahymena after long-lasting starvation G. Csaba*, P. Kova´cs, E´va Pa´llinger Department of Genetics, Cell and Immunobiology, Semmelweis University and Immunogenomical Research Group, Hungarian Academy of Sciences, Budapest, Hungary Received 24 October 2006; revised 29 January 2007; accepted 19 February 2007
Abstract 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 24 h; (2) starved for 24 h and re-fed for 30 min or (3) starved for 30 min. Amount and localization of vertebrate-like hormones, produced by Tetrahymena, b-endorphin, adrenocorticotropin (ACTH), serotonin, histamine, insulin and triiodothyronine (T3) 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 T3 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 lifethreatening effect of stress. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Tetrahymena; Stress; Starvation; ACTH; Serotonin; Histamine; T3
1. Introduction The ability of the protozoan Tetrahymena to bind histamine, serotonin, insulin, ACTH, endorphin, epidermal growth factor, melatonin and opioids (Csaba, 1980, 1985, 1993; Csaba and Lantos, 1973, 1975a,b; Csaba and Kova´cs, 1991, 1999; Csaba et al., 2004; KThidai et al., 1995, 2003; Shemarova et al., 2004; Zipser et al., 1988; O’Neill et al., 1988) lead to identification of the presence of vertebrate hormone-like molecules (insulin, ACTH, endorphin, histamine, serotonin and melatonin) (Csaba, 1980, 1984, 1985, 2000; LeRoith et al., 1980, 1982, 1983; KThidai et al., 2002). Recently, insulin receptors of Tetrahymena was characterized and found to be similar to its mammalian counterpart (Christopher and Sundermann, 1995; Leick et al., 2001; Christensen et al., 2003). However, functional significance of these hormones in the protozoa
* Corresponding author. Tel./fax: þ36 1 210 2950. E-mail address: [email protected] (G. Csaba).
is far from clear. It is noted that the recognition capabilities of Tetrahymena is so fine that very low concentrations of hormones are enough to activate hormone-dependent metabolic pathways and production of other hormones (Csaba et al., 2006, 2007a). Furthermore, prolonged deprivation of food significantly increased the insulin levels of the cells (Csaba et al., 2007b). We hypothesize that prolonged deprivation of food may influence production of other hormones. To test the hypothesis, we have monitored the endogeneous expression of b-endorphin, ACTH, serotonin, histamine, insulin and T3 by Tetrahymena pyriformis GL following prolonged (24 h) starvation with or without re-feeding. 2. Materials and methods 2.1. Cells and culturing T. pyriformis GL strain was used in the logarithmic phase of growth. The cells were cultured at 28 C in tryptone medium (Sigma, St. Louis, USA) containing 0.1% yeast extract, for 48 h. The density of Tetrahymena cultures studied was 104 cell/ml. Other cultures were cultivated for 24 h in the medium
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.02.007
G. Csaba et al. / Cell Biology International 31 (2007) 924e928 mentioned above and for further 24 h were kept in Losina-Losinsky salt solution (starved cells). Some cultures kept in medium or salt solution were fed (F) or starved (S) after the cultivation for 30 min. Considering the above mentioned facts, the experimental groups were as follows:
925
Table 1 Endogeneous b-endorphin after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
43.54 3.15 59.98 1.62 43.33 3.35 42.09 3.1
Significance p< to 1
1. 48 h culture in medium þ 30 min in fresh medium (F þ F þ F) 2. 24 h culture in medium þ 24 h in salt solution þ 30 min in fresh salt solution (F þ S þ S) 3. 24 h culture in medium þ 24 h in salt solution þ 30 min in fresh medium (F þ S þ F) 4. 48 h culture in medium þ 30 min in fresh salt solution (F þ F þ S)
0.001 n.s. n.s.
to 2
to 3
to 4
0.001
n.s. 0.001
n.s. 0.001 n.s.
0.001 0.001
n.s.
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
2.2. Flow cytometric analysis
Table 2 Endogeneous ACTH after long-time fasting or feeding in Tetrahymena
2.2.1. Preparation of cells for the measurement of endogeneous hormones Samples of cells were fixed with 4% paraformaldehyde solution (dissolved in pH 7.2 phosphate buffered saline [PBS]) for 5 min, and then washed twice in wash buffer (0.1% BSA; 20 mM TriseHCl; 0.9% NaCl; 0.05% Nonidet NP40; pH 8.2). To block nonspecific binding of antibodies the cells were treated with blocking buffer (1% bovine serum albumin [BSA] in PBS) for 30 min at room temperature. Aliquots from cell suspensions (50 ml) were transferred into tubes, and 50 ml primary antibody (anti b-endorphin [E1520], anti-ACTH [A1927], anti-serotonin [S5545], anti-histamine [H7403], anti-T3 [T2777; produced in rabbit] and monoclonal anti-insulin [I2018 produced in mouse], each purchased from Sigma (St. Louis, USA), diluted 1:200 in antibody buffer [1% BSA in wash buffer]) was added for 30 min at room temperature. Negative controls were carried out with 50 ml 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 antibody [F9887] except in case of insulin, where monoclonal anti-mouse IgG [F8771] (Sigma) was used, dilution 1: 50 with antibody buffer) for 30 min at room temperature. For controlling the specificity, autofluorescence of the cells and aspecificity of the secondary antibodies were detected. This latter means that the fluorescence of cells treated only with the secondary antibody (without the specific first antibody) was also measured in the antibody-treated series. This value was not subtracted from the value of 1st and 2nd antibody treated specimens; however, these latter values were 50e100% more in general, than the values of the only 2nd antibody treated ones.
No.
2.2.2. Measurement The measurement was done in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, USA), using 5000 cells for each measurement. In the cell populations the hormone content (concentration) had been compared. For the measurement and analysis CellQuest Pro program was used. The numerical comparison of detected values was done by the comparison of percentual changes of geometric mean channel values (Geo-mean, which represent relative fluorescent intensity and always those groups were compared which were measured together) to the appropriate control groups (line no. 1 in the Tables) and between the experimental groups by using Origin program and Student’s t-test. The experiments were done at least twice with similar results and the Tables demonstrate one of these experiments.
Group
Geo-mean
Significance p< to 1
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
51.07 3.28 84.35 4.76 70.91 7.82 54.21 1.6
0.001 0.001 n.s.
to 2
to 3
to 4
0.001
0.001 0.05
n.s. 0.001 0.01
0.05 0.001
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
Table 3 Endogeneous serotonin after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
35.56 1.29 59.19 0.82 44.73 1.88 38.98 2.4
Significance p< to 1 0.001 0.001 0.03
to 2
to 3
to 4
0.001
0.001 0.001
0.03 0.03 0.01
0.002 0.001
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
Table 4 Endogeneous histamine after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
19.95 1.03 26.53 1.38 20.71 0.55 21.24 0.76
Significance p< to 1 0.01 n.s. n.s.
to 2
to 3
to 4
0.01
n.s 0.01
n.s. 0.01 n.s.
0.01 0.01
n.s.
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
2.3. Confocal microscopic analysis After the flow cytometric analysis the cells were subjected to confocal microscopic analysis in a BioRad MRC 1024 confocal laser scanning microscope, equipped with kryptoneargon mixed gas-laser as a light source, at an excitation wavelength of 480 nm line.
3. Results The amount of six endogeneous hormones of the Tetrahymena was studied (1) after 24 h starvation (long starvation),
Table 5 Endogeneous insulin after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
Significance p< to 1
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
25.27 1.33 37.92 2.96 35.62 2.24 23.83 1.97
0.01 0.01 n.s.
to 2
to 3
to 4
0.01
0.01 n.s.
n.s. 0.01 0.01
n.s. 0.01
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
G. Csaba et al. / Cell Biology International 31 (2007) 924e928
926
Table 6 Endogeneous T3 after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
Significance p< to 1
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
44.65 1.48 66.69 2.52 63.53 5.06 46.39 3.94
4. Discussion
0.01 0.01 n.s.
to 2
to 3
to 4
0.01
0.01 n.s.
n.s. 0.01 0.01
n.s. 0.01
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s. : not significant.
(2) re-fed for 30 min after 24 h starvation and (3) starved for 30 min only (short starvation). Prolonged starvation significantly elevated the level of each hormone (Tables 1e6). The elevation was about 50% of the basal level and the p-value of significance was at least 0.01. Short starvation did not influence the hormone levels, except serotonin which significantly ( p < 0.03) increased (Table 3/4). However, the re-feeding of the cells (for 30 min) was not so uniform. Endorphin and histamine returned to the basal level (Tables 1/3 and 4/3) after refeeding, ACTH and serotonin became significantly less, than after short starvation without re-feeding, however, significantly more than the basal level (Tables 2/3 and 3/3). Insulin and T3 remained similar to the level after long starvation (Tables 5/3 and 6/3). The confocal microscopic studies supported the quantitative data. The hormones were not specifically localized, they were demonstrated spread in the cytoplasm, around the oral field and nucleus and under the plasma membrane, with two exceptions. These are ACTH, which after long starvation extremely enriches in the cortical zone and oral field (Fig. 1a, b) and serotonin which also shows an enrichment around the nucleus and oral field (Fig. 2a, b). The cells after long starvation are smaller than after complete feeding.
The unicellular Tetrahymena contains vertebrate-like hormones and receptors as well as a signal transduction system functioning by the use of cAMP, cGMP, Ca-calmodulin and inositol phosphates (Csaba, 1980, 1984, 1985, 2000). These data are internationally justified and accepted (O’Neill et al., 1988; LeRoith et al., 1980, 1983; Christopher and Sundermann, 1995; Leick et al., 2001; Christensen et al., 1998, 2003; Wheatley and Christensen, 1999; Shemarova et al., 2004), however, it is not clearly known, what is the functional role of this endocrine-like system in a unicellular organism. At this level there are three possibilities: (1) the cell uses the hormones for self-regulation (intracine or autocrine regulation), (2) there is an intercellular communication by them between the members of the Tetrahymena population and (3) the hormones are only side-products of the synthesis of other proteins, however, this not explains the presence of receptors and signal transduction pathways. It was cleared previously that extremely low concentrations of hormones are enough for provoking changes in the insulin content and binding of Tetrahymena (Csaba et al., 2006, 2007a), which points to the possibility of intercellular communication by these hormones. Now we wanted to know whether the worsening of life conditions by restricting the food supply can influence the unicellular’s hormone content in general, or not. Long starvation is partly a stress for the cells, partly a process, when the deprivation of food does not allow the replacement of amino acids and proteins which had been depleted during physiological processes. The molecules studied were amino acid or polypeptide type hormones. Considering that there is no replacement of proteins for 24 h e which is an extremely long period relative to the individual life of a Tetrahymena (Wheatley and Christensen, 1999) e the hormone level must have been lower after fasting. As all of the hormone levels were much higher than control, it can be supposed
Fig. 1. ACTH in fed (a) and long starved (b) Tetrahymena. After starvation there is a bright fluorescence in the cell body and under the surface. The cells are smaller than in the fed group. Magnification 2000.
G. Csaba et al. / Cell Biology International 31 (2007) 924e928
927
Fig. 2. Serotonin in fed (a) and long starved Tetrahymena. After starvation the fluorescence is stronger, especially around the nucleus and in the oral region. The cells are deformed and smaller than in the normally fed group. Magnification 2000.
that the presence of more hormones is required for the cells to tolerate the strong stress. This can reason the preparation of hormones from internally stored reserve materials. In our previous experiments (Csaba et al., 2007b) the elevation of insulin concentration was observed as a consequence of starvation. It was a possibility to believe that only insulin concentration is increasing, as insulin is known as a vital factor for maintaining Tetrahymena (Christensen et al., 1995, 1998; Wheatley et al., 1993, 1994; Wheatley and Christensen, 1999; Hagemeister et al., 1999; Shemarova et al., 2002). However e considering the present results e it is not exceptional: other hormones could participate in the defense during life-threatening situations. The results bring nearer to the understanding of the role of vertebrate-like hormones in Tetrahymena. However, a later duty to establish whether the accumulation of hormones is specific for starvation, or it is a universal reaction in different stress situations. Acknowledgements This work was supported by the National Research Fund (OTKA-T-037303). The authors thank Ms. Katy Kallay, Marianna Kincses and Angela Koza´k for their expert technical assistance. References Christensen ST, Guerra CF, Awan A, Wheatley DN, Satir P. Insulin receptorlike proteins in Tetrahymena thermophila ciliary membranes. Curr Biol 2003;13:R50e2. Christensen ST, Wheatley DN, Rasmussen MI, Rasmussen L. Mechanisms controlling death, survival and proliferation in a model unicellular eukaryote Tetrahymena thermophila. Cell Death Differ 1995;2:301e8. Christensen ST, Leick V, Wheatley DN. Signaling in unicellular eukaryotes. Internatl Rev Cytol 1998;177:181e253. Christopher GK, Sundermann CH. Isolation and partial characterisation of the insulin binding site of Tetrahymena pyriformis. Biochem Biophys Res Com 1995;212:515e23.
Csaba G, Lantos T. Effect of hormones on Protozoa. Studies on the phagocytotic effect of histamine, 5-hydroxytryptamine and indoleacetic acid in Tetrahymena pyriformis. Cytobiologie 1973;7:361e5. Csaba G, Lantos T. Effect of insulin on the glucose uptake of Protozoa. Experientia 1975a;31:1097e8. Csaba G, Lantos T. Effect of amino acid and polypeptide hormones on the phygocytosis of Tetrahymena pyriformis. Acta Protozool 1975b;37: 409e13. Csaba G, Kova´cs P. Localization of beta-endorphin in Tetrahymena by confocal microscopy. Induction of the prolonged production of the hormone by hormonal imprinting. Cell Biol Int 1999;23:695e702. Csaba G, Kova´cs P. EGF receptor induction and insulin-EGF overlap in Tetrahymena. Experientia 1991;47:718e21. Csaba G, Kova´cs P, Pa´llinger E´. Presence and localization of epidermal growth factor (EGF)- and EGF-receptor-like immunoreactivity in Tetrahymena. Cell Biol Int 2004;28:491e6. Csaba G. Presence in and effects of pineal indoleamines at very low level of phylogeny. Experientia 1993;49:627e34. Csaba G. The present state in the phylogeny and ontogeny of hormone receptors. Horm Metab Res 1984;16:329e35. Csaba G. Hormonal imprinting: its role during the evolution and development of hormones and receptors. Cell Biol Int 2000;24:407e14. Csaba G. Phylogeny and ontogeny of hormone receptors: the selection theory of receptor formation and hormonal imprinting. Biol Rev 1980;55:47e63. Csaba G. The unicellular Tetrahymena as a model cell for receptor research. Internatl Rev Cytol 1985;95:327e77. Csaba G, Kova´cs P, To´thfalusi L, Pa´llinger E´. Effects of extremely low concentrations of hormones on the insulin binding of Tetrahymena. Cell Biol Int 2006;30:957e62. Csaba G, Kova´cs P, Pa´llinger E´. How does the unicellular Tetrahymena utilise the hormones that it produces? Paying a visit to the realm of atto- and zeptomolat concentrations. Cell Tissue Res 2007a;327:199e203. Csaba G, Kova´cs P, Pa´llinger E´. Comparison of the insulin binding, uptake and endogeneous insulin content in long- and short-starved Tetrahymena. Cell Biochem Function 2007b, in press. Hagemeister JJ, Grave M, Kristiansen TB, Assaad F, Hellung-Larsen P. Interface mediated death of unconditioned Tetrahymena cells: effect of medium composition. J Eukaryot Microbiol 1999;46:6e11. KThidai L, Vakkuri O, Keresztesi M, Leppaluoto J, Csaba G. Induction of melatonin synthesis in Tetrahymena by hormonal imprinting e a unicellular ‘‘factory’’ of the indoleamine. Cell Mol Biol 2003;49:521e4. KThidai L, Lovas B, Csaba G. Effect of adrenocorticotropic hormone (ACTH) and insulin on the phagocytic capacity of Tetrahymena. Zool Sci 1995; 12:277e81.
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KThidai L, Vakkuri O, Keresztesi M, Leppaluoto J, Csaba G. Melatonin in the unicellular Tetrahymena pyriformis: effects of different lighting conditions. Cell Biochem Funct 2002;20:269e72. Leick V, Bog-Hansen TC, Juhl HA. Insulin/FGF-binding ciliary membrane glycoprotein from Tetrahymena. J Membr Biol 2001;181:47e53. LeRoith D, Schiloach J, Roth J, Lesniak MA. Evolutionary origins of vertebrate hormones: substances similar to mammalian insulin are native to unicellular eukaryotes. Proc Natl Acad Sci U S A 1980;77:6184e6. LeRoith D, Schiloach J, Berelowitz M, Frohman LA, Roth J. Are messenger molecules in microbes the ancestors of the vertebrate hormones and tissue factors? Fed Proc 1983;42:2602e7. LeRoith D, Liotta AS, Roth J, Shiloach J, Lewis ME, Pert CB, et al. Corticotropin and beta-endorphin-like materials are native to unicellular organisms. Proc Natl Acad Sci U S A 1982;79:2086e90. O’Neill JB, Pert CB, Ruff MR, Smith CC, Higgins WJ, Zipser B. Identification and characterization of the opiate receptor in the ciliated protozoan, Tetrahymena. Brain Res 1988;450:303e15.
Shemarova IV, Selivanova GV, Vlasova TD. The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates. Tsitologia 2002;44:1097e103. Shemarova IV, Selivanova GV, Vlasova TD. A cytophotometric study of the influence exerted by epidermal growth factor on RNA and protein synthesis in the ciliata Tetrahymena pyriformis. Tsitologia 2004;46:993e5. Wheatley DN, Christensen ST. Origins of signalling and memory: matters of life versus death. Acta Biol Hung 1999;50:441e61. Wheatley DN, Rasmussen L, Tiedtke A. Tetrahymena: a model for growth, cell cycle and nutritional studies, with biotechnological potential. Bioessays 1994;16:367e72. Wheatley DN, Christensen ST, Schousbue P, Rasmussen L. Signalling in cell growth and death: adequate nutrition alone may not be sufficient for ciliates. Cell Biol Int 1993;17:817e23. Zipser B, Ruff MR, O’Neill JB, Smith CC, Higgins WJ, Pert CB. The opiate receptor: a single 110 kDa recognition molecule appears to be conserved in Tetrahymena, leech and rat. Brain Res 1988;463:296e304.
Increased hormone levels in Tetrahymena after long-lasting starvation G. Csaba*, P. Kova´cs, E´va Pa´llinger Department of Genetics, Cell and Immunobiology, Semmelweis University and Immunogenomical Research Group, Hungarian Academy of Sciences, Budapest, Hungary Received 24 October 2006; revised 29 January 2007; accepted 19 February 2007
Abstract 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 24 h; (2) starved for 24 h and re-fed for 30 min or (3) starved for 30 min. Amount and localization of vertebrate-like hormones, produced by Tetrahymena, b-endorphin, adrenocorticotropin (ACTH), serotonin, histamine, insulin and triiodothyronine (T3) 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 T3 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 lifethreatening effect of stress. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Tetrahymena; Stress; Starvation; ACTH; Serotonin; Histamine; T3
1. Introduction The ability of the protozoan Tetrahymena to bind histamine, serotonin, insulin, ACTH, endorphin, epidermal growth factor, melatonin and opioids (Csaba, 1980, 1985, 1993; Csaba and Lantos, 1973, 1975a,b; Csaba and Kova´cs, 1991, 1999; Csaba et al., 2004; KThidai et al., 1995, 2003; Shemarova et al., 2004; Zipser et al., 1988; O’Neill et al., 1988) lead to identification of the presence of vertebrate hormone-like molecules (insulin, ACTH, endorphin, histamine, serotonin and melatonin) (Csaba, 1980, 1984, 1985, 2000; LeRoith et al., 1980, 1982, 1983; KThidai et al., 2002). Recently, insulin receptors of Tetrahymena was characterized and found to be similar to its mammalian counterpart (Christopher and Sundermann, 1995; Leick et al., 2001; Christensen et al., 2003). However, functional significance of these hormones in the protozoa
* Corresponding author. Tel./fax: þ36 1 210 2950. E-mail address: [email protected] (G. Csaba).
is far from clear. It is noted that the recognition capabilities of Tetrahymena is so fine that very low concentrations of hormones are enough to activate hormone-dependent metabolic pathways and production of other hormones (Csaba et al., 2006, 2007a). Furthermore, prolonged deprivation of food significantly increased the insulin levels of the cells (Csaba et al., 2007b). We hypothesize that prolonged deprivation of food may influence production of other hormones. To test the hypothesis, we have monitored the endogeneous expression of b-endorphin, ACTH, serotonin, histamine, insulin and T3 by Tetrahymena pyriformis GL following prolonged (24 h) starvation with or without re-feeding. 2. Materials and methods 2.1. Cells and culturing T. pyriformis GL strain was used in the logarithmic phase of growth. The cells were cultured at 28 C in tryptone medium (Sigma, St. Louis, USA) containing 0.1% yeast extract, for 48 h. The density of Tetrahymena cultures studied was 104 cell/ml. Other cultures were cultivated for 24 h in the medium
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.02.007
G. Csaba et al. / Cell Biology International 31 (2007) 924e928 mentioned above and for further 24 h were kept in Losina-Losinsky salt solution (starved cells). Some cultures kept in medium or salt solution were fed (F) or starved (S) after the cultivation for 30 min. Considering the above mentioned facts, the experimental groups were as follows:
925
Table 1 Endogeneous b-endorphin after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
43.54 3.15 59.98 1.62 43.33 3.35 42.09 3.1
Significance p< to 1
1. 48 h culture in medium þ 30 min in fresh medium (F þ F þ F) 2. 24 h culture in medium þ 24 h in salt solution þ 30 min in fresh salt solution (F þ S þ S) 3. 24 h culture in medium þ 24 h in salt solution þ 30 min in fresh medium (F þ S þ F) 4. 48 h culture in medium þ 30 min in fresh salt solution (F þ F þ S)
0.001 n.s. n.s.
to 2
to 3
to 4
0.001
n.s. 0.001
n.s. 0.001 n.s.
0.001 0.001
n.s.
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
2.2. Flow cytometric analysis
Table 2 Endogeneous ACTH after long-time fasting or feeding in Tetrahymena
2.2.1. Preparation of cells for the measurement of endogeneous hormones Samples of cells were fixed with 4% paraformaldehyde solution (dissolved in pH 7.2 phosphate buffered saline [PBS]) for 5 min, and then washed twice in wash buffer (0.1% BSA; 20 mM TriseHCl; 0.9% NaCl; 0.05% Nonidet NP40; pH 8.2). To block nonspecific binding of antibodies the cells were treated with blocking buffer (1% bovine serum albumin [BSA] in PBS) for 30 min at room temperature. Aliquots from cell suspensions (50 ml) were transferred into tubes, and 50 ml primary antibody (anti b-endorphin [E1520], anti-ACTH [A1927], anti-serotonin [S5545], anti-histamine [H7403], anti-T3 [T2777; produced in rabbit] and monoclonal anti-insulin [I2018 produced in mouse], each purchased from Sigma (St. Louis, USA), diluted 1:200 in antibody buffer [1% BSA in wash buffer]) was added for 30 min at room temperature. Negative controls were carried out with 50 ml 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 antibody [F9887] except in case of insulin, where monoclonal anti-mouse IgG [F8771] (Sigma) was used, dilution 1: 50 with antibody buffer) for 30 min at room temperature. For controlling the specificity, autofluorescence of the cells and aspecificity of the secondary antibodies were detected. This latter means that the fluorescence of cells treated only with the secondary antibody (without the specific first antibody) was also measured in the antibody-treated series. This value was not subtracted from the value of 1st and 2nd antibody treated specimens; however, these latter values were 50e100% more in general, than the values of the only 2nd antibody treated ones.
No.
2.2.2. Measurement The measurement was done in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, USA), using 5000 cells for each measurement. In the cell populations the hormone content (concentration) had been compared. For the measurement and analysis CellQuest Pro program was used. The numerical comparison of detected values was done by the comparison of percentual changes of geometric mean channel values (Geo-mean, which represent relative fluorescent intensity and always those groups were compared which were measured together) to the appropriate control groups (line no. 1 in the Tables) and between the experimental groups by using Origin program and Student’s t-test. The experiments were done at least twice with similar results and the Tables demonstrate one of these experiments.
Group
Geo-mean
Significance p< to 1
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
51.07 3.28 84.35 4.76 70.91 7.82 54.21 1.6
0.001 0.001 n.s.
to 2
to 3
to 4
0.001
0.001 0.05
n.s. 0.001 0.01
0.05 0.001
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
Table 3 Endogeneous serotonin after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
35.56 1.29 59.19 0.82 44.73 1.88 38.98 2.4
Significance p< to 1 0.001 0.001 0.03
to 2
to 3
to 4
0.001
0.001 0.001
0.03 0.03 0.01
0.002 0.001
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
Table 4 Endogeneous histamine after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
19.95 1.03 26.53 1.38 20.71 0.55 21.24 0.76
Significance p< to 1 0.01 n.s. n.s.
to 2
to 3
to 4
0.01
n.s 0.01
n.s. 0.01 n.s.
0.01 0.01
n.s.
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
2.3. Confocal microscopic analysis After the flow cytometric analysis the cells were subjected to confocal microscopic analysis in a BioRad MRC 1024 confocal laser scanning microscope, equipped with kryptoneargon mixed gas-laser as a light source, at an excitation wavelength of 480 nm line.
3. Results The amount of six endogeneous hormones of the Tetrahymena was studied (1) after 24 h starvation (long starvation),
Table 5 Endogeneous insulin after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
Significance p< to 1
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
25.27 1.33 37.92 2.96 35.62 2.24 23.83 1.97
0.01 0.01 n.s.
to 2
to 3
to 4
0.01
0.01 n.s.
n.s. 0.01 0.01
n.s. 0.01
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s.: not significant.
G. Csaba et al. / Cell Biology International 31 (2007) 924e928
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Table 6 Endogeneous T3 after long-time fasting or feeding in Tetrahymena No.
Group
Geo-mean
Significance p< to 1
1 2 3 4
FþFþF FþSþS FþSþF FþFþS
44.65 1.48 66.69 2.52 63.53 5.06 46.39 3.94
4. Discussion
0.01 0.01 n.s.
to 2
to 3
to 4
0.01
0.01 n.s.
n.s. 0.01 0.01
n.s. 0.01
0.01
1st and 2nd letters: status in the 1st and 2nd 24 h; 3rd letter: status in the last 30 min. F ¼ fed; S ¼ starved; n.s. : not significant.
(2) re-fed for 30 min after 24 h starvation and (3) starved for 30 min only (short starvation). Prolonged starvation significantly elevated the level of each hormone (Tables 1e6). The elevation was about 50% of the basal level and the p-value of significance was at least 0.01. Short starvation did not influence the hormone levels, except serotonin which significantly ( p < 0.03) increased (Table 3/4). However, the re-feeding of the cells (for 30 min) was not so uniform. Endorphin and histamine returned to the basal level (Tables 1/3 and 4/3) after refeeding, ACTH and serotonin became significantly less, than after short starvation without re-feeding, however, significantly more than the basal level (Tables 2/3 and 3/3). Insulin and T3 remained similar to the level after long starvation (Tables 5/3 and 6/3). The confocal microscopic studies supported the quantitative data. The hormones were not specifically localized, they were demonstrated spread in the cytoplasm, around the oral field and nucleus and under the plasma membrane, with two exceptions. These are ACTH, which after long starvation extremely enriches in the cortical zone and oral field (Fig. 1a, b) and serotonin which also shows an enrichment around the nucleus and oral field (Fig. 2a, b). The cells after long starvation are smaller than after complete feeding.
The unicellular Tetrahymena contains vertebrate-like hormones and receptors as well as a signal transduction system functioning by the use of cAMP, cGMP, Ca-calmodulin and inositol phosphates (Csaba, 1980, 1984, 1985, 2000). These data are internationally justified and accepted (O’Neill et al., 1988; LeRoith et al., 1980, 1983; Christopher and Sundermann, 1995; Leick et al., 2001; Christensen et al., 1998, 2003; Wheatley and Christensen, 1999; Shemarova et al., 2004), however, it is not clearly known, what is the functional role of this endocrine-like system in a unicellular organism. At this level there are three possibilities: (1) the cell uses the hormones for self-regulation (intracine or autocrine regulation), (2) there is an intercellular communication by them between the members of the Tetrahymena population and (3) the hormones are only side-products of the synthesis of other proteins, however, this not explains the presence of receptors and signal transduction pathways. It was cleared previously that extremely low concentrations of hormones are enough for provoking changes in the insulin content and binding of Tetrahymena (Csaba et al., 2006, 2007a), which points to the possibility of intercellular communication by these hormones. Now we wanted to know whether the worsening of life conditions by restricting the food supply can influence the unicellular’s hormone content in general, or not. Long starvation is partly a stress for the cells, partly a process, when the deprivation of food does not allow the replacement of amino acids and proteins which had been depleted during physiological processes. The molecules studied were amino acid or polypeptide type hormones. Considering that there is no replacement of proteins for 24 h e which is an extremely long period relative to the individual life of a Tetrahymena (Wheatley and Christensen, 1999) e the hormone level must have been lower after fasting. As all of the hormone levels were much higher than control, it can be supposed
Fig. 1. ACTH in fed (a) and long starved (b) Tetrahymena. After starvation there is a bright fluorescence in the cell body and under the surface. The cells are smaller than in the fed group. Magnification 2000.
G. Csaba et al. / Cell Biology International 31 (2007) 924e928
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Fig. 2. Serotonin in fed (a) and long starved Tetrahymena. After starvation the fluorescence is stronger, especially around the nucleus and in the oral region. The cells are deformed and smaller than in the normally fed group. Magnification 2000.
that the presence of more hormones is required for the cells to tolerate the strong stress. This can reason the preparation of hormones from internally stored reserve materials. In our previous experiments (Csaba et al., 2007b) the elevation of insulin concentration was observed as a consequence of starvation. It was a possibility to believe that only insulin concentration is increasing, as insulin is known as a vital factor for maintaining Tetrahymena (Christensen et al., 1995, 1998; Wheatley et al., 1993, 1994; Wheatley and Christensen, 1999; Hagemeister et al., 1999; Shemarova et al., 2002). However e considering the present results e it is not exceptional: other hormones could participate in the defense during life-threatening situations. The results bring nearer to the understanding of the role of vertebrate-like hormones in Tetrahymena. However, a later duty to establish whether the accumulation of hormones is specific for starvation, or it is a universal reaction in different stress situations. Acknowledgements This work was supported by the National Research Fund (OTKA-T-037303). The authors thank Ms. Katy Kallay, Marianna Kincses and Angela Koza´k for their expert technical assistance. References Christensen ST, Guerra CF, Awan A, Wheatley DN, Satir P. Insulin receptorlike proteins in Tetrahymena thermophila ciliary membranes. Curr Biol 2003;13:R50e2. Christensen ST, Wheatley DN, Rasmussen MI, Rasmussen L. Mechanisms controlling death, survival and proliferation in a model unicellular eukaryote Tetrahymena thermophila. Cell Death Differ 1995;2:301e8. Christensen ST, Leick V, Wheatley DN. Signaling in unicellular eukaryotes. Internatl Rev Cytol 1998;177:181e253. Christopher GK, Sundermann CH. Isolation and partial characterisation of the insulin binding site of Tetrahymena pyriformis. Biochem Biophys Res Com 1995;212:515e23.
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