Influence of hormone concentration and time factor on development of receptor memory in a unicellular (Tetrahymena) model system

1. Treatment of Tetrahymena pyriformis cells with diiodotyrosine (T2) gave rise to a considerable, concentration-dependent increase of the growth rate within the range of 10(-15) and 10(-9) M, but did not influence it at the level of 10(-18) M. 2. Re-exposure of the cells 1, 2 and 4 weeks later to the hormone concentrations originally used accounted for a marked increase of growth rate at all hormone levels tested, indicating that the extremely low concentration of 10(-18) M, which failed to stimulate growth on first exposure, did nevertheless give rise to hormonal imprinting, which caused the cells to "remember" the hormone, as judged from their increased responsiveness to it on re-exposure. 3. The degree of growth response was concentration-dependent on both first and second exposure: higher levels of treatment gave rise to firmer imprinting, and to greater response on re-exposure. 4. The length of exposure time proved to be more decisive than the level of treatment in respect of the development of hormonal imprinting. 5. Short-term exposures up to 60 min, although they stimulated cell growth by direct effect, gave rise to lasting inhibition of cellular response to re-exposure(s) rather than to hormonal imprinting.     

Effect of inhibitors and activators of tyrosine kinase on insulin imprinting in Tetrahymena.

Primary exposure of Tetrahymena cells to insulin gave rise to hormonal (insulin) imprinting in the offspring generations, as judged from the increase in binding upon reexposure to insulin. Vanadate mimicked the action of insulin, inasmuch as it also induced imprinting for insulin, whereas the other tyrosine kinase activator tested, namely H2O2, had no such effect. However, combined treatment with vanadate+H2O2 + insulin induced a more pronounced imprinting for insulin than either insulin or vanadate on their own. The tyrosine kinase inhibitor genistein, a plant flavonoid, did not change the value for insulin binding significantly relative to the control immediately after exposure, but increased it slightly in the offspring generations after 24 h at high dilution. Upon combination with insulin, 10(-4)M genistein inhibited imprinting by insulin. These experimental observations suggest that there may be a key role for tyrosine kinase activity in the mechanism (development) of imprinting.     

Influence of membrane fluidity changes upon the imprinting of polypeptide hormones in Tetrahymena

Hormonal imprinting is a physiological phenomenon, in which after the first encounter the receptorial and functional responses of a cell change for future occasions. The present experiments demonstrate (using Tetrahymena as a model cell) that the imprinting is very sensitive to the changes in membrane physical state. Cultivation of Tetrahymena cells in 28 or 15 degrees C or in ergosterol-supplemented media caused only quantitative differences in the imprinting; however, the process of cooling (shift-down) or reheating (shift-up) resulted in a false reaction. The combined treatment by ergosterol and cooling completely abolished the imprinting. These results indicate that hormonal imprinting is a membrane-dependent process.     

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.     

Verification of epigenetic inheritance in a unicellular model system: multigenerational effects of hormonal imprinting

The unicellular Tetrahymena has receptors for hormones of higher vertebrates, produces these hormones, and their signal pathways are similar. The first encounter with a hormone in higher dose provokes the phenomenon of hormonal imprinting, by which the reaction of the cell is quantitatively modified. This modification is transmitted to the progeny generations. The duration of the single imprinter effect of two representative signal molecules, insulin and 5-HT (5-hydroxytryptamine), in two concentrations (10-6 and 10-15 M) were studied. The effects of imprinting were followed in 5 physiological indices: (i) insulin binding, (ii) 5-HT synthesis, (iii) swimming behaviour, (iv) cell growth and (v) chemotaxis in progeny generations 500 and 1000. The result of each index was different from the non-imprinted control functions, growth rate, swimming behaviour and chemotactic activity to insulin being enhanced, while others, e.g. synthesis and chemotactic responsiveness of 5-HT and the binding of insulin were reduced. This means that a function-specific heritable epigenetic change during imprinting occurs, and generally a single encounter with a femtomolar hormone concentration is enough for provoking durable and heritable imprinting in Tetrahymena. The experiments demonstrate the possibility of epigenetic effects at a unicellular level and call attention to the possibility that the character of unicellular organisms has changed through to the present day due to an enormous amount of non-physiological imprinter substances in their environment. The results - together with results obtained earlier in mammals - point to the validity of epigenetic imprinting effects throughout the animal world.     

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.     

Insulin uptake, localization and production in previously insulin treated and untreated Tetrahymena. Data on the mechanism of hormonal imprinting

Confocal microscopic experiments demonstrate the presence of insulin in Tetrahymena, observed also in earlier experiments. However, there is a broad spectrum of insulin-containing cells from the immunocytochemically insulin-free, to the strongly antibody-reactive ones. During 1 h of insulin treatment (imprinting) the cells gradually bind and take up insulin, and the process is slow. One minute after the start of treatment there is not difference in the number of insulin antibody-reactive cells and amount of insulin. After 5 or 10 min the cells bind and contain more insulin and after 1 h most of the cells are densely packed with the insulin antibody-reactive material. Insulin imprinting accelerates binding and uptake alike: 48 h after imprinting and 1 min after the start of the second treatment, more insulin is present on the surface and inside the cells, than after 10 min in the first-time treated cells. Theoretically, this effect of hormonal imprinting helps to maintain the species by facilitating molecular recognition and binding as well as uptake of useful molecules. The experiments also support previous observations on the parallel receptor-evoking (strengthening) and hormone-producing effect of hormonal imprinting.     

Receptor 'memory' in Tetrahymena: does it satisfy the general criteria of memory? An experimental study on induction and extinction by retroactive interference in a unicellular organism

Diiodotyrosine induced receptor 'memory' at a concentration as low as 10(-18) M. Repeated exposure enhanced cellular responsiveness. Treatment with diiodotyrosine for 1 h, 4 times, induced receptor 'memory' more efficiently than a single uninterrupted treatment for 4 h. Immediately after induction, the receptor 'memory' is subject to retroactive interference by foreign hormonal stimuli, and can be extinguished completely by combined hormone treatment. Thus, the phenomenon of retroactive interference also takes effect at the unicellular level. The experimental observations indicate that receptor 'memory', induced in Tetrahymena by hormonal imprinting, has certain common features with the neuronal memory of higher organisms.     

Taxon-dependence of receptor level cell-to-cell communication in Tetrahymena: possible explanation for the transmission of hormonal imprinting

Insulin treatment induced in Tetrahymena pyriformis a positive hormonal imprinting, and in Tetrahymena thermophila a negative imprinting, resulting in increased and decreased binding capacity, respectively, at re-exposure to the hormone. The imprinting, or the information associated with it, is transferred by the nutrient medium of the insulin-treated cells to those not treated. The issue of transfer depends on the nature of the receiver taxon, leading always to a positive imprinting in Tetrahymena pyriformis, and to a negative imprinting in Tetrahymena thermophila, regardless of the nature of the 'imprinted' transmitter taxon. The findings substantiate the transferability of hormonal imprinting by the nutrient medium at the unicellular level, the key role of the postreceptorial mechanism in determining the trend of imprinting and may explain the persistence of imprinting in the progeny generations.     

Sex-dependent effect of melatonin on the secretory activity of rat and hamster adrenal gland in vitro

Adrenal quarters from adult male or female hamsters were incubated in the presence of melatonin (10(-7) or 10(-4)M), and cortisol concentration in the incubation medium was assayed by RIA. Melatonin did not change cortisol output by adrenals obtained from the male hamsters, while a slight stimulatory effect was observed in female glands, the lower concentration of melatonin being more effective than the higher one. At both concentrations tested, melatonin notably stimulated corticosterone output by isolated rat adrenocortical cells derived from the males, and lowered corticosterone secretion by the cells obtained from the female glands only at a concentration of 10(-7) M. The lower concentration of melatonin increased ACTH (0.1 mU.ml-1)-stimulated corticosterone output by the cells of male and female rat adrenals. The pineal hormone was ineffective at a concentration of 10(-4) M, as well as in the presence of a higher dose of ACTH (1.0 mU.ml-1). These findings indicate a distinct sex-dependent effect of melatonin on in vitro cortisol and corticosterone production, and demonstrate that the modulatory effect of melatonin of the secretion of steroid hormones is more effective at lower concentrations.     

Insulin pretreatment (imprinting) produces elevated capacity in the insulin binding of Tetrahymena. Different binding by the cilia of the body and oral field

Gold-labeled insulin is bound first of all to the cilia of the oral field of Tetrahymena. A primary treatment (hormonal imprinting) with insulin increases the binding capacity even after 24h and makes it more sensitive for appearance a week later, within a minute of giving insulin-gold. The food vacuoles contain insulin-gold in pretreated cells or without pretreatment as well, though in imprinted situations the label can be found in pinocytotic vesicles at the bases of cilia in the oral field. Altogether, a functional difference can be observed between the cilia of the oral and non-oral surfaces of Tetrahymena and hormonal imprinting has a specifying effect on the binding of labeled hormone.     

Effect of the cAMP level on hormonal imprinting in Tetrahymena

Augmentation of the cAMP level has no positive effect on hormonal imprinting in Tetrahymena. Artificial elevation of the cAMP level may inhibit the development of imprinting or may result in abnormal imprinting. The role of Ca2+ is of great importance in mediation of the imprinting mechanism. Generally, this role is not influenced by an elevated cAMP level but, exceptionally, the latter may effect the mechanism of imprinting.     

Melatonin in the unicellular Tetrahymena pyriformis: effects of different lighting conditions

Melatonin content in the cellular fraction and medium of Tetrahymena pyriformis GL cultures was measured at different time points of light and dark exposures. Tetrahymena produced, stored and secreted immunoreactive melatonin, which in displacement and HPLC studies, behaved like synthetic melatonin. There was not a continuous secretion of melatonin produced by the cells. In contrast to this, storage of melatonin was observed, which was more expressed in dark conditions. Prolonged light exposure suppressed melatonin production and secretion alike, however it did not block it completely.     

Influence of deciliation and ciliary regeneration on hormonal imprinting in Tetrahymena. Observations on the 'localization' of imprinting

Imprinting induced in Tetrahymena with insulin is not abolished by deciliation. No imprinting occurred in deciliated cells exposed to insulin at 1 or 2 h of regeneration. However, imprinting did occur if Tetrahymena was exposed to insulin after 3 h of regeneration. It appears that while presence of cilia is a prerequisite of imprinting, the pertinent information is not, or not exclusively stored in the cilia.     

Discrepancy in hormone binding and information transfer between sister cells of Tetrahymena clones

Tetrahymena cells treated with insulin in mass cultures were separated to single-cell clones or one of the "sister-cells" of dividing Tetrahymena (in single-cell culture) was treated with insulin. In both cases the FITC-insulin binding of sister-cells were compared. The insulin imprinting significantly increased the insulin binding of cells. There was also a significant difference between the imprinted and not imprinted sisters as well as between the not imprinted sisters. This demonstrates the existence of a difference (in hormone binding) between sister-cells and justifies that the information of the first hormone treatment (imprinting) is not equally divided between the sister-cells.     

Inhibition of small conductance K+ -channels attenuated melatonin-induced relaxation of serotonin-contracted rat gastric fundus

The aim of this study was to investigate the effects of melatonin on rat gastric fundus smooth muscle. Melatonin (10(-4) to 10(-3) M) had no effect on the basal tone of gastric smooth muscle. After precontraction with carbachol (10(-6) M) or serotonin (10(-7) M), melatonin caused a concentration dependent inhibitory action. The half maximal effect on serotonin-induced contraction was found with 1.12 +/- 0.86 x 10(-5) M of melatonin. Increasing concentrations of melatonin (10(-5) to 10(-3) M) resulted in a right shift of the serotonin concentration response curve (10(-10) to 10(-5) M). This inhibitory effect of melatonin was partially blocked in the presence of apamin (10(-10) to 10(-7) M), a specific blocker of the small conductance calcium-dependent potassium channel, but not in the presence of other potassium channel blockers like charybdotoxin (10(-8) M), glibenclamide (l0(-5) M), or tetraethylammonium (ODQ, 10(-4) M). The inhibitory effect was not changed in the presence of the neuronal blocker tetrodotoxin (10(-6) M), the selective P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) M), the nitric-oxide synthase inhibitor N-nitro-L-arginine (3 x 10(-4) M), or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (10(-4) M), suggesting that neither the purinergic, nitrergic, nor guanylate cyclase pathways were involved. We further investigated inhibitory responses to electrical field stimulation (EFS) at different frequencies under non-adrenergic, non-cholinergic (NANC) conditions on a serotonin-induced contraction in the presence of melatonin (10)-5 to 10(-4) M). Melatonin significantly reduced these inhibitory NANC responses in higher (8-32 Hz), but not lower (05-4 Hz), frequencies (16 Hz without melatonin, 103 +/- 6.3%; melatonin 10(-5) M, 80.4 +/- 7.5%; melatonin 10(-4) M, 39.1 +/- 17.1%). Melatonin had no effect on contractile responses induced by EFS under basal tone. These results demonstrate that the inhibitory effect of melatonin in rat gastric fundus smooth muscle is apamin sensitive, but is not affected by other potassium channel blockers. This suggests that melatonin may be another transmitter candidate for the apamin sensitive responses within the gastrointestinal tract.     

Hormone evolution studies: multiplication promoting and imprinting ("memory") effects of various amino acids on Tetrahymena

Aromatic, heterocyclic, polar and non-polar amino acids were examined for imprinting potential in a unicellular (Tetrahymena) model system. Serine gave rise to positive, glycine to negative imprinting, whereas valine, tryptophan, tyrosine and phenylalanine had no imprinting effect whatever. However, tyrosine and phenylalanine stimulated the division of Tetrahymena already at primary interaction, the former even for a relatively long time. It follows that amino acids, too, can give rise to imprinting, although their imprinting potentials are dissimilar. These phenomena have attracted attention to possible interrelationships between the supposed amino acid receptors of Tetrahymena and the evolution of amino acids to hormones.     

Prolonged elevation of insulin content in the unicellular tetrahymena after insulin treatment: induction of insulin production or storage?

In the unicellular organism, Tetrahymena, the first encounter with an exogeneously given hormone results in hormonal imprinting. This causes an increase of the binding capacity of receptors and the production of the appropriate hormone in the progeny generations of the treated cell. In the present experiments the quantity (using radioimmunoassay) and localization (using confocal laser scanning microscopy) of the immunologically insulin‐like material (hereafter insulin) were studied for 10 days after 4 h or 24 h 10⁻⁶ m insulin treatment (hormonal imprinting). Forty‐eight hours after both insulin treatments a high quantity of insulin was present in the cells. This value was also significantly increased after 96 h. After 8 days the difference to the control was significant only in the 24 h treated group. Confocal microscopy (using antibody to pig insulin) localized insulin in the cell body. The oral field contained extremely high quantities of the endogeneous hormone. Insulin treatment (after 48 and 96 h) caused an elevation of insulin content in general, and specific accumulation in the posterior sections of the cell, around the nucleus and in the periphery were observed. Ten days after both treatments only the peripheral region of the cell body and the ciliary row contained more insulin than the control. This means that after insulin treatment the quantity of insulin increases for a lengthy time period which is followed by the expression of insulin in the peripheral region. Insulin contained by Tetrahymena 48 h after imprinting stimulated glucose uptake of rat diaphragm. Copyright © 1999 John Wiley & Sons, Ltd.     

Effect of inhibition of endocytosis, recycling and lysosomal activity on the insulin binding capacity and imprintability of Tetrahymena

Dinitrophenol (DNP), an inhibitor of endocytosis of hormone receptors, Tris, an inhibitor of recycling and chloroquine, an inhibitor of lysosomal degradation, all decreased the binding of insulin and inhibited the development of hormonal imprinting in Tetrahymena. The effects of DNP and Tris seemed to be similar even quantitatively. The effect of chloroquine proved to be somewhat different, it appeared later, was more pronounced after 24 hours and more marked when insulin was also administered. Combined administration of Tris + DNP inhibited the binding of insulin but this inhibition was the one which disappeared most completely after 24 hours and the one where the inhibition of imprinting was the most pronounced. Tris + chloroquine led to severe destruction of the cells. The conclusion has been drawn that the inhibition of membrane circulation inhibits not only the hormone binding but also the development of imprinting in Tetrahymena.     

The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates Tetrahymena pyriformis

The influence of the epidermal growth factor (EGE) (10(-8) M), insulin (10(-6) M) and EGF (10(-8) M) in combination with insulin (10(-6) M) on proliferation and DNA synthesis in the nuclei of ciliates Tetrahymena pyriformis GL was studied. Insulin and EGF, known to stimulate growth of many types of mammalian cells revealed a mitogen influence on the unicellular eukaryotes. This effect involves stimulation of DNA synthesis, rising synchronization of cell division (upon the influence of EGF), and increase in cell number during the exponential growth. The mitogen effect may be evoked by cell progression in G1-phase under the action of growth factors and, consequently by earlier entry of cells into S-phase of the first cell cycle. Insulin repressed division of cells that entered into the generative cycle. These cells were delayed in late S-phase and G2-phase of the cycle. Part of these cells perished, while other cells could successively overcome the cell block to start their division by the 4th hours of cultivation. A collateral cytotoxic effect of insulin was found, being most prominent in early periods of Tetrahymena cultivation.