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.     

Experimental observations on the mechanism of hormonal imprinting: influence of actinomycin D, methylamine and colchicine on receptor memory in a unicellular model system

The first interaction between target cell and hormone gives rise to hormonal imprinting, which accounts for greater responsiveness of the cell at later interactions. The mechanism of hormonal imprinting is obscure; we based experimental approach to its closer study on combined treatment of Tetrahymena, as model cells, with diiodotyrosine (T2), which stimulates the division, and cell growth inhibitors, which interfere with different stages of cell reproduction, and methylamine, which inhibits cluster formation in the membrane. Of these, actinomycin D and methylamine inhibited the growth of the Tetrahymena, while colchicine did not, and all three suppressed the division stimulating action of T2, but could not prevent hormonal imprinting, as demonstrated on later re-exposure to T2 of cells preexposed and not preexposed to T2 in combination with the inhibitors. It appears that the underlying mechanism of hormonal imprinting is highly complex, and involves many subcellular mechanisms and structures, but suppression of, or gross interference with, one or another of these cannot delete, only quantitatively reduce, the consequence of the first interaction with the hormone, i.e. hormonal imprinting.     

Influence of hormone treatment applied or begun in different phases of the cell cycle on hormonal imprinting in Tetrahymena

Primary exposure to a hormone (hormonal imprinting) alters--in the case of the Tetrahymena increases--cellular response to re-exposure(s) to the same hormone. The intensity of hormonal imprinting depends on the phase of the cell cycle in which the primary exposure has taken place. The effect of imprinting was greater on the cells exposed to the hormone in phase G1 than on those exposed in phase S or G2. The response pattern of the progeny generations corresponded to that of the primarily exposed (imprinted) ancestor cell, irrespective of their own pre-exposure in phase G1, G2 or S of their cycle.     

The role of Ca2+ in hormonal imprinting of the Tetrahymena

It is known from model experiments on Tetrahymena that primary exposure to a hormone induces receptor formation or amplification, in other words a hormonal imprinting. Substances acting on the intracellular Ca2+ level of the Tetrahymena, such as TMB-8, EDTA, EGTA, NiCl2 and La(NO3)3, interfered with hormonal imprinting of the unicellular to different degrees, and some of them influenced hormone (insulin, TSH) binding also independently of imprinting. Interference with the intracellular Ca-metabolism generally influenced imprinting by insulin and TSH, which were mediated by different mechanisms, to dissimilar degrees, or in opposite directions. On combined application of the agents acting on Ca-metabolism, their effects were additive. It appears that intact Ca-mediation is an essential prerequisite for normal hormonal imprinting.     

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.     

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.     

Suitability of oligopeptides for induction of hormonal imprinting--implications on receptor and hormone evolution

Hormonal imprinting induced in Tetrahymena and in Chang liver cells with di-, tri-, tetra- and pentapeptides (synthetic opioids and their fragments) has shown that both cell types are able to differentiate the related molecules from one another. The dipeptide phenylalanine + proline induced a measurable imprinting in the liver cells, and chain length increase, especially terminal coupling with tyrosine enhanced the imprinting potential enormously. Intra-chain changes in the amino acid sequence had a measurable effect on the intensity of imprinting. The molecules showing the relatively strongest physiological action accounted for the most intensive imprinting in both cell types; this indicates that, in all probability, induction of binding site formation plays a key role in the development of signal molecules, and thereby in hormone evolution.     

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.     

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.     

Involvement of the phosphoinositol (PI) system in the mechanism of hormonal imprinting

Certain components of the phosphoinositol (PI) system are present in the unicellular Tetrahymena. Treatment of Tetrahymena with insulin did not alter the relative proportions of the examined six phospholipid components (PIP2, PIP, phosphatidylcholine, phosphatidylethanolamine, PI, PA), but the primary interaction (imprinting) with insulin accounted for an about 75% decrease in the PIP2-level and an about 20% increase in the phosphatidylethanolamine level. The experimental results strongly suggest that hormonal imprinting accounted for adjustment of the second messenger systems of Tetrahymena to an energy saving level.     

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.     

Induction of melatonin synthesis in Tetrahymena pyriformis by hormonal imprinting--a unicellular "factory" of the indoleamine.

Melatonin is present in Tetrahymena and its synthesis can be enhanced by pretreatment (imprinting) with melatonin. Two days after imprinting melatonin level is elevated in the cells and more elevated in the supematant. Such a minute quantity, as 10(-12) M melatonin for 1 hour is able to provoke imprinting, however the effect is more expressed using 10(-6) M. Maintenance in light conditions further elevated the amount of melatonin in the cells and supematant alike, related to the melatonin content of cells kept in darkness. The experiments call attention to the light-sensitivity of imprinting-provoked melatonin production in Tetrahymena and to the possibility of using this property for important physiological functions in higher grades of phylogeny.     

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.     

The binding of diazepam in the mitochondria of Tetrahymena pyriformis as detected by quantitative high resolution autoradiography

Tritiated diazepam accumulates mainly in the mitochondria of the unicellular Tetrahymena. This is the case in both a single (the first encounter) and a repeated (one day or a week after the first) administration of the drug. When imprinting of Tetrahymena by diazepam (the first encounter) is followed a week later by the administration of the labelled drug, the membranes of the vesicles, too, show the appearance of label. Regarding the studies presented here, the unicellular Tetrahymena also contain diazepam receptors in the mitochondria as suggested for cells of higher rank animals.     

Induction of hormone receptor formation in the unicellular tetrahymena

Studies based on treatment with antibodies to thyrotropic hormone, luteotropic hormone, growth hormone or adrenocorticotropic hormone have shown that although the unicellular Tetrahymena does not possesssui generis receptors to all polypeptide hormones, such binding structures may arise, or become established in the membrane of the unicellular Tetrahymena in the presence of exogenous hormone. The Tetrahymena subjected to hormonal imprinting still contained an increased amount of hormone after six generation changes, which suggested that either hormone production had been induced by treatment, or the internalized hormone had not been degraded intracellularly. Thus the role of hormonal imprinting in receptor formation has also been substantiated by the immunocytochemical approach used in the present study.     

Dissimilar effects of L and D amino acids on the growth of Tetrahymena

Of the L and D configurations of four amino acids (phenylalanine, valine, tryptophan, tyrosine) tested for influence on the growth rate of Tetrahymena, only L-tyrosine was able to induce imprinting in Tetrahymena pyriformis Zeuthen. D-valine stimulated the division of T.pyriformis NT-1, but failed to induce imprinting. The experiments have substantiated the selectivity of the amino acid receptors of Y.pyriformis, and the extraordinary imprinting potential of tyrosine as well, as judged by its influence on the growth rate.     

Phylogenetic changes in sensitivity to Anemonia sulcata toxin (ATX II), and impact of first interaction with the toxin (imprinting) on later response to it

The Anemonia sulcata toxin ATX II is cardiotoxic and neurotoxic, and--at a high dose level--even lethal for the mouse, neurotoxic, but non-lethal for the frog, and has no adverse influence whatever on the Planaria and Tetrahymena; it even stimulates the growth of the Tetrahymena at a low dose level. It also induces imprinting in the Tetrahymena, as judged from the altered response of the latter to ATX II on re-exposure. No similar imprinting effect was demonstrable in mice.     

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.     

Induction of steroid binding sites (receptors) and presence of steroid hormones in the unicellular Tetrahymena pyriformis

The unicellular Tetrahymena does not normally possess a steroid hormone (dehydroepiandrosterone, DHEA) or a glucocorticoid (dexamethasone) receptor, but both kinds of receptor can be induced in it by pretreatment (imprinting) with the adequate hormone. The specific receptors which arise are demonstrable experimentally. Examination of Tetrahymena cells for endogenous steroids by the radioimmunoassay (RIA) technique detected an appreciable concentration of DHEA and DHEA sulphate, and lesser concentrations of testosterone and estradiol in this unicellular organism.     

Influence of interference with the cyclic AMP-adenylcyclase-phosphodiesterase system on TSH-induced hormonal imprinting in the Tetrahymena

Primary interaction of TSH with the unicellular Tetrahymena accounted for an increase in TSH binding capacity on reexposure, i.e. for a regular hormonal imprinting. TSH in itself did not give rise to a faulty imprinting (for insulin). Combination of TSH with dibutyryl cAMP reduced the intensity of imprinting, whereas theophylline or lithium ions not only reduced the efficacy of normal imprinting, but also gave rise to faulty imprinting (for insulin instead of TSH).