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 vanadate and ouabain on insulin binding and insulin imprinting in Tetrahymena.

Na-metavanadate and ouabain that act on Na+K(+)-ATPase had no influence on insulin binding to Tetrahymena immediately after treatment, but after 24 h considerably enhanced the binding capacity of generations of progeny. The increase in binding was of a similar magnitude to that elicited by insulin imprinting. Vanadate failed to increase the imprinting potential of insulin while ouabain even prevented insulin imprinting when administered together with insulin, but, did not affect imprinting when administered after insulin. By analogy with higher organisms it appears that inhibition of Na+K(+)-ATPase plays no role in the insulin-like effect of vanadate on the unicellular Tetrahymena, as judged also from the capacity to bind insulin of the generations of offspring.     

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 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).     

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.     

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.     

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.     

Influence of neonatal insulin imprinting on the insulin binding capacity of rat erythrocytes in adulthood

A single neonatal insulin treatment decreased considerably the insulin binding capacity of erythrocytes in adult rats, by analogy of the behaviour of the hepatic insulin receptors in response to insulin exposure during the perinatal period, or during liver regeneration in adulthood. These observations substantitate earlier conclusions on the mechanism of imprinting and strongly suggest the universality of perinatal imprinting in living organisms. In vitro insulin exposure of the erythrocytes of adult rats depressed 48 h later the erythrocytic insulin binding capacity to a similar degree in individuals treated and not treated with insulin when newborn, from which it follows that neonatal exposure had no influence on erythrocytic response to later in vitro treatment. In the light of the present study the use of erythrocytes as model cells for imprinting studies deserves consideration.     

Effect of insulin imprinting on the 3H-amino acid uptake of the Tetrahymena

Insulin imprinting given to the unicellular Tetrahymena considerably increases the uptake and intracellular storage of amino acids even many generations after the actual contact with the hormone. On the other hand, both the first and the second contacts with insulin increase the rate of the excretion of the stored amino acids. On the basis of the results obtained it seems to be possible that both protein synthesis and exocytosis of the Tetrahymena change as an effect of imprinting, either in general or specifically due to the formation of new hormone receptors.     

Interrelationships between the imprinting potential and biological activity of insulin derivatives: studies on Tetrahymena and Chang liver cells

Insulin dimers deprived of biological activity by linking with suberic acid symmetrically in position B29 or B1 were not able to induce imprinting. Lack of N-terminal phenylalanine or even of five C-terminal amino acids did not interfere with imprinting, regardless of whether or not it was associated with an activity loss. It appears that while hormonal imprinting is closely associated with the hormone's ability to bind to the receptor, it may be related as well as unrelated to the hormone's biological activity. The imprinted Tetrahymena and Chang cells bound the insulin and its derivatives in a similar manner.     

Localization of beta-endorphin in tetrahymena by confocal microscopy. Induction of the prolonged production of the hormone by hormonal imprinting

The unicellular Tetrahymena has hormone receptors and hormones which are characteristic of higher vertebrates, as well as similar signal transduction pathways. In the present experiments, immunocytochemistry and confocal microscopy were used to study the presence and localization of beta-endorphin in Tetrahymena pyriformis GL. Endorphin (or endorphin-like material) was localized in the cortical structures, oral field, cilia and nuclear envelope. One-hour treatment with beta-endorphin ('hormonal imprinting') increased the presence of immunocytochemically demonstrable endorphin immediately and after 24 h, and was especially strong after 96 h of treatment. Simultaneous treatment with naloxone, an opioid antagonist, did not inhibit endorphin effect, but had an additive effect on endorphin production. Naloxone alone induced a very intensive accumulation of endorphin 96 h after treatment. The results support the possibility of a hormone production being induced by the imprinting procedure, but the imprinter-like effect of naloxone also points to the importance in this case of non-discriminatory receptors also being involved in the process.     

Study of the imprinting and overlap of insulin and concanavalin-A at the receptor level in a protozoan (Tetrahymena) model system

In a protozoan (Tetrahymena) model system, insulin treatment produced a long-term imprinting which upon re-exposure to the hormone resulted in an enhanced binding of the hormone. Insulin pretreatment produced similar effect with regard to the binding of concanavalin-A. Concanavalin-A could only induce a short-term imprinting for itself and was not capable at all of inducing imprinting for insulin. Based on the results of this study it appears that the binding of the sugar component of the receptor, which can be achieved also by lectin, is not sufficient to induce imprinting but the whole (hormone) molecule is needed.     

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.     

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.     

The regulatory role of calmodulin-dependent guanylate cyclase in association with hormonal imprinting in Tetrahymena

The primary interaction with insulin accounted for considerable increases in both the calmodulin content and guanylate cyclase activity of Tetrahymena. Both activities were still elevated after 24 h (6-8 generations), but while the calmodulin level showed a decrease, guanylate cyclase activity showed a further significant increase relative to the immediate response. A second treatment with insulin decreased rather than increased both activities, but to dissimilar degrees, in that the calmodulin content returned to the control level, whereas guanylate cyclase activity still increased over the level measured after the first treatment. It appears that insulin imprinting altered the calmodulin-dependent guanylate cyclase regulation in Tetrahymena, and caused a switch-over to an 'energy-saving' system through decelerating the breakdown of cGMP by phosphodiesterase.     

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.     

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.     

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.     

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.     

Influence of low and high temperature on diiodotyrosine imprinting in Tetrahymena

Hormonal imprinting takes place at the primary interaction between target cell and hormone, and alters cellular response to the hormone for lifetime (at the unicellular level in many subsequent generations). Imprinting induced in Tetrahymena cells by diiodotyrosine at the optimum temperature of 25 degrees C took effect on re-exposure to the hormone at 25 degrees C and 15 degrees C, but failed to take effect if the cells were first exposed to the hormone at 15 degrees C or 32 degrees C.