Effect of modification of membrane saccharides on hormonal imprinting in Tetrahymena

Concanavalin-A (Con-A) competes with insulin for the insulin binding sites of the receptor, an event resulting in overlapping bonds. If the saccharide components of the receptor are subjected to periodate treatment the binding of insulin decreases and so does the imprinting evoked by it. Simultaneously, the binding of Con-A increase immediately after the treatment and 24 hours following the insulin imprinting. This phenomenon indicates that though the two ligands (i.e. insulin and Con-A) overlap on the intact receptor, alterations in the saccharide component influence their binding though not in the same manner and not in the same direction. Periodate treatment, similarly as observed in mammalian lymphocyte cultures, enhanced the division of Tetrahymenas and the peak of Con-A binding occurred parallel to the peak of division after periodate treatment of equal duration. Periodate treatment disturbed not only the binding of Con-A but that of other lectins as well. It has been concluded that hormonal imprinting requires a physiological condition of the membrane and any disturbance of the membrane will lead to a decrease in the efficacy of imprinting.     

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.     

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.     

Impact of starvation on hormone binding and hormonal imprinting in Tetrahymena.

Tetrahymena cells maintained (starved) in a physiological salt solution showed a considerable decrease in insulin binding capacity. The cells previously imprinted with insulin showed a comparable relative binding decrease after a similar exposure. This change was reversible by prolonged maintenance in plain nutrient medium after which the binding capacity of the imprinted cells increased appreciably over the control. The cells maintained (starved) in salt solution for 2 h were no longer imprintable with insulin; it follows that prolonged starvation not only reduced the recognition potential, but also extinguished the imprintability of Tetrahymena cells.     

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 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).     

Cytochemical investigation into the Ca-dependence of positive and negative hormonal imprinting in Tetrahymena

Insulin gives rise to positive imprinting in Tetrahymena pyriformis, but to negative imprinting in T. thermophila, as revealed by the respective increases and decreases in the insulin-binding capacity of these organisms observed during later interactions with this hormone. We found that changes in insulin-binding capacity exhibited parallelism with fluctuations of the levels of free, intracellular Ca2+ detectable by Quin-2 labeling. An exception was the second interaction of T. thermophila with insulin, which although showing a positive trend, produced a relatively small increase in the level of intracellular Ca2+. These observations suggest an interrelationship between hormone-binding capacity and the fluctuation of intracellular Ca2+ levels. Either hormone binding depends on the availability of Ca2+, or, alternatively, the latter depends on the binding capacity. Further studies are required to elucidate the true nature of this interdependence.     

Cytochemical investigation into the Ca-dependence of positive and negative hormonal imprinting in Tetrahymena

Summary Insulin gives rise to positive imprinting in Tetrahymena pyriformis, but to negative imprinting in T. thermophila, as revealed by the respective increases and decreases in the insulin-binding capacity of these organisms observed during later interactions with this hormone. We found that changes in insulin-binding capacity exhibited parallelism with fluctuations of the levels of free, intracellular Ca²⁺ detectable by Quin-2 labeling. An exception was the second interaction of T. thermophila with insulin, which although showing a positive trend, produced a relatively small increase in the level of intracellular Ca²⁺. These observations suggest an interrelationship between hormone-binding capacity and the fluctuation of intracellular Ca²⁺ levels. Either hormone binding depends on the availability of Ca²⁺, or, alternatively, the latter depends on the binding capacity. Further studies are required to elucidate the true nature of this interdependence.     

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.     

Studies into hormonal imprinting in a Tetrahymena thermophila mutant incapable of lysosomal enzyme secretion

Reexposure to insulin after primary interaction (hormonal imprinting) was followed by a binding increase in T. pyriformis and by a binding decrease in T. thermophila. The sec. mutant, MS-1 strain of T. thermophila, which is unable of lysosomal enzyme secretion, also showed a binding increase on a second exposure to insulin, from which it follows that alteration of the enzyme secretion, or other factors associated with mutation, accounted for reversion of the trend of imprinting. Thyrotropic hormone (TSH) also gave rise to a negative imprinting in T. thermophila, but did not alter the binding relations of the MS-1 mutant strain.     

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.     

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.     

Digoxin effect and imprinting in the unicellular Tetrahymena

Treatment with digoxin increased, whereas treatment with ouabain decreased, the growth rate of Tetrahymena, but the progeny generations showed an increased mitotic rate after both treatments. A single exposure to digoxin or ouabain accounted for a more than twofold increase in the incorporation (binding) of the cardiac glycosides in the progeny generations.     

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.     

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.     

Influence of enkephalins on the ACTH-induced hormonal imprinting in tetrahymena

Both adrenocorticotrop hormone (ACTH) and the synthetic enkephalins investigated evoked imprinting in Tetrahymena and led to increased hormone binding at further contact with ACTH. Neither molecule evoked, however, imprinting for the enkephalins. The pentapeptide enkephalin containing also proline had the most pronounced imprinting effect and, when given together with ACTH, it increased the imprintatory effect of ACTH considerably. In all the situations investigated the enkephalin tetrapeptide inhibited the positive effect of the enkephalin pentapeptide, whereas it did not influence the imprintatory effect of ACTH. Similarities can be found between the pharmacological and imprinting effects of enkephalin in mammals, and the effects seen in the present investigations.     

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.