Evidence of the receptor nature of the binding sites induced in Tetrahymena by insulin treatment. A quantitative cytofluorimetric technique for the study of binding kinetics

Tetrahymena pyriformis GL cells pretreated (imprinted) and not pretreated with insulin showed dissimilar quantitative relations of FITC-insulin binding. Displacement of FITC-insulin by unlabelled insulin was considerably less in the control than in the imprinted series. The curve for saturation of the binding sites with FITC-insulin resembled a true saturation curve. The imprinted cells bound considerably more hormone in a shorter time than the control cells at identical levels of exposure. The dissociation of bound hormone from the imprinted cells increased over the control at 23 degrees C, and to a still greater degree at 4 degrees C. The effect of the pH of the medium on the dissociation of bound FITC-insulin also differed between the imprinted and not imprinted cells. Thus the proposed cytofluorimetric assay of binding kinetics demonstrated the actual conditions of receptor activity, and indicated that the induced insulin binding sites of Tetrahymena behaved similarly to 'classical' receptors.     

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.     

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 starvation on insulin production and insulin binding in Tetrahymena

Tetrahymena pyriformis GL was starved for 24 h and then the immunologically demonstrable insulin content and FITC-insulin binding were measured by flow cytometry and localization was studied by confocal microscopy. The amount of endogeneous insulin as well as FITC insulin binding, was highly significantly elevated. Glucose feeding for 30 min abolished the elevation of FITC-insulin binding. In starved cells, insulin-binding sites disappeared from the surface and FITC-insulin was bound inside the cells, within large food vacuoles. Endogeneous insulin was dispersed in the cytoplasm both in the control and starved cells and food vacuoles did not contain it. The results call attention to the stimulatory effect of starvation on insulin production in Tetrahymena, in parallel with the internal storage of insulin receptors, which points to an autocrine mechanism.     

Comparison of the insulin binding, uptake and endogenous insulin content in long- and short-term starvation in Tetrahymena

FITC-insulin binding and endogenous insulin content of Tetrahymena pyriformis, that had been 24 h or 30 min starved, continuously fed or re-fed after starvation was studied by flow cytometry and confocal microscopy. Long starvation elevated both insulin binding and endogenous insulin content of the cells. Short re-feeding after long starvation or short starvation after continuous feeding does not change the situation. Fixed cells also bind FITC-insulin, however, in this case long starvation reduces, and re-feeding after long starvation elevates, the binding, which means that hormone binding by receptors only differs from receptor binding and engulfment (in living cells). The increase of FITC-insulin content in living cells seems to be due to engulfment, rather than by receptor binding. The results point to the unicellular organism's requirement for insulin production and binding in a life-threatening stress situation.     

Starved Tetrahymena Synthesize and Maintain an Excess of 5s rRNA in Their Cytoplasm1

We have measured the rate of accumulation of newly synthesized 5s ribosomal RNA (5s rRNA) in Tetrahymena thermophila cells in early log phase growth and in cells that had been starved in a dilute salt solution. From these measurements we have determined the rates of synthesis and levels of accumulation of 5s rRNA relative to 5.8s rRNA in these two different cell populations. In growing cells 5s rRNA is transcribed and accumulated in a 1:1 molar ratio when compared with 5.8s rRNA. In contrast, in starved cells, 5s rRNA is produced at a rate which is about 15% higher than that seen for 5.8s rRNA. This excess 5s rRNA accumulates in the cytoplasm in a non-ribosomal form and is maintained in the cell as long as the cell remains in a starved condition. The role this excess 5s rRNA may play in the control of 5s rRNA gene expression is discussed.     

Comparison of the effect of hormones on the hormone synthesis of Tetrahymena in medium or salt solution

Tetrahymena pyriformis was maintained in TYM (tryptone‐yeast medium) as well as in Losina salt solution. One hour treatment of 10^?15 M histamine, serotonin or insulin was given before the histamine, serotonin, triiodothyronine and adrenocorticotropin contents of the cells were measured by flow cytometry after immunocytochemical staining. Maintenance in salt solution increased the hormone level in the cells, and use of the treatment hormone treatments further increased the endogenous hormone content relative to that in medium. The cells in salt mimic better the natural conditions, which means that the effects of hormones under more natural conditions are expressed to a greater extent than the exogenously given hormones in TYM typically used under laboratory conditions. Intercellular hormonal communication between the cells of a Tetrahymena population might assist in the survival of the individual cells.     

Increased hormone levels in Tetrahymena after long-lasting starvation

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 24h; (2) starved for 24h and re-fed for 30min or (3) starved for 30min. Amount and localization of vertebrate-like hormones, produced by Tetrahymena, beta-endorphin, adrenocorticotropin (ACTH), serotonin, histamine, insulin and triiodothyronine (T(3)) 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 T(3) 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 life-threatening effect of stress.     

Presence and localization of histidine decarboxylase enzyme (HDC) and histamine in Tetrahymena pyriformis.

Histidine decarboxylase (HDC) enzyme and its function under hormonal influences were studied in a low level of phylogeny. HDC protein is present in the unicellular ciliate Tetrahymena and its expression was not altered by insulin or histamine treatment. Starvation for 24 h enormously decreased the quantity of histamine in the cells. However, insulin influenced the activity of the HDC enzyme, demonstrated by the seven-fold quantity of histamine in the starved cells after insulin treatment. Insulin also increased the uptake of histamine from the tryptone-yeast extract medium. HDC was found in different parts of the cytoplasm, mainly in the periphery (epiplasm) of the cells. The experiments demonstrated the uptake and synthesis of histamine by Tetrahymena as well as the possibility of hormonal regulation of HDC activity.     

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.     

Impact of histamine treatment and feeding on the hydrolytic enzyme level of Tetrahymena in culture and in conditions of starvation

• 1.1. The unicellular Tetrahymena pyriformis contains and also produces hydrolytic enzymes, such as glucosidase, phosphatase and glucosaminidase.
• 2.2. Return of Tetrahymena to plain medium after treatment with bacteria alone, histamine alone or bacteria plus histamine was equally followed by persistence of the hydrolytic enzyme activity around the control value and an activity increase at about 60 min.
• 3.3. Incubation of Tetrahymena in salt (Losina-Losinsky) solution after the applied treatment accounted for reduction to practically zero of the glucosidase and glucosaminidase activities, whereas the phosphatase activity tended to increase rather than to decrease in both the bacterium-treated and histamine-treated cultures.
• 4.4. The enzyme activity patterns of the Tetrahymena cells pretreated (imprinted) with histamine did not differ from the control pattern either after re-exposure to histamine or after feeding with bacteria, but showed a uniformization of the activity pattern and a considerable decrease in enzyme activity on incubation (starvation) in salt (Losina-Losinsky) solution.

     

Impact of combined hormonal pretreatment (insulin+TSH) on the imprinting of hormones administered in combination to Chinese hamster ovary cell culture.

Cultured Chinese hamster ovary (CHO) cells were treated (imprinted) with insulin and with thyrotropin (TSH) related to gonadotropins (FSH+LH). When one week later the treatment was repeated with one of the hormones, considerable differences could be observed in the binding capacity of the cells. In the hormone combination TSH was able to evoke persistent imprinting only to a markedly lesser degree than insulin, meanwhile the imprintatory effect of insulin was of greater extent even on the cell regarded to be unspecific for insulin. Hormone treatment of one hour duration--when investigated immediately after--did not extinct the binding capacity to TSH but enhanced that to insulin. With the deterioration of the conditions of culturing, the enhanced binding capacity disappeared.     

Insulin treatment (hormonal imprinting) increases the insulin production of the unicellular Tetrahymena long term. Is there a simultaneous formation of hormone receptor and hormone?

Insulin treatment of Tetrahymena pyriformis resulted in a long-lasting increase in the insulin content of the cells. After about 200 generations, insulin levels significantly higher than in the control were demonstrated by using a quantitative immunocytochemical method. Although the insulin content fluctuated from day to day, it was always higher in the insulin pre-treated (imprinted) cells than in the controls. The results emphasise the simultaneous stimulation of hormone and receptor formation by hormonal imprinting.     

Studies into secretions of Tetrahymena: enzymes secreted into inorganic medium.

The peptides secreted by Tetrahymena cells into inorganic medium were chromatographed. Six fractions showing a marked enzyme-like activity were examined for influence on certain physiological parameters of Tetrahymena. The enzymatically active fractions increased the phagocytic activity of Tetrahymena and decreased its binding capacity for lectins and hormone (insulin), but enhanced insulin imprinting at primary interaction. It remains to be clarified whether these effects were due to the enzymatic or other components of the fractions investigated, or to lack of the compensatory influence of the fractions not studied.     

Impact of 5-azacytidine on insulin binding and insulin-induced receptor formation in tetrahymena.

The unicellular Tetrahymena is able to bind the vertebrate hormone insulin, and the binding sites presented by it become amplified under hormonal influence. The increased binding capacity for insulin reappears in many offspring generations. 5-azacytidine inhibits insulin binding and the insulin-induced formation of binding sites as well in the cell generation directly involved in interaction, but enhances insulin binding in the daughter cell generations. The nutrient medium of the cells whose binding capacity was enhanced by azacytidine treatment transmitted the information accounting for increased binding to "virgin" cells not previously treated with azacytidine.     

Increase of Growth Rate and Phagocytic Activity of Tetrahymena Induced By Pseudomonas Lectins

Galactosephilic and mannosephilic lectins from Pseudomonas aeruginosa interact with Tetrahymena pyriformis GL. Specific adsorption of these lectins onto the Tetrahymena can be shown by inhibition of hemagglutination and by peroxidase binding to the cells mediated by the mannosephilic lectins. Interaction with the lectins does not agglutinate the protozoa even after immobilization by Na fluoride, formaldehyde, and glutaraldehyde or after papain treatment. However, inclusion of the lectins in the growth medium increases the growth rate of Tetrahymena and their presence in the medium supplied to starved ciliates increases phagocytosis of Chinese ink and vacuolization.     

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.     

Hormonal effects on Tetrahymena: change in case of combined treatment

In order to approach their natural conditions, populations of Tetrahymena were kept in Losina-Losinky's salt solution for 1 h, than in the tryptone+yeast medium. During this time they were treated with histamine, serotonin or insulin, or with the combinations of these hormones. Effect of the combined treatments on the production of serotonin (5HT), or adrenocorticotropic hormone (ACTH) or triiodothyronine (T?) by the cells was compared to the effect of single-hormone treatments. Significant differences were seen between the results obtained following the single or combined treatments. There was no summation of the effects, however an elevation or diminution of the hormone production was observed after the combined treatment, as compared with the untreated controls or with the use of one of the hormones in the samples. The experiments demonstrate that there is a hormonal regulation between the Tetrahymena cells and the hormones influence each other's effect.     

Chemical reception mechanisms at a low level of phylogeny. Influence of polypeptide hormones and non-hormone polypeptides on the growth of Tetrahymena

The polypeptide hormones insulin, glucagon, thyrotropin (TSH), pregnant mare serum gonadotropin (PMSG) and adrenocorticotropin (ACTH) stimulated the growth of the Tetrahymena, and the non-hormone polypeptides (bovine serum albumin (BSA), protamine) had a similar effect. Re-exposure after 24 h accounted for a greater growth stimulation than pre-exposure alone in cultures treated with TSH and PMSG, and re-exposure after 7 days had such effect in all polypeptide-treated cultures. It follows that the non-hormone polypeptides had a similar imprinting potential to the polypeptide hormone. The non-hormone polypeptides were also able to cross-imprint for one another, i.e. pre-exposure to one enhanced the binding capacity of the cells for the other on re-exposure, and vice versa. A single treatment with a polypeptide hormone or a non-hormone polypeptide did in itself stimulate the growth of the Tetrahymena for as long as 1 week.     

Permanence of the cell-to-cell transmission of insulin induced hormonal imprinting

When insulin-treated (imprinted) Chang liver cell cultures were mixed with cultures which did not receive insulin treatment the information of imprinting was transmitted to the cultures which were not in direct contact with insulin. The ability of the cells to transmit imprinting was long lasting and could be detected even after four weeks, when it was nearly of the same degree as at the first measurement. Difference was found between the binding capacity of the receptors of the plasma membrane and those of the nuclear membrane.