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.     

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.     

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.     

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.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.     

Effect of insulin on the incorporation of3H-inositol into the inositol phospholipids (PI,PIP, PIP2) and Glycosyl-phoshatidylinositols (GPIs) of Tetrahymena pyriformis

The unicellular tetrahymena contains inositol phospholipids (PI, PIP, PIP₂) and GPIs. Treatment with 10^–5M insulin decreases the total³H-inositol incorporation and incorporation into PI. 24 h after 10^–6M insulin treatment there is an elevation of these parameters. Second treatment with 10^–6M insulin doubles and 10^–5M decreases these levels. This means that the effect on phosphoinositide turnover by insulin in Tetrahymena is rather concentration dependent. Inositol incorporation into GPIs is also influenced by insulin.     

Long-Term Maintenance of Tetrahymena Spp.

SYNOPSIS. Simple media for Tetrahymena, using rat gut or soybean as the nutrient source are described. Cultures can be maintained in these media up to one year at room temperature.     

Inhibition of insulin receptors by vanadate and ouabain

Insulin binding studies were performed, using cells from 5 non-obese, non-diabetic subjects, on four separate days: 2 were paired control studies to demonstrate precision, and 2 other sets were binding studies in which one incubation solution was a control and the other contained either vanadate, (10(-4) M) or ouabain (10(-4) M). For both substances tracer binding of 125I insulin was reduced significantly, 27% by vanadate and 30% by ouabain. Furthermore, at all points on the binding curve these substances inhibited binding by 18-98%, in a pattern consistent with reduced receptor number. The concentrations of vanadate or ouabain which we used did not change cell volume or inhibit trypan blue dye exclusion, as an index of cell viability. Because vanadate and ouabain inhibit Na+K+ATPase and have largely dissimilar effects on a variety of cell systems, our observations may reflect specific involvement of Na+K+ATPase in binding or closely related processes.     

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.     

Influence of imprinting with A and B chains of insulin on binding and functional changes in tetrahymena

Insulin and its A and B chain increased the quantity of intracellular PAS-positive material (glycogen) in tetrahymena, whereas the combined A+B chains decreased it. Imprinting—previous interaction—with insulin, its A and B chains in themselves and with the A+B chain increased the hormone binding capacity of tetrahymena, but the functional effect of imprinting (storage or breakdown of glycogen) showed a different tendency with insulin and A+B chain on the one hand, and A chain and B chain on the other. Since the imprinting potential of a molecule promotes the induction of receptor formation, the fact remains that both component chains of insulin were able to act as potential imprinters, although the A chain was superior to the B chain in this respect throughout, and combined treatment with the A+B chain ultimately induced the formation of a similar binding site as insulin itself.     

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.     

Insulin antagonizes the phagocytosis stimulating action of histamine in Tetrahymena

Histamine increased specifically the phagocytic activity of the unicellular Tetrahymena, whereas insulin had no influence on it. Insulin antagonized the phagocytosis stimulating action of histamine after simultaneous exposure and after preexposure two days earlier as well, although in the latter case to a lesser degree. Double exposure to a combination of histamine + insulin didn't influence the phagocytic activity at all, demonstrating the histamine antagonizing effect of insulin in this model.     

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.     

Molecularly imprinted copolymer membranes functionalized by phase inversion imprinting for uracil recognition and permselective binding

Uracil (URA) was selected as a template for preparing molecularly imprinted membranes of poly(acrylonitrile-co-methylacrylic acid) [P(AN-co-MAA)] using the phase inversion technique. This study used Fourier transform infra-red (FT-IR) and (1)H nuclear magnetic resonance (NMR) spectroscopic studies to characterize the polymer-template interaction and scanning electron microscopy (SEM) and atomic force microscopy (AFM) for morphology of the URA imprinted membrane. Resultant membranes had typical ultrafiltration structure with porous morphology and showed a permeation flux of 3.5 x 10 9-5)m(3)/(m(2)s) for 32 microM URA aqueous solution. Permselective binding to the target molecule was observed in permeation experiments with 7.9 micromol/g binding capacity of URA. Binding selectivity was discussed for URA and its analogs, dimethyluracil (DMURA) and caffeine (CAF), with 0.6 and 0.8 micromol/g binding capacity, respectively.     

Imprinting Effects of Proline Containing Dipeptides (Proline-Glycine, Proline-Leucine, Proline-Valine and Their Retro Variants) in Tetrahymena. Evolutionary Conclusions

Proline-glycine, proline-leucine and proline-valine dipeptides and their retro variants were used in the experiments to study the effects of pretreatment (imprinting) in Tetrahymena, by investigating fluorescein isothiocyanate (FITC)-conjugated peptide binding. The protozoan organism could differentiate between the proline-dipeptides containing different partner amino-acids and between the dipeptides having the amino acids in reversed positions. The effect of imprinting was positive or negative and this was dependent on the type of the partner amino acid and on its position. Pro-Gly and Pro-Leu induced positive imprinting (elevated FITC-dipeptide binding) and Pro-Val induced negative imprinting (decrease of FITC-peptide binding). There was positive imprinting induction in two cases for the retro FITC-peptide and in one case for the FITC-conjugate of the imprinter peptide itself. The highest positive imprinting (almost 60% increase) was induced by Pro-Gly for FITC-Gly-Pro. Considering earlier—chemotaxis—experiments, the results of the present—binding—studies run parallel with the physiological effects. The experiments call attention to the sharp differentiating ability of small peptides at a unicellular level, that could have some role in the selection of molecules for hormone formation, during evolution.     

Control of insulin receptor affinity by a Ca2+-sensitive binding site

Calcium (Ca²⁺) increased insulin-receptor binding in both membrane and solubilised receptor preparations. Ca²⁺ increased both receptor affinity and initial rate of association of [¹²⁵I]insulin to the receptor preparations. Ca²⁺ had no effect on insulin receptor number in either receptor preparation. The effect of Ca²⁺ on affinity could be mimicked by ions with similar ionic radii and properties (e.g., Ba²⁺, Mg²⁺ and Sr²⁺). EDTA and oleic acid reduced insulin binding and receptor affinity and these effects were reversed by the addition of Ca²⁺. These studies suggest that Ca²⁺ and Ca²⁺-like ions may bind to a site on or near the receptor and may be responsible for a conformational change with a consequent increase in receptor affinity.     

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Subchronic treatment with vanadate does not potentiate the toxicity of cardiac glycosides

Since it has been claimed that vanadate is an endogenous regulator of Na/K-ATPase activity and that it potentiates the toxicity of cardiac glycosides, we were alarmed to discover that certain Finnish physicians were prescribing vanadate in combination with other trace minerals to elderly patients for many different chronic diseases (e.g., cancer, rheumatism). To study the interaction of vanadate and cardiac glycosides, we fed vanadate in the drinking water (25 μg/mL) to guinea pigs for 20 d, and studied either their sensitivity to the acute toxicity of the cardiac glycoside ouabain or whether the vanadate would influence the subacute toxicity of ouabain. Vanadate had no influence on the toxicity of ouabain either acute or subchronically administered, nor was there any sign of inhibition of Na/K-ATPase activity as measured by⁸⁶Rb-uptake into intact erythrocytes (RBCs), RBC content of sodium or potassium or Na/K-ATPase activity in RBC membranes prepared from the vanadate-treated guinea pigs. Vanadate had been absorbed in substantial quantities from the gastrointestinal tract, since serum, heart, liver, and especially kidney contained measurable amounts of vanadium in contrast to controls, but it is concluded that this vanadate is not in a biologically active form.