Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Light microscopic autoradiographic visualization of [3H]-arginine vasopressin binding sites in rat brain

Specific [³H]-arginine vasopressin ([³H]-AVP) binding sites were identified in the rat brain by light microscopic autoradiography. Discrete intrahypothalamic nuclei were densely labelled by [³H]-AVP. High specific binding was observed in the paraventricular and supraoptic nuclei. These binding sites may represent specific receptors for AVP, postulated to exist in the mammalian central nervous system.     

Localization of binding sites for atrial natriuretic factor and angiotensin II in the central nervous system of the clawed toad Xenopus laevis

Summary The distribution of binding sites for atrial natriuretic factor (ANF) and angiotensin II (A II) was investigated in the central nervous system (CNS) of the clawed toad Xenopus laevis by means of in vitro autoradiography using [¹²⁵I]-rat ANF(99–126) or [¹²⁵I] [Val⁵] A II and [¹²⁵I]human A II as labeled ligands. The highest densities of specific ANF-binding were detected in the nucleus habenularis, thalamic regions, hypophyseal pars nervosa and nucleus interpeduncularis. Moderate ANF-binding was found in the bulbus olfactorius, pallium, septum, striatum, lateral forebrain bundle, nucleus infundibularis, hypophyseal pars distalis and tectum. The highest levels of specific A II binding sites were observed in the nucleus praeopticus, nucleus habenularis, hypophyseal pars nervosa and pars distalis, whereas the amygdala contained moderate A II binding. The existence of specific binding sites for ANF and A II in the CNS of Xenopus laevis suggests that both peptides act as neurotransmitters or neuromodulators in the amphibian CNS. The co-localization of dense binding sites for both peptides in the nucleus habenularis, hypophyseal pars nervosa and pars distalis supports the view that ANF and A II have opposite regulatory functions in these regions.     

The structure of the tetanus toxin reveals pH‐mediated domain dynamics

The tetanus neurotoxin (TeNT) is a highly potent toxin produced by Clostridium tetani that inhibits neurotransmission of inhibitory interneurons, causing spastic paralysis in the tetanus disease. TeNT differs from the other clostridial neurotoxins by its unique ability to target the central nervous system by retrograde axonal transport. The crystal structure of the tetanus toxin reveals a “closed” domain arrangement stabilised by two disulphide bridges, and the molecular details of the toxin's interaction with its polysaccharide receptor. An integrative analysis combining X‐ray crystallography, solution scattering and single particle electron cryo‐microscopy reveals pH‐mediated domain rearrangements that may give TeNT the ability to adapt to the multiple environments encountered during intoxication, and facilitate binding to distinct receptors.     

Reduction of Bacillus thuringiensis Cry1Ac toxicity against Helicoverpa armigera by a soluble toxin-binding cadherin fragment

A cadherin-like protein has been identified as a putative receptor for Bacillus thuringiensis (Bt) Cry1Ac toxin in Helicoverpa armigera and plays a key role in Bt insecticidal action. In this study, we produced a fragment from this H. armigera Cry1Ac toxin-binding cadherin that included the predicted toxin-binding region. Binding of Cry1Ac toxin to this cadherin fragment facilitated the formation of a 250-kDa toxin oligomer. The cadherin fragment was evaluated for its effect on Cry1Ac toxin-binding and toxicity by ligand blotting, binding assays, and bioassays. The results of ligand blotting and binding assays revealed that the binding of Cry1Ac to H. armigera midgut epithelial cells was reduced under denaturing or native conditions in vitro. Bioassay results indicated that toxicities from Cry1Ac protoxin or activated toxin were reduced in vivo by the H. armigera cadherin fragment. The addition of the cadherin fragment had no effect on Cry2Ab toxicity.     

Affinity purified tetanus toxin binds to isolated chromaffin granules and inhibits catecholamine release in digitonin-permeabilized chromaffin cells

Tetanus toxin, a potent neurotoxin which blocks neurotransmitter release in the CNS, also inhibits Ca2+-induced catecholamine release from digitonin-permeabilized, but not from intact bovine chromaffin cells. In searching for intracellular targets for the toxin we studied the binding of affinity-purified tetanus toxin to bovine adrenal chromaffin granules. Tetanus toxin bound in a neuraminidase-sensitive fashion to intact granules and to isolated granule membranes, as assayed biochemically and visualized by electron microscopic techniques. The binding characteristics of the toxin to chromaffin granule membranes are very similar to the binding of tetanus toxin to brain synaptosomal membranes. We suggest that the toxin-binding site is a glycoconjugate of the G1b type (a polysialoganglioside or a glycoprotein-proteoglycan) which is localized on the cytoplasmic face of the granule membrane and might directly be involved in exocytotic membrane fusion.     

MEMBRANE POTENTIAL DEPENDENT BINDING OF SCORPION TOXIN TO THE ACTION POTENTIAL SODIUM IONOPHORE. STUDIES WITH A 3-(4-HYDROXY 3-[125I] IODOPHENYL) PROPIONYL DERIVATIVE

Abstract— A polypeptide toxin purified 80-fold from the venom of the scorpion Leiurus quinquestriatus enhances activation of the action potential Na⁺ ionophore by the alkaloid neurotoxins veratridine, batrachotoxin and aconitine in electrically excitable neuroblastoma cells. The purified toxin can be labelled with [¹²⁵I] by reaction with N-succinimidyl 3-(4-hydroxy 3-[¹²⁵I] iodophenyl) propionate. The [¹²⁵I] labelled toxin obtained from carboxymethyl Sephadex ion exchange chromatography appears homogeneous by gel electrophoresis and isoelectric focusing. The [¹²⁵I] labelled toxin binds to a single class of saturable binding sites and also activates the action potential Na⁺ ionophore in electrically excitable neuroblastoma cells showing identical concentration dependence for both the binding and the activation effects. The labelled toxin does not show any saturable binding or activation of the action potential Na⁺ ionophore in variant neuroblastoma clones that specifically lack the action potential Na⁺ ionophore. The results indicate that scorpion toxin binds specifically to the action potential Na⁺ ionophore. The binding sites have a mean equilibrium dissociation constant of 3 IIH, a mean binding capacity of 46fmol toxin per mg cell protein and a mean density of 24 sites per μm² of cell surface membrane. A single action potential Na⁺ ionophore transports 1 × 10⁸ ions per min and has a conductance of 3 psiemens at physiologic ion concentrations. Depolarization of cells by elevated K⁺ concentration inhibits the saturable binding. Depolarization of cells by incubation in high Na⁺ medium (130mm -Na⁺, 5mm -K⁺) with gramicidin A or batrachotoxin also inhibits the saturable toxin binding. These results suggest that scorpion toxin binds specifically to a regulatory component (gate) of the Na⁺ ionophore. whose conformation is dependent on membrane potential.     

SV2 mediates entry of tetanus neurotoxin into central neurons

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.     

Temperature-Mediated Interaction of Tetanus Toxin with Cerebral Neuron Cultures: Characterization of a Neuraminidase-Insensitive Toxin-Receptor Complex

Abstract: Energy-dependent internalization of ¹²⁵I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0–4°C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0–4°C; the effect is further amplified at 37°C. Addition of disialoganglioside G_D1b (1–5 μg/ml) also lowers the amount of cell-associated toxin. The fraction of ¹²⁵I-labeled toxin retained by the cells after exposure to high-salt medium at 0–4°C or after addition of G_D1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37°C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.     

Preliminary histochemical and electron microscopic observations on the nervous system of hydra

The nervous system of a primitive Coelenterate (Chlorohydra viridissima) has been studied using ultrastructural and histochemical techniques. The authors confirm the ultrastructural pattern of nerve cells described by Lentz and coworkers. Reserpine treatment fails to induce any reduction of catechol- and indoleamine content visible to histochemical observation. In vivo treatment with tetanus toxin does not induce behavioural changes and no specific binding of toxin is revealed by immunocytological analysis. This suggests that neuron tetanus toxin receptor sites are absent in hydra. Hydra nerve cells must therefore be considered as extremely primitive elements, which the Authors consider to support the hypothesis of neurons having originated as a gradual differentiation of myoepithelial cells, as proposed by Pantin (1956) and by Passano (1963).     

Synaptic binding sites in brain for [3H]β-bungarotoxin — A specific probe that perturbs transmitter release

A neurotoxic, [³H]labelled derivative of β-bungarotoxin, known to inhibit neurotransmitter release and to be free of phospholipase activity, was used to demonstrate autoradiographically the distribution and ultrastructural location of its saturable binding component in brain. Light-microscope autoradiography of rat cerebellum and hippocampus showed that it resides primarily in synaptic-rich areas, with much lower densities of sites in other regions containing cell bodies; also, little binding was associated with myelinated tracts. Ultrastructural localisation and sub-fractionation studies on purified cerebrocortical synaptosomes showed that [³H]toxin binding sites are located predominantly on brain synaptosomal membranes, consistent with their possible association with transmitter release.     

Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations

A recently described new method for determination of killer toxin activity was used for kinetic measurenments of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (K _T1=2.6x10⁹ L.U./ml, V _max1=0.19 s^-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (K _T2=3.2x10⁷ L.U./ml, V _max2=0.03 s^-1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occured within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.     

Tetanus toxin binds with high affinity to neuroblastoma x glioma hybrid cells NG 108-15 and impairs their stimulated acetylcholine release

Differentiated neuroblastoma x glioma hybrid cells NG 108-15 express on their surface specific binding sites for tetanus toxin. 450 sites/cell with a KD of 2 x 10(-11) M were found under "physiological" conditions of pH and salt concentrations. A Hill coefficient of 1.1 indicated noncooperative binding. Specific binding of 125I-toxin to its sites could be prevented either by preincubation of the toxin with a neutralizing monoclonal antibody or by pretreatment of the cells with neuraminidase (Vibrio cholerae). To quantify the action of tetanus toxin on the stimulated release of 14C activity from differentiated cells preincubated with [14C]choline, a new type of perfusion device was designed which could be filled with cells growing in monolayers on Cytodex-3 microbeads. Tetanus toxin inhibited the stimulated 14C release in a time- and dose-dependent manner. A greater than 50% inhibition was found after 2 h of incubation with 10(-12) M toxin. The inhibitory action of tetanus toxin could be prevented with a monoclonal antibody to the toxin or with neuraminidase treatment of the cells. These results suggest that the neuraminidase-sensitive 2 x 10(-11) KD receptors are the productive receptors for tetanus intoxication in differentiated NG 108-15 cells. The possible chemical composition of these receptors is discussed. Differentiated NG 108-15 cells provide a useful model in which picomolar tetanus concentrations produce both measurable saturable binding and inhibition of potassium-evoked, acetylcholine release under physiological conditions of pH and salt concentrations.     

α-Bungarotoxin binding in the central nervous system of Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin in the central nervous system of the horseshoe crab, Limulus polyphemus, was investigated. Comparative binding studies in various tissues of L. polyphemus demonstrated a selective association of the toxin with nervous tissues. The greatest enrichment of toxin binding in subcellular fractions of brain tissue was observed in a fraction enriched in mitochondria and acetylcholinesterase-containing membranes. Autoradiographic studies revealed the localization of α-bungarotoxin binding to the longitudinal connectives and neuropile regions of the abdominal ganglia. Three toxin binding components with approximate sedimentation coefficients of 9 S, 15.4 S and 17.4 S were present in solubilized extracts of brain tissue. ¹²⁵I-labeled α-bungarotoxin binding to these components was inhibited 72%, 47%, 9% and 0% by 10 μM concentrations of (+)-tubocurarine, nicotine, scopolamine and pilocarpine, respectively. The apparent formation of the 15.4 S and 17.4 S proteins from the 9 S protein was obtained. The 15.4 S and 17.4 S components are suggested as aggregates of the 9 S protein. This 9 S protein is proposed as an acetylcholine receptor from the central nervous system of L. polyphemus.     

Altered Glycosylation of 63- and 68-Kilodalton Microvillar Proteins in Heliothis virescens Correlates with Reduced Cry1 Toxin Binding, Decreased Pore Formation, and Increased Resistance to Bacillus thuringiensis Cry1 Toxins

The binding and pore formation abilities of Cry1A and Cry1Fa Bacillus thuringiensis toxins were analyzed by using brush border membrane vesicles (BBMV) prepared from sensitive (YDK) and resistant (YHD2) strains of Heliothis virescens. ¹²⁵I-labeled Cry1Aa, Cry1Ab, and Cry1Ac toxins did not bind to BBMV from the resistant YHD2 strain, while specific binding to sensitive YDK vesicles was observed. Binding assays revealed a reduction in Cry1Fa binding to BBMV from resistant larvae compared to Cry1Fa binding to BBMV from sensitive larvae. In agreement with this reduction in binding, neither Cry1A nor Cry1Fa toxin altered the permeability of membrane vesicles from resistant larvae, as measured by a light-scattering assay. Ligand blotting experiments performed with BBMV and ¹²⁵I-Cry1Ac did not differentiate sensitive larvae from resistant larvae. Iodination of BBMV surface proteins suggested that putative toxin-binding proteins were exposed on the surface of the BBMV from resistant insects. BBMV protein blots probed with the N-acetylgalactosamine-specific lectin soybean agglutinin (SBA) revealed altered glycosylation of 63- and 68-kDa glycoproteins but not altered glycosylation of known Cry1 toxin-binding proteins in YHD2 BBMV. The F1 progeny of crosses between sensitive and resistant insects were similar to the sensitive strain when they were tested by toxin-binding assays, light-scattering assays, and lectin blotting with SBA. These results are evidence that a dramatic reduction in toxin binding is responsible for the increased resistance and cross-resistance to Cry1 toxins observed in the YHD2 strain of H. virescens and that this trait correlates with altered glycosylation of specific brush border membrane glycoproteins.     

Agonist-induced affinity alterations of a central nervous system alpha-bungarotoxin receptor

Abstract— The ability of cholinergic agonists to block the specific interaction of α-bungarotoxin (α-Bgt) with membrane-bound sites derived from rat brain is enhanced when membranes are preincubated with agonist. Thus, pretreatment of α-Bgt receptors with agonist (but not antagonist) causes transformation of sites to a high-affinity form toward agonist. This change in receptor state occurs with a half-time on the order of minutes, and is fully reversible on dilution of agonist. The results are consistent with the identity of α-Bgt binding sites as true central nicotinic acetylcholine receptors. Furthermore, this agonist-induced alteration in receptor state may represent an in vitro correlate of physiological desensitization. As determined from the effects of agonist on toxin binding isotherms, and on the rate of toxin binding to specific sites, agonist inhibition of toxin binding to the high-affinity state is non-competitive. This result suggests that there may exist discrete toxin-binding and agonist-binding sites on central toxin receptors.     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Localization of Bacillus thuringiensis Cry1A toxin-binding molecules in gypsy moth larval gut sections using fluorescence microscopy

The microbial insecticide Bacillus thuringiensis (Bt) produces Cry toxins, proteins that bind to the brush border membranes of gut epithelial cells of insects that ingest it, disrupting the integrity of the membranes, and leading to cell lysis and insect death. In gypsy moth, Lymantria dispar, two toxin-binding molecules for the Cry1A class of Bt toxins have been identified: an aminopeptidase N (APN-1) and a 270 kDa anionic glycoconjugate (BTR-270). Studies have shown that APN-1 has a relatively weak affinity and a very narrow specificity to Cry1Ac, the only Cry1A toxin that it binds. In contrast, BTR-270 binds all toxins that are active against L. dispar larvae, and the affinities for these toxins to BTR-270 correlate positively with their respective toxicities. In this study, an immunohistochemical approach was coupled with fluorescence microscopy to localize APN-1 and BTR-270 in paraffin embedded midgut sections of L. dispar larvae. The distribution of cadherin and alkaline phosphatase in the gut tissue was also examined. A strong reaction indicative of polyanionic material was detected with alcian blue staining over the entire epithelial brush border, suggesting the presence of acidic glycoconjugates in the microvillar matrix. The Cry1A toxin-binding sites were confined to the apical surface of the gut epithelial cells with intense labeling of the apical tips of the microvilli. APN-1, BTR-270, and alkaline phosphatase were found to be present exclusively along the brush border microvilli along the entire gut epithelium. In contrast, cadherin, detected only in older gypsy moth larvae, was present both in the apical brush border and in the basement membrane anchoring the midgut epithelial cells. The topographical relationship between the Bt Cry toxin-binding molecules BTR-270 and APN-1 and the Cry1A toxin-binding sites that were confined to the apical brush border of the midgut cells is consistent with findings implicating their involvement in the mechanism of the action of Bt Cry toxins.     

Uptake,distribution and binding of vertebrate and invertebrate steroid hormones and time-dependence of ponasterone A binding in Calliphora vicina

Summary The presence of specific binding sites for radiolabelled vertebrate-type and arthropod-type steroid hormones was investigated in several organs including salivary gland, and central nervous system of third instar Calliphora vicina larvae by thaw-mount autoradiography. Ponasterone A, a 20-hydroxyecdysone agonist and 20-hydroxyecdysone are the only steroids which bind to nuclear high affinity binding sites. These binding sites are DNA associated while nucleoli show no tracer binding. Ecdysone, an endogenous 20-hydroxyecdysone precursor, is taken up by target cells but no significant nuclear binding occurs. 1,25-Dihydroxyvitamin D₃ concentrates in cytoplasm only and its uptake is highest compared to all other steroids. Progesterone and testosterone show weak accumulation in the cytoplasm, while for cholesterol, corticosterone, cortisol, dexamethasone, dihydrotestosterone and estradiol-17, no noticeable uptake occurs. For ponasterone A, a clear time dependence of uptake and intracellular distribution is visible, suggesting the existence and involvement of specific ecdysteroid uptake and transport mechanisms. These results suggest the presence of binding sites for various mammalian steroids in insects. Whether vertebrate steroid hormones or metabolites of them play a role in insects or whether the uptake and binding is based on chemical similarities alone without biological significance remains to be further investigated.