High affinity, specific binding of [125I] beta nerve growth factor to glass beads

The binding of [¹²⁵I] β nerve growth factor to glass beads was studied. It was found that [¹²⁵I] β nerve growth factor exhibited high affinity, specific binding to glass beads. This binding cannot be explained as radioactivity being occluded in the spaces between the glass beads. The binding appears to be nonsaturable under the conditions used. Binding is complete in less than ten minutes with a half-time of two minutes. The binding appears similar to that seen for receptor binding on responsive cells.     

2[125I]iodomelatonin binding sites in spleens of guinea pigs.

2-[125I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8 +/- 4.12 pmol/l and binding site density (Bmax) of 0.69 +/- 0.082 fmol/mg protein at mid-light (n = 10). There was no significant change in the Kd (41.8 +/- 3.16 pmol/l) or the Bmax (0.58 +/- 0.070 fmol/mg protein) at mid-dark (n = 10). Kinetic analysis showed a Kd of 23.13 +/- 4.81 pmol/l (mean +/- SE, n = 4), in agreement to that derived from the saturation studies. The 2-[125I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than N-acetylserotonin, 6-hydroxymelatonin greater than 5-methoxytryptamine, 5 methoxytryptophol greater than serotonin, 5-methoxyindole-3-acetic acid greater than 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan greater than tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction (65.5%), the rest are distributed in the microsomal fraction (17.4%), mitochondrial fraction (14.7%) and cytosolic fraction (0.3%). The demonstration of 2-[125I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system.     

HPLC-purified 2-[125I]iodomelatonin labels multiple binding sites in hamster brain

Binding of 2-[125I]iodomelatonin in hamster brain synaptosomal membranes at 0 degrees C is rapid, saturable, reversible and sensitive to heat and trypsin treatment. Computer resolution of curvilinear Scatchard plots yielded high- and low-affinity components as follows: Kd1 = 0.32 +/- 0.14 nM, Bmax1 = 5.6 +/- 1.7 fmol/mg protein and Kd2 = 10.5 +/- 3.2 nM, Bmax2 = 123 +/- 33 fmol/mg protein (n = 3). Competition experiments indicated that 2-iodomelatonin and prazosin are the most potent inhibitors of high-affinity binding. Unlike prazosin, several alpha-adrenergic agents and various neurotransmitters were ineffective. These findings suggest that prazosin may be a potent antagonist at a unique, non-alpha-adrenergic, high-affinity binding site for melatonin.     

Characterization of picomolar affinity binding sites for [125I]-human calcitonin gene-related peptide in rat brain and heart

We have characterized picomolar affinity binding sites for human calcitonin gene-related peptide (CGRP) in rat brain and heart (atria and ventricle) membranes. By saturation analysis, apparent dissociation constant (KD) values of high affinity sites for [125I]-human CGRP are 9 approximately 15 pM (brain), 34 pM (ventricle) and 85 pM (atria). Low affinity sites with KD values of about 50 nM are found in rat brain and ventricle, but not in atria. Human and rat CGRP potently inhibited [125I]-human CGRP binding to these high affinity sites with apparent inhibition constant (Ki) values comparable to their KD values. Salmon calcitonin marginally inhibited these binding with Ki values between 0.1 microM and 1 microM. Extremely potent cardiovascular and gastrointestinal actions of CGRP might be mediated through CGRP binding sites with picomolar affinity which are similar to those we characterized in this study.     

125I][D-Ala2]deltorphin-I: a high affinity, delta-selective opioid receptor ligand.

The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.     

Radiotoxicity of 5-[123I]iodo-2'-deoxyuridine in V79 cells: a comparison with 5-[125I]iodo-2'-deoxyuridine

The toxic effects of the short-lived (T 1/2 = 13.2 h) Auger-electron-emitting isotope 123I, incorporated in the form of 123IUdR into the DNA of V79 cells in vitro, have been investigated and compared to those of 125IUdR. For the concentrations tested, the rate of incorporation of 123IUdR at any time is proportional to the concentration of extracellular radioactivity. The curve for survival of clonogenic cells decreases exponentially and exhibits no shoulder at low doses. The mean lethal dose (D37) to the nucleus is 79 +/- 9 cGy and is about the same as that obtained previously with 125IUdR. However, the total number of decays needed to produce this D37 with 123IUdR is about twice that required with 125IUdR, approximately equal to the ratio of the energy deposited in microscopic volumes by 125I and 123I, respectively. This correlation suggests that nuclear recoil, electronic excitation, and chemical transmutation are probably of minor importance to the observed biological toxicity with either isotope. The results also indicate that there are no saturation effects in the decay of 125IUdR in the DNA of V79 cells (i.e., all of the emitted energy is biologically effective) and that each of the two steps involved in the 125I decay is equally effective in causing biological damage.     

Photolabeling of human and murine multidrug resistance protein 1 with the high affinity inhibitor [125I]LY475776 and azidophenacyl-[35S]glutathione

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent transporter of structurally diverse organic anion conjugates. The protein also actively transports a number of non-conjugated chemotherapeutic drugs and certain anionic conjugates by a presently poorly understood GSH-dependent mechanism. LY475776is a newly developed (125)I-labeled azido tricyclic isoxazole that binds toMRP1 with high affinity and specificity in a GSH-dependent manner. The compound has also been shown to photolabel a site in the COOH-proximal region of MRP1's third membrane spanning domain (MSD). It is presently not known where GSH interacts with the protein. Here, we demonstrate that the photactivateable GSH derivative azidophenacyl-GSH can substitute functionally for GSH in supporting the photolabeling of MRP1 by LY475776 and the transport of another GSH-dependent substrate, estrone 3-sulfate. In contrast to LY475776, azidophenacyl-[(35)S] photolabels both halves of the protein. Photolabeling of the COOH-proximal site can be markedly stimulated by low concentrations of estrone 3-sulfate, suggestive of cooperativity between the binding of these two compounds. We show that photolabeling of the COOH-proximal site by LY475776 and the labeling of both NH(2)- and COOH- proximal sites by azidophenacyl-GSH requires the cytoplasmic linker (CL3) region connecting the first and second MSDs of the protein, but not the first MSD itself. Although required for binding, CL3 is not photolabeled by azidophenacyl-GSH. Finally, we identify non-conserved amino acids in the third MSD that contribute to the high affinity with which LY475776 binds to MRP1.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Characterization of the [125I]endomorphin-2 binding sites in the MCF7 breast cancer cell line

In the present study, the expression of the micro-opioid receptor on protein level has been demonstrated in MCF7 breast cancer cells. Binding of the [125I]-labeled micro-opioid receptor selective ligand endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) was examined in vitro using a cross-linking assay followed by a Western blot technique. The radioactive complex had a molecular weight of about 65 kDa and was detectable by anti-micro-opioid receptor antibody, indicating the presence of micro-opioid receptors in MCF7 cell membranes. Characterization of endomorphin-2 binding to the membranes obtained from MCF7 cells was performed. Cold saturation experiments with [125I]endomorphin-2 showed biphasic binding curves in Scatchard coordinates. One component represents a high affinity and low capacity, and the other low affinity and higher capacity binding sites. The obtained Bmax values for [125I]endomorphin-2 binding to MCF7 membranes were much higher than those obtained for mouse brain. Pharmacological characterization of the [125I]endomorphin-2 binding sites was made using endomorphin-2 and two other micro selective ligands, morphiceptin, and [D-1-Nal3]morphiceptin on MCF7 cell membrane preparations and whole MCF7 cells. In both cases, the rank order of potency was [D-1-Nal3]morphiceptin>endomorphin-2>morphiceptin, but in case of whole MCF7 cells the IC50 values were about 40 times higher.     

Calcium-induced phosphorylations and [125I]calmodulin binding in renal membrane preparations

Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from th renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [¹²⁵I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of $\text{20 μ}\text{M La}{}^{\mathrm{2+}}$ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.     

Temperature-sensitive high affinity [3H]tryptamine binding sites in rat brain

The influence of prior incubation on [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. A 55 min preincubation of the membranes at 37 degrees C induced an approx. 2.4-fold increase in the specific binding of [3H]ligand to the subsequently washed preparations and this phenomenon was quite temperature-dependent. On the other hand, the proportion of nonspecific binding sites was significantly decreased by 70% of the original sites within 20 min of the start of preincubation. Pargyline, ascorbic acid, EGTA, metal ions (Ca2+, Mg2+, Na+) and guanine nucleotides, included in the preincubation buffer, were all inactive on the stimulation of [3H]tryptamine binding, while the pretreatment of membranes with glutaraldehyde antagonized the augmentation of this binding. Furthermore, it was revealed that the Scatchard plot of the [3H]tryptamine binding preincubated at 0 degree C conformed to a straight line (KD = 33.1 nM, Bmax = 543 fmoles/mg protein), whereas a curvilinear Scatchard plot was obtained at 37 degrees C preincubation. Nonlinear regression analysis of the latter resulted in apparent KD (nM) & Bmax (fmoles/mg protein) values of 0.45 & 102.7 and 33.7 & 603.4 for the high and low affinity sites, respectively. All these observations lead to the inference that the preincubation-induced increase in [3H]tryptamine binding (i.e., nearly high affinity proportion of sites) may occur as a result of temperature-sensitive interconvertible conformational changes.     

The potassium channel opener BRL 38227 inhibits binding of [125I]-labelled endothelin-1 to rat cardiac membranes.

Binding of [125I]-labelled endothelin-1 (ET-1) to rat cardiac membranes and the effects of endothelin-1 (ET-1), endothelin-3 (ET-3), the calcium channel antagonist nifedipine, and both enantiomers of the potassium channel opener cromakalim (BRL 34915) on binding have been examined. Specific binding of [125I]-ET-1 was inhibited in a concentration dependent manner by both unlabelled ET-1 (10(-12)-10(-7) M) and ET-3 (10(-12)-10(-6) M). Nifedipine (10(-11)-10(-5) M) did not affect [125I]-ET-1 binding. However, BRL 38227 (10(-11)-10(-5) M), the biologically active isomer of cromakalim, significantly inhibited [125I]-ET-1 binding. The inactive isomer, BRL 38226 (10(-11)-10(-5) M) had no effect. These findings provide the first evidence for a stereospecific interaction between BRL 38227 and an ET-1 binding site in rat cardiac membranes.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

The effect of oxidation of the Met27 residue of [125I]monoiodoglucagon on receptor-binding affinity

When glucagon is iodinated by the chloramine-T method, the Met27 residue is oxidized. This is not the case when the iodination is performed by the lactoperoxidase method. The two preparations can be purified to the same specific activity using QAE-Sephadex A-25 ion exchange chromatography. Receptor-binding studies in isolated rat adipocytes or hepatocytes revealed that the oxidized from possessed an average-binding affinity which is only about two thirds of that of the non-oxidized form. The reduced affinity of the oxidized tracer cannot be explained by an increased rate of dissociation.     

The opioid growth factor, [Met5]-enkephalin, inhibits DNA synthesis during recornification of mouse tail skin

Opioid peptides serve as tonically active negative growth regulators in renewing and regenerating epithelia. To examine the involvement of opioids in renewal of the stratum corneum after tape stripping of tail skin, C57BL/6 J mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX, 20 mg/kg i.p.) following injury. Blockade of opioidreceptor interaction by NTX for 4 h resulted in an elevation of 36–;66% in basal cell DNA synthesis measured 24 h after injury. Injection of the endogenous opioid peptide, [Met⁵]-enkephalin (OGF, 10 mg/kg i.p.) 4 h before termination, suppressed radiolabelled thymidine incorporation in the basal cell layer by 37–46%at 24 h after wounding. The magnitude of the effects on DNA synthesis of OGF, but not NTX, depended on the timing of administration with respect to injury. OGF maximally depressed basal cell labelling (72%) when given 16 h after tape stripping. Concomitant administration of naloxone (10 mg/kg) with OGF blocked the inhibition of DNA synthesis; naloxone alone at the dosage utilized had no effect on cell labelling. Both OGF and its receptor, OGFr, were detected by immunocytochemistry in the basal and suprabasal cell layers, but not the cornified layer of tape stripped and uninjured tail skin. These results indicate: (a) a native opioid peptide and its receptor are expressed in epidermal cells of injured and uninjured mouse tail skin; (b) removal of the stratum corneum by tape stripping does not disrupt the function of the endogenous opioid growth system; (c) the proliferative response to wounding of the tail is tonically inhibited by the receptor-mediated action of an endogenous opioid peptide; and (d) DNA synthesis by basal cells can be elevated by disrupting opioid peptidereceptor interactions.     

Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes

Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified binding in a dose-dependent manner, leading to 80% decrease with 1 × 10(-4)M concentration. On the other hand, the single addition of 1 × 10(-6)M, 1 × 10(-5)M and 1 × 10(-4)M SR 48692 (Sanofi-Aventis, U.S., Inc.) decreased [(3)H]-ouabain binding (in %) to 87 ± 16; 74 ± 16 and 34 ± 17, respectively. Simultaneous addition of neurotensin and SR 48692 led to additive or synergic effects. Partial NTS2 agonist levocabastine inhibited [(3)H]-ouabain binding likewise. Saturation assays followed by Scatchard analyses showed that neurotensin increased K(d) value whereas failed to modify B(max) value, indicating a competitive type interaction of the peptide at Na(+), K(+)-ATPase ouabain site. At variance, SR 48692 decreased B(max) value whereas it did not modify K(d) value. [(3)H]-ouabain binding was also studied in cerebral cortex membranes obtained from rats injected i. p. 30 min earlier with 100 μg and 250 μg/kg SR 48692. It was observed that the 250 μg/kg SR 48692 dose led to 19% decrease in basal [(3)H]-ouabain binding. After SR 48692 treatments, addition of 1 × 10(-6)M led to additive or synergic effect. Results suggested that [(3)H]-ouabain binding inhibition by neurotensin hardly involves NTS1 receptor.     

Characterization of rat brain opioid receptors by [Tyr-3,5-3H]1,d-Ala2, Leu5-enkephalin binding

[Tyr-3,5-³H]¹,d-Ala², Leu⁵-enkephalin ([³H]DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from [Tyr-3,5-diiodo]¹,d-Ala², Leu⁵-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting [Tyr-3,5-³H]¹,d-Ala², Leu⁵-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24°C within 45 min. The pseudo first order association rate constant was 0.1 min^–1. The dissociation of the receptor-ligand complex was biphasic with k_–1-s of 0.009 and 0.025 min^–1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed aK _D=0.7 nM andB _max=60 fmol/mg protein; the low affinity site had aK _D=5 nM andB _max=160 fmol/mg protein. A series of opioid peptides inhibited [³H]DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds preferentially to the subtype of opioid receptors. Modification of the original peptides either at the C or N terminal ends of the molecules resulted in a decrease in their affinity.     

(125)I]EYF: a new high affinity radioligand to neuropeptide FF receptors

[(125)I]EYF ([(125)I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [(125)I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K(D) = 0.041 and 0.019 nM, respectively).Competition studies showed that [(125)I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity.Autoradiographic studies demonstrated that [(125)I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [(125)I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn.Non specific binding reached 5-10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%.These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [(125)I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.     

Radiotoxicity of 5-[125I]iodo-2'-deoxyuridine in mammalian cells following treatment with 5-fluoro-2'-deoxyuridine.

We have enhanced the uptake of 5-[125I]iodo-2'-deoxyuridine (125IUdR) in Chinese hamster V79 cells with 5-fluoro-2'-deoxyuridine (FUdR) and have examined the combined toxicity of these agents. Although the uptake of 125IUdR increases approximately 3.2 +/- 0.5-fold in the presence of 1 microM FUdR, when cell survival fraction is plotted as a function of intranuclear 125IUdR content, the biphasic curve obtained reaches a plateau at a higher survival fraction than with control cells not exposed to FUdR. The results suggest that a greater number of cells were prevented from entering the S phase and consequently from incorporating 125IUdR. An FUdR- 125IUdR combination, therefore, does not seem to enhance the therapeutic potential of 125IUdR. Such observations are also of importance when FUdR and other inhibitors are used to enhance cold IUdR uptake in an effort to obtain an increase in radiosensitization effects.