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.     

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     

A rat kidney neutral peptidase that degrades B chain of insulin, glucagon, and ACTH: purification by affinity chromatography and some properties.

A metallo-endopeptidase that catalyzes at near neutral pH the hydrolysis of certain polypeptides was purified from rat kidney microsomes by a simplified procedure using affinity chromatography on Sepharose 4B coupled with insulin B chain. The purified enzyme showed a single component by chromatography on diethylaminoethyl cellulose and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 213,000. Studies on its substrate specificity showed that the purified enzyme rapidly degrades insulin B chain, glucagon, adrenocorticotropin, and, at a significantly lower rate, insulin A chain. The enzyme has a very weak or no activity toward ribonuclease and vasopressin. In contrast, the enzyme does not degrade denatured hemoglobin, bovine serum albumin, insulin (nano- or micromolar), oxytocin, furylacryloylglycyl-leucine amide (FAGLA), synthetic substrates of cathepsin C (β-napthalamides of glycine-l-arginine and l-histidine-l-serine), or synthetic substrates of aminopeptidases (l-arginine- or l-glutamic acid-β-napthylamide). The enzyme degrades reduced or oxidized B chain at about the same rate, but S-sulfonated B chain is degraded at a markedly lower rate. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. Activity of the enzyme is markedly inhibited by chelating agents (EDTA and o-phenanthroline) and, modestly, by high concentrations of citrate and histidine. Activity of the enzyme is also markedly inhibited by simple thiol compounds (dithiothreitol, glutathione, and mercaptoethanol), but not by sulfhydryl reagents (N-ethylmaleimide or iodoacetate). The inactive apoenzyme, prepared by treatment of the enzyme with EDTA followed by dialysis, was reactivated by Zn²⁺ > Ca²⁺, minimally by Cu²⁺, but not by Hg²⁺. Some anions (phosphate, borate, and bicarbonate) were strongly inhibitory, but chloride had no effect. The following agents were found to have no effect: soybean and lima bean trypsin inhibitors, N^?-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N^α,?-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin (Trasylol), phenylmethylsulfonyl fluoride (a serine protease inhibitor), 1-methyl histidine, 3-methyl histidine, histamine, imidazole, and heparin.     

Haloperidol-succinylglycyl[125I]iodotyrosine, a novel iodinated ligand for dopamine D2 receptors

A novel radioiodinated ligand of the butyrophenone type has been synthesized for the quantification and characterization of dopamine D2 receptors. This haloperidol-derived ligand, haloperidol-succinylglycyl[125I]iodotyrosine ([125I]HSGTI), binds rapidly (equilibrium is reached within 30 min, at 10 pM and 37 degrees C) and with high affinity (Kd = 0.3 nM) to bovine striatal membranes. Its pharmacology, determined by competitive displacement with dopaminergic and non-dopaminergic drugs, is characteristic of binding to dopamine D2 receptors.     

The endosomal compartment of rat hepatocytes. Its characterization in the course of [125I]insulin internalization

When freshly isolated hepatocytes are incubated with [125I]insulin in the presence of the microtubule-disrupting agent colchicine, internalization of the labelled hormone is not significantly altered. However, the drug limits the endocytosis of the labelled material to a peripheral band of cytoplasm extending 1 micron beyond the plasma membrane. Both in the presence and absence of colchicine, internalized [125I]insulin preferentially associates with clear vesicles (endosomes) and lysosome-like structures, but the relative amount of labelled material associated with clear vesicles is higher in the presence of the drug than in its absence. An inverse pattern is observed for the lysosome-like structures. As demonstrated by cytochemical methods, clear vesicles do not contain the lysosomal enzyme aryl sulfatase. Moreover, colchicine induces an increase of the clear vesicle diameter without affecting their frequency, while it perturbs multivesicular bodies and dense bodies in an opposite way by increasing their frequency without affecting their size. By reducing and/or delaying the fusion between internalized endocytotic vesicles and lysosomes, colchicine allows better characterization of the endosomal compartment of isolated rat hepatocytes and allows it to be distinguished from other compartments, such as multivesicular bodies and the Golgi apparatus.     

Ornithine decarboxylase activity and: [125I]iododeoxyuridine incorporation in rat prostate.

The relationship between ornithine decarboxylase activity and [125I]iododexyuridine incorporation was studied in prostates from castrated rats (aged 5, 26 and 80 weeks) injected daily with testosterone for up to 10 days. The results suggest that ornithine decarboxylase activity is a parameter of secretory activity, rather than growth, in the ventral prostate. In the dorsolateral prostate, ornithine decarboxylase activity tends to parallel [125I]iododeoxyuridine incorporation.     

Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with [125I]-alpha-bungarotoxin.

Acetylcholine receptors have been purified from junctional regions of normal rat diaphragm muscle and from extrajunctional regions of denervated diaphragm. The reaction of purified receptors with [122I]-alpha-bungarotoxin has been investigated by kinetic methods. The toxin-receptor complexes dissociated in a biphasic manner at 35 degrees with a rapidly dissociating component (t1/2 = 4 hr) and a slowly dissociating component (t1/2 is greater than or equal to 100 hr). The association reaction between toxin and receptor did not obey simple second-order kinetics but could be analyzed in terms of two classes of binding sites corresponding to the two rates of dissociation. This treatment of the data allowed derivation of association rate constants for the two sites. Value obtained for the dissociation constants were 3.7 times 10(-10) and less than or equal to 0.4 times 10(-10) M for the junctional receptor and 1.7 times 10(-10) and is less than or equal to 0.2 times 10(-10) M for the extrajunctional receptor. In each case it is the more tightly binding component that associates and dissociates more slowly. Receptors present in crude preparations were comparable to purified receptors in their reaction with [125I-alpha-bungarotoxin. The validity of the two site model is discussed in relation to the kinetic studies.     

Treatment of neuroblastoma with [125I]metaiodobenzylguanidine.

To find a treatment that may be effective against micrometastases of advanced, stage III or IV neuroblastoma, [125I]metaiodobenzylguanidine (125I-MIBG) was used in a phase I toxicity trial. In seven patients, thrombocytopenia was encountered with absorbed whole body doses of 85-135 rad from 125I-MIBG, but the dosimetry was imprecise in predicting bone marrow injury. Three patients survived for over one year, results that may indicate efficacy of 125I-MIBG therapy.     

[125I]MIBG uptake and release in different regions of the rat brain

Radioiodinated m-iodobenzylguanidine ([¹²⁵I]MIBG) and tritiated norepinephrine ([³H]NE]) uptake and release were compared, in different regions of the brain of the rat. The classification of the regions according to uptake was the same for both tracers: striatum > hypothalamus > hippocampus > cortex > brainstem. Tetrabenazine (TBZ), a granular monoamine uptake inhibitor reduced the uptake in the different regions. The inhibition rate was higher for [³H]NE uptake than for [¹²⁵I]MIBG. The spontaneous release was the same for [¹²⁵I]MIBG and [³H]NE and was the lowest in the striatum. The K⁺ stimulated release of [³H]NE was more complete than the release of [¹²⁵I]MIBG and was the most important in the striatum. From these results, it is inferred that MIBG enters the brain tissue via NE uptake mechanisms. It appears that MIBG is stored in the chromaffin granules, as NE, but also in the cytoplasm. A modified molecule derived from MIBG which would cross the blood-brain barrier, would then appear as a potential scintigraphic marker of monoamine uptake, storage and release.     

Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

A rapid, high-yield method of producing mono-[125I]A14 iodoinsulin

We have developed a rapid method for producing homogeneous mono-[125I]A14 iodoinsulin with high specific activity and yield. After iodination by the lactoperoxidase method, the labeled peptides were applied to a C18 Porasil pre-column, washed with aqueous buffer to eliminate the free [125I]-iodide and placed "in-line" with a C-18 HPLC column; mono-[125I]A14 iodoinsulin was then eluted isocratically with 29% acetonitrile in 16 minutes. The labeled hormone was extremely stable, and proved suitable for various biological studies.     

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

Blood glucose lowering assay proved that [B16Ala]insulin and [B26Ala]insulin exhibit potency of acute blood glucose lowering in normal pigs, which demonstrates that they are fast- acting insulin. Single-chain precursor of [B16Ala]insulin and [B26Ala]insulin is [B16Ala]PIP and [B26Ala]PIP, respectively, which are suitable for gene expression. Secretory expression level of the precursors in methylotrophic yeast Pichia pastoris was quite high, 650 mg/L and 130 mg/L, respectively. In vivo biological assay showed that the two fast-acting insulins have full or nearly full biological activity. So both [B16Ala]insulin and [B26Ala]insulin can be well developed as fast-acting insulin for clinic use.     

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.     

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

Insulin has been successfully used in clinic treatment of diabetes for more than 80 years. However, the clinic practice has shown that regular insulin preparation used in clinic exhibits several intrinsic problems that have existed for a long time. One of the major problems is that insulin has a potency of self-association when its concentration is higher than physiological concentration (10-8—10-10 mol/L)[1,2]. The concentration of the regular insulin is higher than 10-4 mol/L. At such a hi…     

Binding, internalization, and degradation of [125I]insulin by cultured bovine aortic endothelial cells: effects of serotonin

Cultured bovine aortic endothelial cells bind and internalize [125I]insulin and down regulate insulin receptors. Internalized insulin was not degraded significantly and diffused from the endothelial cells. Neither 5-hydroxytryptamine, methylamine, nor dansylcadaverine have any observable effect on insulin binding, internalization, metabolism, or down regulation of insulin receptors. Transglutaminase activity, however, is inhibited by 5-hydroxytryptamine and methylamine. These data indicate that transglutaminase is not required for insulin receptor-mediated endocytosis by bovine aortic endothelial cells in culture.     

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

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.