Reversed-phase high-performance liquid chromatography of radioiodinated salmon calcitonins

Reversed-phase HPLC conditions for simultaneous separation of salmon calcitonin, mono- and di-radioiodinated salmon calcitonins and their tryptic digested fragments have been developed. Salmon calcitonin was radioiodinated with Na¹²⁵I by the iodo-beads method. After solid-phase extraction from the reaction mixtures using C₁₈ Bond Elut cartridges, mono- and di-radioiodinated salmon calcitonins were separated from each other, as well as from unlabeled salmon calcitonin, on a Bondclone 10 C₁₈ column (300×7.8 mm I.D.) by isocratic elution with 0.1% trifluoroacetic acid in 34% aqueous acetonitrile. The characteristics of either iodinated peptides or unlabeled salmon calcitonin were evaluated on the basis of UV absorbance (215 and 280 nm), fluorescence (λ_ex=282 nm, λ_em=310 nm) and measurement of specific radioactivity by means of a flow-through radio-isotope detector. HPLC separation of a tryptic digest of iodinated salmon calcitonin fraction on a W-porex 5 C₁₈ 300 Å column (250×4.6 mm I.D.) and subsequent amino acid analysis, led to the conclusion that radioiodination took place at the Tyr residue and not at the His moiety.     

Evidence for a new subclass of calcitonin/ calcitonin gene-related peptide binding site in rat brain

Binding sites for calcitonin and calcitonin gene-related peptide are widely distributed in the central nervous system. In this study, binding of [¹²⁵I]-alpha-rat calcitonin gene-related peptide and [¹²⁵I]-salmon calcitonin in adjacent sections of rat brain revealed clearly distinct patterns of binding in most regions although in some restricted areas such as parts of the ventral striatum, including the nucleus accumbens, there was some overlap in the patterns of binding. In the primary olfactory cortex, which bound only calcitonin gene-related peptide, salmon calcitonin was very weak in inhibiting the binding of calcitonin gene-related peptide. In the nucleus accumbens, high affinity binding of calcitonin and calcitonin gene-related peptide at their homologous receptors was observed, with affinity constants for calcitonin and calcitonin gene-related peptide of 1.4 × 10⁹ M⁻¹ and 1.2 × 10⁹ M⁻¹ respectively. Cross competition studies in this nucleus demonstrated that salmon calcitonin was able to compete for [¹²⁵I]-rat calcitonin gene-related peptide labelled sites with high affinity, with an affinity constant of 0.8 × 10⁹ M⁻¹. However, rat calcitonin gene-related peptide was less potent in inhibiting the binding of [¹²⁵I]-salmon calcitonin labelled sites with only 28% inhibition at 10⁻⁶M. Further characterization of the calcitonin sensitive calcitonin gene-related peptide labelled sites demonstrated that a range of calcitonin analogs inhibited the binding of [¹²⁵I]-rat calcitonin gene-related peptide with the same order of potency as the analogs competed for [¹²⁵I]-salmon calcitonin labelled sites. Digital substraction mapping revealed calcitonin-sensitive calcitonin gene-related peptide binding sites over parts of the ventral striatum, including mid-caudal nucleus accumbens and fundus striati; over the lateral border of the lateral bed nucleus of the stria terminalis; part of the central amygdaloid nucleus; the organum vasculosum of the lamina terminalis and area postrema and over the wings of the dorsal raphe.These results demonstrate the existence of a new subtype of calcitonin/calcitonin gene-related peptide binding site, which has high affinity for the two otherwise biochemically distinct peptides.     

Differential Calcitonin Gene-Related Peptide (CGRP) and Amylin Binding Sites in Nucleus Accumbens and Lung: Potential Models for Studying CGRP/Amylin Receptor Subtypes

Abstract: Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[¹²⁵I]iodohistidyl¹⁰)humanCGRPα ([¹²⁵I]hCGRPα) binding (IC₅₀ = 0.4–7.7 nM), which was displaced by hCGRP_8–37α with equally high affinity (IC₅₀ = 0.4–7.3 nM). High-affinity binding for [¹²⁵I]Bolton-Hunter human amylin ([¹²⁵I]BH-h-amylin) was also observed in these tissues (IC₅₀ = 0.2–6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC₅₀ = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin_8–37 competed for [¹²⁵I]hCGRPα binding with higher affinity (IC₅₀ = 5.4 nM) compared with [¹²⁵I]BH-h-amylin binding (IC₅₀ = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC₅₀ values for rat amylin_8–37 were 117 and 12 nM against [¹²⁵I]hCGRPα and [¹²⁵I]BH-h-amylin, respectively. Also, functional studies evaluating the activation of adenylate cyclase and generation of cyclic AMP in response to these agonists indicated that hCGRPα (EC₅₀ = 0.3 nM), h-amylin (EC₅₀ = 150 nM), and salmon calcitonin (EC₅₀ = 1,000 nM) activated adenylate cyclase, resulting in increased cyclic AMP production in porcine lung membranes that was antagonized by hCGRP_8–37α. The affinity of hCGRP_8–37α was similar for all three peptides. The cyclic AMP responses to amylin and salmon calcitonin were significantly (p < 0.05) lower than that of hCGRPα and not additive, suggesting that they are acting as partial agonists at the same CGRP1-type receptor in porcine lung membranes. Similar observations were made for guinea pig lung membranes. However, human amylin and salmon calcitonin were weaker than hCGRPα in activating lung adenylate cyclase. None of these peptides activated adenylate cyclase in membranes prepared from the nucleus accumbens of both species. The data from these studies demonstrate both species and tissue differences in the existence of distinct CGRP and amylin binding sites and present a potential opportunity to study further CGRP and amylin receptor subtypes.     

Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells

Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor.     

Mitochondrial matrix granules in soft tissues. II. Isolation and initial characterization of a calcium-precipitable, soluble lipoprotein subfraction from brown fat and liver mitochondria

Degradation of ¹²⁵I-labelled HDL ([¹²⁵I]HDL) was measured in isolated rat hepatocytes that had been preincubated with [¹²⁵I]HDL and then reincubated in fresh medium without [¹²⁵I]HDL. About 5 % of the [¹²⁵I]HDL associated with the cells in advance were degraded per hour at 37 °C. This in vitro degradation was inhibited about 50% by lysosomal inhibitors such as chloroquine, ammonia and leupeptin. Depolymerization of microtubuli by colchicine inhibited the degradation of [¹²⁵I]HDL to about 65–75 % of the control cells. Cytochalasin B (CB), a destabilizer of microfilaments, had a less marked effect on the degradation in vitro. Degradation of [¹²⁵I]HDL associated with cells in vivo after intravenous injection was also studied in isolated cells. About 8.5% of the [¹²⁵I]HDL associated with the cells in vivo were degraded per hour in the isolated cells. The effects of ammonia, chloroquine, leupeptin and colchicine on HDL degradation were similar for [¹²⁵I]HDL taken up in vivo and in vitro. Subcellular fractionation by centrifugation in sucrose gradients indicated that [¹²⁵I]HDL associated with hepatocytes in vivo are primarily accumulated in lysosomes. [¹²⁵I]HDL associated with the cells in vitro are located in organelles whose distribution coincides with that of 5′-nucleotidase. These organelles may be endocytic vesicles. It is concluded that the internalization of [¹²⁵I]HDL in rat hepatocytes is relatively slow. The intracellular degradation of the apoproteins of HDL is at least partly lysosomal.     

Characterization of specific calcitonin gene related peptide receptors present in hamster pancreatic β cells

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of¹²⁵I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound¹²⁵I-[His]hCGRP I could be induced in the presence of 1 M chicken CGRP (cCGRP). The specific¹²⁵I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC₅₀) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0–2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of¹²⁵I-[His]hCGRP I and sCT was only effective at a high concentration (1 M). Binding of¹²⁵I-[His]hCGRP I was dose dependently inhibited by guanosine-5-O-(3-thiotriphosphate) or (GTPS) and a 70% reduction of binding was obtained with 0.1 mM GTPS. The IC₅₀ value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTPS. Human CGRP I and cCGRP at 2.5 M did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 M) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.Abbreviations CGRP calcitonin gene-related peptide - IAPP islet amyloid polypeptide - IC₅₀ half-maximal inhibitory concentration - GTPS guanosine-5-O-(3-thiotriphosphate) - ¹²⁵I [His]hCGRP I, (2[¹²⁵I]iodohistidyl¹⁰) human CGRP I     

Endocytosis and degradation of serglycin in liver sinusoidal endothelial cells

We have previously reported that liver sinusoidal endothelial cells (LSECs) are responsible for the clearance of monocyte chondroitin sulfate proteoglycan serglycin from the circulation (øynebråten et al.(2000) J. Leukocyte Biol. 67; 183–188). The aim of the present study was to investigate the kinetics of degradation of endocytosed serglycin in primary cultures of LSECs. The final degradation products of serglycin labelled biosynthetically in the glycosaminoglycan (GAG) chains with [³H] in the acetyl groups of N-acetyl galactosamine residues, [¹⁴C] in the pyranose rings, or [³⁵S] in the sulfate groups were identified as[³H]-acetate, [¹⁴C]-lactate and [³⁵S]-sulfate. Comparison of the rate of release of degradation products from the cells after endocytosis of serglycin labelled chemically with ¹²⁵I in the tyrosine residues, or biosynthetically with [³⁵S] or [³H] in the sulfate or acetyl groups, respectively, showed that ¹²⁵I appeared more rapidly in the medium than [³⁵S]-sulfate and [³H]-acetate. Judging from the speed of appearance of free ¹²⁵I both intracellularly and in the medium, the core protein is degraded considerably more rapidly than the GAG chains.Desulfation of the GAG chains starts after the GAG chains are released from the core protein. Generation of lactate and acetate as the final products from degradation of the carbon skeleton of the GAG chains indicates that catabolism of endocytosed macromolecules in LSECs proceeds anaerobically.     

Determination of catecholamines by radioenzymatic assay using ion-pair liquid chromatography

A simple radioenzymatic method for the simultaneous determination of norepinephrine epinephrine and dopamine in human serum was developed. Catecholamines were converted to normetanephrine (NMN), metanephrine (MN), 3-methoxytyramine (3-MT) and their 4-O-methyl isomers by catechol O-methyl transferase with S-adenosylmethionine-(³H-methyl) ([³H]SAM). After addition of unlabelled NMN, MN and 3-MT as carriers, the methyl derivatives were isolated from the reaction mixture by passing through a small boric acid gel column and separated from each other by ion-pair liquid chromatography. Interference by tailing of radioactivity of [³H]SAM and its degradation products eluted earlier was minimized by the use of a precolumn and a branched flow path.     

Characterization and regional distribution of calcitonin binding sites in the rat brain

Binding sites for calcitonin (CT), as assayed by the displacable binding of [125-I] iodo salmon CT ([125-I]sCT), were found on a membrane fraction prepared from rat brain. The half times of association varied between 23 and 7 min as a function of the temperatures used in the incubation medium, ranging from 6° to 37°C. Salmon CT in amounts as low as 10^?10 M inhibited the binding of [125-I]sCT to the membranes, whereas the virtually biologically inactive free acid of human CT and human CT sulfone did not affect the binding. The specific binding of [125-I]sCT to the membranes was directed to structural and/or conformational features in the COOH-terminal half of salmon CT. 133 to 8,900 times higher amounts of porcine CT and human CT and analogues thereof were required to achieve an inhibition of binding equal to that produced by salmon CT. Sixty-seven percent of specific binding of labeled hormone was not dissociable, even after 6 h of incubation with an excess of unlabeled hormone. [125-I]sCT extracted from the membranes was not degraded, as judged by gel permeation chromatography, and retained binding activity. Specific binding was highest in the hypothalamus, followed by the brainstem. It was intermediate in the midbrain-thalamus and the striatum, lower in the cortex and negligible in the hippocampus, and cerebellum and the spinal cord.     

Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells.

The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis.     

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     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK₁ pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced ‘permanent’ stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK₁ cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK₁ cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

Degradation of microinjected proteins: Effects of lysosomotropic agents and inhibitors of autophagy

HeLa cells, injected with radioiodinated proteins by fusion with RBC ghosts, were exposed to inhibitors of lysosomal proteolysis and autophagy. The degradation of injected [¹²⁵I]bovine serum albumin (BSA) was unaffected by chloroquine, NH₄Cl, nocodazole, colcemid, puromycin, cycloheximide, or enucleation. Although degradation of [¹²⁵I]lactate dehydrogenase (LDH) and [¹²⁵I]pyruvate kinase (PK) was inhibited one-third by chloroquine or ammonia, their degradation was unaffected by the other compounds. In contrast, enhanced degradation of ¹²⁵I-PK resulting from depriving injected HeLa cells of amino acids and serum was inhibited 70% by colcemid and abolished by chloroquine or ammonia. Similarly, degradation of [¹⁴C]sucrose-labeled BSA-polylysine conjugates that entered HeLa cells by endocytosis was inhibited as much as 80% by chloroquine and ammonia. Sensitivity of both enhanced proteolysis and degradation of exogenous proteins to ammonia or chloroquine indicates they are effective inhibitors of lysosomal proteolysis in HeLa cells. Failure of ammonia or chloroquine to inhibit degradation of injected ¹²⁵I-BSA and the modest inhibition of degradation of injected ¹²⁵I-LDH or ¹²⁵I-PK indicates that virtually all BSA molecules and most PK or LDH molecules are degraded by a nonlysosomal proteolytic system. Components of this degradative system are present in vast excess or are long lived, since inhibition of protein synthesis for 20 hr had no effect on the degradation of injected proteins.     

Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation

Disulfide links of the IgG2ak anti-ovarian carcinoma antibody, 5G6.4, were site-specifically biotinylated [≈2 biotins/ IgG2a] using a novel crosslinking procedure using the biotin derivatized ETAC (equilibrium transfer alkylation crosslink reagent) 1a. Complexation of ETAC 1a biotinylated 5G6.4 on a column of immobilized protein A at high dilution, followed by passage of [¹²⁵I]streptavidin, washing and pH change leads to elution of a streptavidin-free product with a molecular mass in the 200–300 kDa range. By contrast, direct mixing with [¹²⁵I]streptavidin rapidly gave larger oligomers of ⪢669 and ≈440–669 kDa molecular mass, respectively. The biodistribution of the 200–300 kDa complex showed significantly diminished liver, kidney and spleen uptake as well as higher blood activity than the 440–669 kDa complex. The methodology represent the first application of ETAC chemistry to disulfide-bond directed biotinylation of antibodies and the synthesis of streptavidin antibody conjugates which minimizes their polymerization.     

VPg unlinkase,the phosphodiesterase that hydrolyzes the bond between VPg and picornavirus RNA: a minimal nucleic moiety of the substrate

VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5"-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg–RNA complex were used: [¹²⁵I]Kp–pUpUpGp, [¹²⁵I]Kp–pUp, and [¹²⁵I]Kp–pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg–RNA). [¹²⁵I]K-peptides were unlinked from [¹²⁵I]Kp–pUpUpGp and [¹²⁵I]Kp–RNA with similar velocity, but [¹²⁵I]Kp–pUp was split much slower. Under the same conditions [¹²⁵I]Kp–pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [¹²⁵I]VPg–pU, [¹²⁵I]VPg–pUp, and [¹²⁵I]VPg–pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [¹²⁵I]VPg–RNA and [¹²⁵I]VPg–pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.     

Comparative Affinities of Adrenomedullin (AM) and Calcitonin Gene-Related Peptide (CGRP) for [125I] AM and [125I] CGRP Specific Binding Sites in Porcine Tissues

Abstract

We have investigated the binding characteristics of rat [¹²⁵I] adrenomedullin (AM) and human [¹²⁵I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [¹²⁵I] rat AM and [¹²⁵I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [¹²⁵I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [¹²⁵I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [¹²⁵I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [¹²⁵I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

Calcitonin binding and degradation by two cultured human breast cancer cell lines (MCF 7 and T 47D).

Two human breast cancer cell lines (MCF 7 and T 47D) possess calcitonin-responsive adenylate cyclase systems. Suspended cells of both lines specifically bound 125I-labelled salmon calcitonin with mean dissociation constants of 1.7 nM (MCF 7) and 1.4 nM (T 47D); mean receptor numbers were 5300 and 24400 per cell respectively. Measurement of specific binding to MCF 7 cells was obscured by rapid and substantial degradation of the labelled hormone. Degradation of 125I-labelled salmon calcitonin: (i) was of high capacity; (ii) lacked the specificity displayed by 125I-labelled salmon calcitonin binding to the same cells; and (iii) was not related to binding since cell incubation supernatants retained full degrading activity. The degrading activity was inhibited by corticotropin (1-24)-tetracosapeptide, insulin and bacitracin. Inclusion of bacitracin in the incubation resulted in apparently fewer numbers of lower affinity receptors on MCF 7 cells, whereas these parameters were identical to T 47D cells incubated in the presence or absence of bacitracin. Eel [2-aminosuberic acid 1,7]-calcitonin was resistant to proteolysis in the presence of either cell line. Analysis of hormone-receptor interactions with calcitonin-responsive cells should take account of potent calcitonin-degrading activities in some cell lines.     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Synthesis and evaluation of novel radioiodinated PSMA targeting ligands for potential radiotherapy of prostate cancer

Radioligand therapy (RLT) using prostate-specific membrane antigen (PSMA) targeting ligands is an attractive option for the treatment of Prostate cancer (PCa) and its metastases. We report herein a series of radioiodinated glutamate-urea-lysine-phenylalanine derivatives as new PSMA ligands in which l-tyrosine and l-glutamic acid moieties were added to increase hydrophilicity concomitant with improvement of in vivo targeting properties. Compounds 8, 15, 19a/19b and 23a/23b were synthesized and radiolabeled with ¹²⁵I by iododestannylation. All iodinated compounds displayed high binding affinities toward PSMA (IC₅₀ = 1–13 nM). In vitro cell uptake studies demonstrated that compounds containing an l-tyrosine linker moiety (8, 15 and 19a/19b) showed higher internalization than MIP-1095 and 23a/23b, both without the l-tyrosine linker moiety. Biodistribution studies in mice bearing PC3-PIP and PC3 xenografts showed that [¹²⁵I]8 and [¹²⁵I]15 with higher lipophilicity exhibited higher nonspecific accumulations in the liver and intestinal tract, whereas [¹²⁵I]19a/19b and [¹²⁵I]23a/23b containing additional glutamic acid moieties showed higher accumulations in the kidney and implanted PC3-PIP (PSMA+) tumors. [¹²⁵I]23b displayed a promising biodistribution profile with favorable tumor retention, fast clearance from the kidney, and 2–3-fold lower uptake in the liver and blood than that observed for [¹²⁵I]MIP-1095. [^125/131I]23b may serve as an optimal PSMA ligand for radiotherapy treatment of prostate cancer over-expressing PSMA.     

Disposition of125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [¹²⁵I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. Afteri.v. administration, [¹²⁵I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [¹²⁵I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [¹²⁵I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [¹²⁵I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [¹²⁵I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.