A technique for the electron microscopic autoradiography of Al adenosine receptors in brain tissue. Ligand-receptor fixation by osmium tetroxide

A procedure for the electron microscopic autoradiography of Al adenosine receptors is described. Fresh tissue slices from rat hippocampus were incubated with the radioactive adenosine analogs: Cyclohexyl[3H]adenosine, 5'-N-ethylcarboxamido[3H]adenosine or or [125I]-iodohydroxyphenylisopropyladenosine. Various fixation agents were tested with respect to the retention of these ligands by the tissue. While most of the ligands were lost in aldehyde fixation they were retained by osmium tetroxide probably via a crosslinking reaction. The final method of choice was an aldehyde prefixation (in the case of [125I]-iodohydroxyphenylisopropyladenosine with 4% buffered paraformaldehyde) during which more than 90% of the nonspecifically bound ligands were washed out while 40% of the specifically bound ligands remained. Subsequent fixation with osmium tetroxide (1%) allowed a standard protocoll for dehydration and embedding to be used with only minimal (less than 5%) further loss of the ligands. Electron microscopic autoradiography provided evidence for a specific distribution of the binding sites for [125I]-iodohydroxyphenylisopropyladenosine.     

In Vivo Differences between Two Optical Isomers of Radioiodinated o-iodo-trans-decalinvesamicol for Use as a Radioligand for the Vesicular Acetylcholine Transporter

Purpose

To develop a superior VAChT imaging probe for SPECT, radiolabeled (-)-OIDV and (+)-OIDV were isolated and investigated for differences in their binding affinity and selectivity to VAChT, as well as their in vivo activities.

Procedures

Radioiodinated o-iodo-trans-decalinvesamicol ([¹²⁵I]OIDV) has a high binding affinity for vesicular acetylcholine transporter (VAChT) both in vitro and in vivo. Racemic [¹²⁵I]OIDV was separated into its two optical isomers (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV by HPLC. To investigate VAChT binding affinity (Ki) of two OIDV isomers, in vitro binding assays were performed. In vivo biodistribution study of each [¹²⁵I]OIDV isomer in blood, brain regions and major organs of rats was performed at 2,30 and 60 min post-injection. In vivo blocking study were performed to reveal the binding selectivity of two [¹²⁵I]OIDV isomers to VAChT in vivo. Ex vivo autoradiography were performed to reveal the regional brain distribution of two [¹²⁵I]OIDV isomers and (-)-[¹²³I]OIDV for SPECT at 60 min postinjection.

Results

VAChT binding affinity (Ki) of (-)-[¹²⁵I]OIDV and (+)-[¹²⁵I]OIDV was 22.1 nM and 79.0 nM, respectively. At 2 min post-injection, accumulation of (-)-[¹²⁵I]OIDV was the same as that of (+)-[¹²⁵I]OIDV. However, (+)-[¹²⁵I]OIDV clearance from the brain was faster than (-)-[¹²⁵I]OIDV. At 30 min post-injection, accumulation of (-)-[¹²⁵I]OIDV (0.62 ± 0.10%ID/g) was higher than (+)-[¹²⁵I]OIDV (0.46 ± 0.07%ID/g) in the cortex. Inhibition of OIDV binding showed that (-)-[¹²⁵I]OIDV was selectively accumulated in regions known to express VAChT in the rat brain, and ex vivo autoradiography further confirmed these results showing similar accumulation of (-)-[¹²⁵I]OIDV in these regions. Furthermore, (-)-[¹²³I]OIDV for SPECT showed the same regional brain distribution as (-)-[¹²⁵I]OIDV.

Conclusion

These results suggest that radioiodinated (-)-OIDV may be a potentially useful tool for studying presynaptic cholinergic neurons in the brain.     

Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.     

Synthesis and biological evaluation of indole-chalcone derivatives as β-amyloid imaging probe

A series of chaclone derivatives containing an indole moiety were evaluated in competitive binding assays with Aβ_1-42 aggregates versus [¹²⁵I]IMPY. The affinity of these compounds ranged from 4.46 to >1008 nM, depending on the substitution on the phenyl ring. Fluorescent staining in vitro showed that one compound with a N,N-dimethylamino group intensely stained Aβ plaques within brain sections of AD transgenic mice. The radioiodinated probe [¹²⁵I]-(E)-3-(1H-indol-5-yl)-1-(4-iodophenyl)prop-2-en-1-one, [¹²⁵I]4, was prepared and autoradiography in sections of brain tissue from an animal model of AD showed that it labeled Aβ plaques specifically. However, experiments with normal mice indicated that [¹²⁵I]4 exhibited a low uptake into the brain in vivo (0.41% ID/g at 2 min). Additional chemical modifications of this indole-chalcone structure may lead to more useful imaging agents for detecting β-amyloid plaques in the brains of AD patients.     

Synthesis and biological evaluation of radio-iodinated benzimidazoles as SPECT imaging agents for NR2B subtype of NMDA receptor

In this study, the benzimidazole derivatives were synthesized and evaluated as imaging agents for the NR2B subtype of NMDA receptor. Among these ligands, 2-{[4-(4-iodobenzyl)piperidin-1-yl]methyl}benzimidazol-5-ol (8) and N-{2-[4-(4-iodobenzyl)-piperidin-1-ylmethyl]benzoimidazol-5-yl}-methanesulfonamide (9) exhibited high affinity for the NR2B subunit (K_i values; 7.28 nM for 8 and 5.75 nM for 9). In vitro autoradiography experiments demonstrated high accumulation in the forebrain regions but low in the cerebellum for both [¹²⁵I]8 and [¹²⁵I]9. These regional distributions of the radioligands correlated with the expression of the NR2B subunit. The in vitro binding of these ligands was inhibited by NR2B antagonist but not by other site ligands, which suggested the high selectivity of [¹²⁵I]8 and [¹²⁵I]9 for the NR2B subunit. In mice, the regional brain uptakes of [¹²⁵I]8 and [¹²⁵I]9 at 5–180 min after administration were 0.42–0.56% and 0.44–0.67% dose/g, respectively. The brain-to-blood ratio of [¹²⁵I]8 at 180 min was reduced by 34% in the presence of non-radioactive ligands and by 59% in the presence of the NR2B ligand Ro-25,6981. These results indicated that [¹²⁵I]8 could be partially bound to the NR2B subunit in vivo. Although the brain uptake of these benzimidazole derivatives was too low to allow for in vivo SPECT imaging, these compounds might be useful scaffolds for the development of imaging probes specific for the NMDA receptors.     

Detection of a 65 kDaras binding protein in rat and sheep brain cytosol using a chemical cross linking agent

The ability of aras protein to associate with proteins present in rat brain cytosolin vitro was investigated using chemical cross-linking agents and the¹²⁵I-labelled v-H-ras protein. Two iodinated protein complexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [¹²⁵I]ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-PAGE. Formation of the [¹²⁵I] 85 kDa complex was enhanced by a high concentration of EDTA while generation of the 40 kDa species was abolished by this treatment. Formation of the [¹²⁵I] 85 kDa complex was inhibited by unlabelledras protein, GTP, GTPS, and GDP but not by ATPS and GMP.Chromatography of the cross-linked brain cytosol-[¹²⁵I]ras mixture on DEAE cellulose partially resolved the [¹²⁵I] 85 kDa complex from the [¹²⁵I]ras protein. The [¹²⁵I] 85 kDa complex (formed using ethyleneglycolbis (succinimidylsuccinate) as the cross-linking agent) could be immunoprecipitated using a rabbit anti-ras polyclonal antibody. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [¹²⁵I]-labelledras. A substantial enrichment of the proportion of the [¹²⁵I] 85 kDa complex in the cross-linked extract was achieved by preparative SDS-PAGE. It is concluded that thein vitro chemical cross-linking approach employed here has detected tworas binding proteins in rat brain cytosol: a 65 kDa heat-sensitive and a 20 kDa heat-stable protein. The possibility that the 65 kDaras binding protein is aras regulatory orras effector protein which has not so far been characterised is briefly discussed.Abbreviations DSS disuccinimidyl suberate - EGS ethyleneglycolbis (succinimidylsuccinate) - GTPS guanosine 5-[-thio] triphosphate - ATPS adenosine 5-[-thio] triphosphate     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

Summary To determine if rat kidney Na⁺, K⁺-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na⁺, K⁺-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na⁺, K⁺-ATPase and rat kidney microsomes were treated with antiserum (1200), followed by [¹²⁵I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the -subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the -subunit. When the Na⁺, K⁺-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na⁺, K⁺-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na⁺, K⁺-ATPase concentration in the rat kidney.Supported by Grant AM 17047 from NIH and by the Veterans Administration     

Conversion and binding of tetraiodothyronine in developing rat brain

In this study we have examined whether rat brain nuclear thyroid hormone receptors bind T₄ or metabolites of T₄ and whether there is a developmental change in brain T₄ metabolism and binding. Developing animals were injected with trace [¹²⁵I]3,5-tetraiodothyronine ([¹²⁵I]T₄) and after sacrifice brain nuclear and cytoplasmic fractions were examined to determine whether their radioactivity was represented by the injected [¹²⁵I]T₄ or any of its metabolites. Of the radiothyronines specifically bound to the nucleus, 90% was found to be triiodothyronine ([¹²⁵I] T₃) and 10% was [¹²⁵I]T₄. Of the cytoplasmic, protamine sulfate-precipitable fraction, 40% was [¹²⁵I]T₄ and 60% [¹²⁵I]T₃. Inasmuch as the percentage of [¹²⁵I] T₃ found in plasma during the same postinjection interval was similar to that present as contaminant of the injected material, it was concluded that brain [¹²⁵I] T₃ derives from local monodeiodination of T₄ to T₃. The main developmental change observed was a marked decline in the total cytoplasmic and nuclear [¹²⁵I] T₄ uptake. However, with development, the T₃/T₄ ratio remained constant in the nuclear fraction while it decreased in the cytoplasmic fraction. It is concluded that although T₃, deriving from monodeiodianation of T₄, is the main form of thyroid hormone that regulates brain development by its binding to brain nuclear receptors, the fact that T₄ is the most available from during the critical period makes it, indirectly, very important to brain development. Further, the decline observed with development in T₄ uptake and monodeiodination to T₃, may contribute to the concomitantly declining role of thyroid hormones on brain tissue.     

The alveolar-lining layer in the lung of the axolotl,Ambystoma mexicanum

Summary Lungs of neotenic larvae of Ambystoma mexicanum were prepared for maintaining the air-tissue boundary during aldehyde fixation. Four methods of postfixation were applied: 1) osmium tetroxide followed by en-bloc staining with uranyl acetate and phosphotungstic acid, 2) ruthenium redosmium tetroxide, 3) osmium tetroxide-ferrocyanide, and 4) tannic acidosmium tetroxide.Three types of cells line the inner surface of the axolotl lung: 1) pneumocytes, covering the capillaries with flat cellular extensions and containing two types of granules: the osmiophilic lamellar bodies, precursors of extracellular membranous material, and apical granules of unknown significance; 2) ciliated cells, also containing osmiophilic lamellar bodies; and 3) goblet cells filled with secretory granules as well as osmiophilic bodies.The extracellular material forms membranous whorls as well as tubular myelin figures, consisting of membranous backbones combined with an intensely stained substance. This material strikingly resembles the surfactant of amphibian lungs.     

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.     

Species differences and mechanism of action of A<Subscript>3</Subscript> adenosine receptor allosteric modulators

Activity of the A₃ adenosine receptor (AR) allosteric modulators LUF6000 (2-cyclohexyl-N-(3,4-dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A₃AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[³⁵S]thio]triphosphate ([³⁵S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A₃ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A₃ARs but provided only weak activity at mouse A₃ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A₃AR agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N ⁶-(4-amino-3-[¹²⁵I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([¹²⁵I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A₃AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [¹²⁵I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between species-variable EL1 and agonist-contacting EL2. These results indicate that A₃AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.     

Centrioles and Microtubules in <Emphasis Type="Italic">Chlorella</Emphasis>

IN keeping with its widespread use in biochemical studies, the ultrastructure of the unicellular green alga Chlorella has been investigated many times^1–3, yet at least two components—microtubules and centrioles—have escaped detection, doubtless because of the use of inadequate fixation techniques. We report here on the presence and behaviour of these subcellular components during the cell cycle in C. pyrenoidosa 211-8p grown autotrophically in cultures synchronized⁴ by alternating light (time 0–15 h) and dark (15–24 h) periods and sampled for examination by electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde (pH 6.7, 25° C, 2 h) and post-fixation in 2% osmium tetroxide (pH 7, 0° C, 2 h).     

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     

Synthesis and evaluation of novel benzothiazole derivatives based on the bithiophene structure as potential radiotracers for β-amyloid plaques in Alzheimer’s disease

In this study, six novel benzothiazole derivatives based on the bithiophene structure were developed as potential β-amyloid probes. In vitro binding studies using Aβ aggregates showed that all of them demonstrated high binding affinities with K_i values ranged from 0.11 to 4.64 nM. In vitro fluorescent staining results showed that these compounds can intensely stained Aβ plaques within brain sections of APP/PS1 transgenic mice, animal model for AD. Two radioiodinated compounds [¹²⁵I]-2-(5′-iodo-2,2′-bithiophen-5-yl)-6-methoxybenzo[d]thiazole [¹²⁵I]10 and [¹²⁵I]-2-(2,2′-bithiophen-5-yl)-6-iodobenzo[d]thiazole [¹²⁵I]13 were successfully prepared through an iododestannylation reaction. Furthermore, in vitro autoradiography of the AD model mice brain sections showed that both [¹²⁵I]10 and [¹²⁵I]13 labeled the Aβ plaques specifically with low background. In vivo biodistribution studies in normal mice indicated that [¹²⁵I]13 exhibited high brain uptake (3.42% ID/g at 2 min) and rapid clearance from the brain (0.53% ID/g at 60 min), while [¹²⁵I]10 showed lower brain uptake (0.87% ID/g at 2 min). In conclusion, these preliminary results of this study suggest that the novel radioiodinated benzothiazole derivative [¹²⁵I]13 may be a candidate as an in vivo imaging agent for detecting β-amyloid plaques in the brain of AD patients.     

AN ELECTRON MICROSCOPIC CHARACTERIZATION OF CLASSES OF SYNAPTIC VESICLES BY MEANS OF CONTROLLED ALDEHYDE FIXATION

Examination of variables of aldehyde fixation that may affect the shape of agranular synaptic vesicles has revealed that even brief storage of aldehyde-perfused nervous tissue pieces in cacodylate buffer, prior to hardening in osmium tetroxide, has an unusually severe flattening effect on agranular vesicles of a particular type. These are the vesicles of peripheral cholinergic axon endings, and of certain central synaptic bulbs. Types of synaptic bulbs can now be further defined on the basis of shape of agranular synaptic vesicles under controlled conditions of aldehyde fixation. Previously described "S" bulbs in the spinal cord contain uniformly spheroid vesicles, which are wholly resistant to flattening. Previously described "F" bulbs contain somewhat smaller agranular vesicles that are flattened after aldehyde fixation, even when this is followed by prompt hardening in osmium tetroxide solution. A third type, previously characterized as having irregularly round agranular vesicles after the above treatment, contains only severely flattened vesicles when the osmium tetroxide hardening is preceded by even a brief wash with sodium cacodylate buffer containing sucrose. Moreover, the "third" type is characteristic of all cholinergic peripheral axon endings examined, as well as the large axosomatic ("L") synaptic bulbs of the spinal cord.     

The use of homologous and hetebologous 125 I-radioligands in the radioimmunoassay of progesterone

Eight homologous and heterologous¹²⁵ I-radioligand systems for the radioimmunoassay of progesterone were examined. Using an antiserum raised to 11α-hydroxyprogesterone 11-succinyl-bovine serum albumin, standard curves were set up with the homologous radioligands, 11α-hydroxyprogesterone 11-succinyl-[¹²⁵I]-iodotyramine, -[¹²⁵I]-iodohistamine and -[¹²⁵I]-iodotyrosine methyl ester. Heterologous bridge systems were represented by progesterone-11α-oxycarbonyl-[¹²⁵I]-iodotyrosine methyl ester and 11α-hydroxyprogesterone 11-phthalyl-[¹²⁵I]-iodotyrosine methyl ester, and heterologous site systems by progesterone-3-(O-carboxymethyl)oxime-[¹²⁵I]-iodotyramine, progesterone-12-(O-carboxymethyl) oxime-[¹²⁵ I]-iodotyramine, and progesterone-20-(O-carboxymethyl) oxime-[¹²⁵I]-iodohistamine. The preparation of the steroid derivatives and iodination by a two-phase method are described. The curves obtained from the homologous radioligands were relatively insensitive compared with a tritiated system, with the tyrosine methyl ester derivative providing a more sensitive assay than the corresponding tyramine or histamine analogues. The heterologous bridge systems gave more sensitive curves than the homologous tracers whilst the 3- and 12-(O-carboxymethyl) oxime derivatives of progesterone furnished curves as sensitive as the tritiated reference. Progesterone-20-(O-carboxymethyl)oxime-[¹²⁵I]-iodohistamine was not bound by the antibody.     

Assessment of [125I]WYE-230949 as a Novel Histamine H3 Receptor Radiopharmaceutical

Histamine H₃ receptor therapeutics have been proposed for several diseases such as schizophrenia, attention deficit hyperactivity disorder, Alzheimer''s disease and obesity. We set out to evaluate the novel compound, [¹²⁵I]WYE-230949, as a potential radionuclide imaging agent for the histamine H₃ receptor in brain. [¹²⁵I]WYE-230949 had a high in vitro affinity for the rat histamine H₃ receptor (K_d of 6.9 nM). The regional distribution of [¹²⁵I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H₃ receptor. Rat brain uptake of intravenously injected [¹²⁵I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [¹²⁵I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [¹²⁵I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [¹²⁵I]WYE230949 is not a potential radiotracer for imaging rat histamine H₃ receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.     

Characteristics of [125I]ω-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain

Characteristic of [¹²⁵I]-conotoxin (-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]: DSP) was systematically investigated in crude membranes from chick whole brain. [¹²⁵I]-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [¹²⁵I]-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [¹²⁵I]-CgTX labeling of the 216 kDa band and specific [¹²⁵I]-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [¹²⁵I]-CgTX using DSP involves the specific binding sites of [¹²⁵I]-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.     

Characterization of [I]GLP-1(9-36), a novel radiolabeled analog of the major metabolite of glucagon-like peptide 1 to a receptor distinct from GLP1-R and function of the peptide in murine aorta

Aims

Glucagon-like peptide 1 (GLP-1) is an insulin secretagogue, released in response to meal ingestion and efficiently lowers blood glucose in Type 2 diabetic patients. GLP-1(7-36) is rapidly metabolized by dipeptidyl peptidase IV to the major metabolite GLP-1(9-36)-amide, often thought to be inactive. Inhibitors of this enzyme are widely used to treat diabetes. Our aim was to characterize the binding of GLP-1(9-36) to native mouse tissues and to cells expressing GLP1-R as well as to measure functional responses in the mouse aorta compared with GLP-1(7-36).

Main methods

The affinity of [¹²⁵I]GLP-1(7-36) and [¹²⁵I]GLP-1(9-36) was measured in mouse tissues by saturation binding and autoradiography used to determine receptor distribution. The affinity of both peptides was compared in binding to recombinant GLP-1 receptors using cAMP and scintillation proximity assays. Vasoactivity was determined in mouse aortae in vitro.

Key findings

In cells expressing GLP-1 receptors, GLP-1(7-36) bound with the expected high affinities (0.1 nM) and an EC₅₀ of 0.07 nM in cAMP assays but GLP-1(9-36) bound with 70,000 and 100,000 fold lower affinities respectively. In contrast, in mouse brain, both labeled peptides bound with a single high affinity, with Hill slopes close to unity, although receptor density was an order of magnitude lower for [¹²⁵I]GLP-1(9-36). In functional experiments both peptides had similar potencies, GLP-1(7-36), pD₂ = 7.40 ± 0.24 and GLP-1(9-36), pD₂ = 7.57 ± 0.64.

Significance

These results suggest that GLP-1(9-36) binds and has functional activity in the vasculature but these actions may be via a pathway that is distinct from the classical GLP-1 receptor and insulin secretagogue actions.