Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of iodothyronines with apolipoprotein A-1 in high density lipoprotein particle isolated from human serum by affinity chromatography on thyroxine-sepharose

The kinetic and equilibrium characteristics of interaction of thyroxine (T4) and its structural analogs with a high density lipoprotein (HDL) fraction isolated from human serum by T4-Sepharose affinity chromatography and containing apolipoprotein A-I (apo A-I) as a sole protein component, were studied. The binding of [125I]T4 to apo A-I-HDL reached a maximum after 40 min and did not change during the next 80 min of incubation at 0 degrees--22 degrees C. Dissociation of [125I]T4 induced by the addition of excess unlabeled T4 to the complex solution proceeded more intensely on a time scale at 0--2 degrees C than at 22 degrees C. Incubation of apo A-I-HDL with increasing concentrations of T4 showed that the binding is saturable. The data analysis using different computer programs revealed the presence in apo A-I-HDL of a single class of binding sites with K alpha = (4.0 +/- 2.1).10(-7) M- and Bmax = 1.7 +/- 0.8 nmol T4/mg of protein. Naturally occurring iodothyronines, their analogs and D-isomers of thyroid hormones competed with [125I]T4 for the binding sites on apo A-I-HDL with the following inhibitory potencies: L-T4 = D-T4 greater than or equal to 3,3',5-triiodo-L-thyronine = 3,3',5-triiodo-D-thyronine greater than 3,5-diiodo-L-thyronine = 3,3',5- triiodothyroacetic acid greater than 3,3',5-triiodothyropropionic acid greater than or equal to 3,5-diiodo-L-thyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.     

Peroxidatic degradation and ether link cleavage of thyroxine in a particulate fraction of human thyroid

The present study was undertaken to investigate degradation of thyroxine (T4) mediated by thyroid peroxidase in man. A particulate fraction (1,000-100,000 x g) of normal human thyroid tissue was prepared and used as crude enzyme. 125I-T4 and unlabeled T4 were incubated with the particulate fraction in buffer containing glucose and glucose oxidase for generation of H2O2. After incubation, iodoamino acids were extracted with ethanol and the products of T4 degradation were analyzed by thin layer chromatography. In this system, T4 was degraded in time-, temperature- and pH-dependent manners, but not in the absence of the H2O2-generating system. The rate of degradation was related to concentration of the particulate fraction. The reaction was inhibited by methimazole, propylthiouracil and catalase. When [3',5'-125I] T4 was used as a tracer, major labeled products of T4 degradation were inorganic iodide and ethanol-unextracted fraction and no detectable labeled 3,5,3'-triiodothyronine (T3) or 3,3',5'-triiodothyronine (rT3) was generated. From a kinetic study by adding various doses of unlabeled T4, the apparent Km value for T4 was 30 microM and the Vmax value was 230 pmol/mg protein/min. When [3,5-125I] T4 was incubated with enzyme preparation, one third of degraded T4 was recovered as diiodotyrosine (DIT) and half of 125I-DIT was degraded in parallel incubation. No formation of radiolabeled DIT was observed in incubation with Na- 125I done in tandem. These findings suggest that thyroid hormones can be metabolized by peroxidase in human thyroid by pathways that include cleavage of ether linkage.     

Analysis of photoaffinity label derivatives to probe thyroid hormone receptor in human fibroblasts, GH1 cells, and soluble receptor preparations

The regulation of growth hormone gene expression by thyroid hormone in cultured GH1 cells is mediated by a chromatin-associated receptor. We have previously described a photoaffinity label derivative of 3,5,3'-triiodo-L-thyronine (L-T3) in which the alanine side chain was modified to form N-2-diazo-3,3,3-trifluoropropionyl-L-T3 (L-[125I]T3-PAL). On exposure to 254 nm UV light, L-[125I]T3-PAL generates a carbene which covalently modifies two thyroid hormone receptor forms in intact GH1 cells; an abundant 47,000 Mr species and a less abundant 57,000 Mr form. We have now synthesized similar photoaffinity label derivatives of 3,5,3',5'-tetraiodo-L-thyronine (L-T4) and 3,3',5'-triiodo-L-thyronine (L-rT3). Both compounds identify the same receptor forms in intact cells and in nuclear extracts in vitro as L-[125I]T3-PAL. Labeling by L-[125I]rT3-PAL was low and consistent with the very low occupancy of receptor by L-rT3. Underivatized L-[125I]T3 and L-[125I]T4 labeled the same receptor forms at 254 nm but at a markedly lower efficiency than their PAL derivatives. In contrast, N-bromoacetyl-L-[125I]T3, a chemical affinity labeling agent, did not derivatize either receptor form in vitro. The relative efficiency of coupling to receptor at 254 nm was L-[125I]T4-PAL greater than L-[125I]T3-PAL greater than L-[125I]T4 greater than L-[125I]T3. Although L-[125I]T4-PAL has a lower affinity for receptor than L-[125I]T3-PAL, its coupling efficiency was 5-10-fold higher. This suggests that the alanine side chain of L-[125I]T4-PAL is positioned in the ligand binding region near a residue which is efficiently modified by photoactivation. With L-[125I]T4-PAL we were able to identify three different molecular weight receptor species in human fibroblast nuclei.     

Formation and metabolism of 3',5'-diiodothyronine and 3,5-diiodothyronine by cultured monkey hepatocarcinoma cells.

Cultured monkey hepatocarcinoma cells were incubated with [3',5'-125I] diiodo-L--thyronine and with [3,5-125I] diiodo-L-thyronine. In both instances monodeiodination as well as sulfoconjugation took place. [3.-125I] iodothyronine and [3',5'-125I] diiodothyronine were identified as metabolites of [3'-5'-125I]-L-thyroxine in the cells, but neither [3-125I]-iodothyronine nor [3,5-125I] diiodothyronine was detected after incubation of the cells with ]3,5-125I]-L-thyroxine. No products of diphenyl ether splitting were observed in the medium after incubation of the cells with either [3,5-125I] diiodo-L-thyronine or [3,5-125I]-L-thyroxine.     

Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.     

Evidence for a close link between the thyroid hormone transport system and the aromatic amino acid transport system T in erythrocytes

The transport of [125I]triiodothyronine ([125I]T3) and [3H]tryptophan ([3H]Trp) by washed rat erythrocytes was studied at 25 degrees C in the presence of leucine in order to block the neutral amino acid transport system L. Eadie-Hofstee plots of initial velocity data gave the following values of Km (micromolar) and Vmax (nanomole/min/10(8) cells): 0.122 +/- 0.007 and 0.140 +/- 0.021 for T3, and 558 +/- 28 and 17.4 +/- 2.3 for Trp (n = 5). The Trp transport system in rat erythrocytes is similar to the human erythrocyte aromatic amino acid-specific system T described by Rosenberg et al. (Rosenberg, R., Young, J. D., and Ellory, J. C. (1980) Biochim. Biophys. Acta 598, 375-384). Unlabeled aromatic amino acids (e.g. Trp, phenylalanine, tyrosine) competitively inhibited [125I]T3 uptake and unlabeled iodothyronine analogues (e.g. T3, D-T3, thyroxine, thyronine) competitively inhibited [3H]Trp uptake. The inhibition constants of these competitors measured with each labeled substrate were highly correlated. N-Ethylmaleimide irreversibly inhibited T3 and Trp transport and each substrate protected the transport system of the other from inactivation by N-ethylmaleimide. The Vmax of T3 and Trp transport by human erythrocytes were 500 and 120 times lower, respectively, than those of rat erythrocytes (0.30 and 126 pmol/min/10(8) cells, respectively). The T3 and Trp transport activities of sheep erythrocytes were undetectable. These results indicate that T3 and Trp either share a common multi-specific transport system or are transported by closely linked systems which interact in the erythrocyte membrane.     

Influence of chronic treatment with iopanoic acid on thyroxine metabolism in the newborn rat

Parameters of the peripheral metabolism of thyroxine (T4) were studied in the early postnatal period. Iopanoic acid (IOP) was administered to newborn rats that were either euthyroid or rendered hypothyroid in utero by propylthiouracil (PTU) or methimazole (MMI) administration to the mothers during gestation and injected with thyroxine on postnatal days 6 and 7. In euthyroid newborn rats given IOP from postnatal day 6, the plasma T4 level increased (+50%) while the plasma 3,3',5'-triiodothyronine (T3) level slightly decreased (-18%). Peripheral deiodination of T4 was also reduced (about -50%) as estimated by thyroid 125I uptake after injection of 125I (3'-5')L-T4. In the newborn rats rendered hypothyroid in utero and given T4 on postnatal days 6 and 7, IOP treatment started on day 4 decreased the constant rate of elimination (-50%), the distribution volume (-43%) and the metabolic clearance (-74%) of plasma T4. The results were the same in PTU- and MMI-treated newborn rats. The differences between newborn and adult animals under IOP treatment are discussed.     

Phenolic and nonphenolic ring deiodinations of iodothyronines in cultured hepatocarcinoma cell homogenate from monkey.

Iodothyronine monodeiodinase activities in homogenates of cultured monkey hepatocarcinoma cells were measured by the deiodination of [3.5-(125)I]-diiodo-L-thyronine or 3-[3',5'-(125)I]triiodo-L-thyronine (phenolic ring-labeled 'reverse' triiodothyronine). The assay system utilized a small ion-exchange column (AG50W-X4, O.9 X approximately 1 cm) to measure 125I-. Both deiodinases were destroyed by boiling for 1 min. Maximal nonphenolic ring deiodination was observed at pH 7.9 whereas maximal phenolic ring deiodination was at pH 6.3. Both reactions were enhanced strongly by dithiothreitol (0.1-5mM), and slightly by 5 mM beta-mercaptoethanol. Phenolic ring deiodination was strongly inhibited by 0.1 mM propylthiouracil. Nonphenolic ring deiodination was accelerated by EDTA (1.2 MM) and inhibited by Mg(2+) (5mM). Methylmercaptoimidazol and Mg(2+), Ca(2+) and Mn(2+) (0.1-1.0 mM) had little or no effect on either reaction, but Zn(2+) (0.1 mM) strongly inhibited both. Both reactions were inhibited by excess iodothyronine analogues at 10 mM to 10 micron M, and thyroxine was shown to be a competitive inhibitor in both cases. On the basis of relative affinities and inhibitory effects, it appears that the order of affinity for the phenolic ring deiodinase is 3,3',5'-triiodo-L-thyronine(rT3) greater than L-thyroxine(T4) greater than 3,5,3'-triiodo-L-thyronine(T3), whereas for the nonphenolic ring deiodinase the order is T3 greater than T4 greater than rT3. Diiodotyrosine did not affect their deiodination.     

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.     

In vivo evaluation of 7 alpha-[11-(4-[125I]iodophenoxy)undecyl]-17 beta-estradiol: a potential vector for therapy of adrenal and estrogen receptor-positive cancers

7 alpha-[11-(4-[125I]Iodophenoxy)undecyl]-17 beta-estradiol ([125I]IPUE2) was synthesized and its tissue distribution studied in immature female Fischer rats. Upon intravenous administration, [125I]IPUE2 was shown to accumulate in the adrenals and, to some extent, in the uterus and the ovaries. Coinjection with estrogen receptor (ER)-saturating quantities of unlabeled 17 beta-estradiol did not significantly reduce the uptake of [125I]IPUE2 in these tissues. The high adrenal uptake of [125I]IPUE2 is most likely associated with the lipophilic nature of the 7 alpha-substituted estradiol. The potential to use the 7 alpha-undecylestradiol as a vector to direct therapeutic groups to adrenal and ER-positive cancers is discussed.     

Diprenylated chalcones and other constituents from the twigs of Dorstenia barteri var. subtriangularis

The twigs of Dorstenia barteri var. subtriangularis yielded three diprenylated chalcones: (-)-3-(3,3-dimethylallyl)-5'-(2-hydroxy-3-methylbut-3-enyl)-4,2',4'-trihydroxychalcone, (+)-3-(3,3-dimethylallyl)-4',5'-[2'-(1-hydroxy-1-methylethyl)-dihydrofurano]-4,2'-dihydroxychalcone and 3,4-(6",6"-dimethyldihydropyrano)-4',5'-[2',-(1-hydroxy-1-methylethyl)-dihydrofurano]-2'-hydroxychalcone for which the names bartericins A, B and C, respectively, are proposed. Stipulin, beta-sitosterol and its 3-beta-D-glucopyranosyl derivative were also isolated. The structures of these secondary metabolites were determined on the basis of spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, HMQC and HMBC. The structural relationship of bartericins B and C was further established by the chemical cyclization of one to the other.     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

The murine IgA-secreting plasmacytoma MOPC-315 expresses somatostatin receptors

We have previously shown that some neuropeptides had a profound effect on in vitro Ig synthesis (especially IgA) and mitogen-driven murine lymphocyte proliferation. MOPC-315, an IgA-secreting plasmacytoma line, has been extensively used in studies of the regulation of IgA synthesis. In this report we show that the neuropeptide somatostatin (SOM) inhibits proliferation ([3H]thymidine uptake) of MOPC-315 and also inhibits IgA synthesis in vitro. MOPC-315 cells bind both fluorescent SOM and [125I]SOM specifically. On cytofluorimetric analysis, 68 +/- 6.8% (mean +/- SE, n = 7) of MOPC 315 cells labeled with fluorescent SOM and this staining was compatible by incubation with an excess of unlabeled peptide. Specific [125I]SOM binding increased linearly with cell concentration, was rapid and achieved equilibrium after 20 min at 4 degrees C. It was temperature-dependent, readily reversible, and under equilibrium conditions demonstrated a dissociation constant of 1.6 +/- 0.7 nM (mean +/- SE, n = 5). Scatchard analysis showed that MOPC-315 cells had 40,733 +/- 16,050 (mean +/- SE) binding sites for SOM per cell. The characteristics of the interactions of SOM with MOPC-315 cells suggest a specific receptor-mediated mechanism whereby this neuropeptide may modulate lymphocyte function.     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.     

Photoaffinity labeling of platelet activating factor binding sites in rabbit platelet membranes

A photoreactive, radioiodinated derivative of platelet activating factor (PAF), 1-O-(4-azido-2-hydroxy-3-iodobenzamido)undecyl-2-O-acetyl-sn- glycero-3-phosphocholine ([125I]AAGP), was synthesized and used as a photoaffinity probe to study the PAF binding sites in rabbit platelet membranes. The nonradioactive analog, IAAGP, induced rabbit platelet aggregation with an EC50 value of 3.2 +/- 1.9 nM as compared to 0.40 +/- 0.25 nM for PAF. Specific binding of [125I]AAGP to rabbit platelet membranes was saturable with a dissociation constant (Kd) of 2.4 +/- 0.7 nM and a receptor density (Bmax) of 1.1 +/- 0.2 pmol/mg protein. Photoaffinity labeling of platelet membranes with [125I]AAGP revealed several 125I-labeled components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein species with apparent molecular weight of 52,000 was consistently observed and inhibited significantly by unlabeled PAF at nanomolar concentrations. The labeling was specific since the PAF antagonists, SRI-63,675 and L-652,731, at 1 uM also blocked the appearance of this band; whereas lysoPAF was not effective at the same concentration. These results suggest that the binding sites of PAF receptor in rabbit platelets reside in the polypeptide of Mr = 52,000.     

Synthesis and evaluation of glycosylated octreotate analogues labeled with radioiodine and 211At via a tin precursor

Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-[(3-tri-n-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 +/- 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 +/- 4.9% and 46.8 +/- 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 +/- 0.8% of input dose compared to 9.67 +/- 0.43% for N(alpha)-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 +/- 4.0% ID/g, 18.8 +/- 7.7% ID/g, and 0.9 +/- 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 +/- 1.2% ID/g, 4.7 +/- 1.4% ID/g, and 0.8 +/- 0.5% ID/g. Both in vitro and in vivo catabolism studies did not suggest the severance of the lys0 along with its appendages from the peptide. Taken together, although GIBLO maintained affinity to SSTR2, its tumor uptake both in vitro and in vivo was substantially lower than that of I-Gluc-TOCA suggesting other factors such as net charge and overall geometry of the peptide may be important.     

Inhibitory effects of calcium channel blockers on thyroid hormone uptake in neonatal rat cardiomyocytes

The effects of the Ca2+ channel blockers verapamil, nifedipine, and diltiazem on triiodothyronine (T3) and thyroxine (T4) uptake were tested in cultured cardiomyocytes from 2-day-old rats. Experiments were performed at 37 degrees C in medium with 0.5% BSA for [125I]T3 (100 pM) or 0.1% BSA for [125I]T4 (350 pM). The 15-min uptake of [125I]T3 was 0.124 +/- 0.013 fmol/pM free T3 (n = 6); [125I]T4 uptake was 0.032 +/- 0.003 fmol/pM free T4 (n = 12). Neither T3 nor T4 uptake was affected by 1% DMSO (diluent for nifedipine and verapamil). Uptake of [125I]T3 but not of [125I]T4 was dose dependently reduced by incubation with 1-100 microM verapamil (49-87%, P < 0.05) or nifedipine (53-81%, P < 0.05). The relative decline in [125I]T3 uptake after 4 h of incubation with 10 microM verapamil or nifedipine was less than after 15 min or 1 h, indicating that the major inhibitory effect of the Ca2+ channel blockers occurred at the level of the plasma membrane. The reduction of nuclear [125I]T3 binding by 10 microM verapamil or nifedipine was proportional to the reduction of cellular [125I]T3 uptake. Diltiazem (1-100 microM) had no dose-dependent effect on [125I]T3 uptake but reduced [125I]T4 uptake by 45% (P < 0.05) at each concentration tested. Neither the presence of 20 mM K+ nor the presence of low Ca2+ in the medium affected [125I]T3 uptake. In conclusion, the inhibitory effects of Ca2+ channel blockers on T3 uptake in cardiomyocytes are not secondary to their effects on Ca2+ influx but, rather, reflect interference with the putative T3 carrier in the plasma membrane.