Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.     

Reciprocal modulation of the binding of angiotensin agonists and antagonists to angiotensin receptors in smooth muscle

1. Direct ligand binding studies have shown that the agonist 125I-[Sar1]Ang II and the antagonist 125I-[Sar1Ile8]Ang II bind to bovine uterus smooth muscle membranes in a time-dependent, reversible and saturable manner; both ligands had the same number of high affinity sites. 2. [Sar1Ile8]Ang II inhibited the binding of 125I-[Sar1]Ang II in a non-competitive manner by decreasing the number of high affinity sites without changing the binding affinity of the radioligand. 3. [Sar1]Ang II also inhibited the binding of 125I-[Sar1Ile8]Ang II in a non-competitive manner. 4. Dissociation of both radioligands from their receptor sites was fast enough that pseudo irreversible occupancy of the binding sites could not account for the observed non-competitive inhibition. 5. Displacement studies using 125I-[Sar1Ile8]Ang II as the radioligand provided evidence for the existence of two binding sites when the displacing ligand was [Sar1]Ang II but not when the displacing ligand was [Sar1Ile8]Ang II. 6. GTPS gamma S had no discernible effect on the binding of either 125I-[Sar1]Ang II or 125I-[Sar1Ile8]Ang II to bovine uterine membranes. 7. The present findings are consistent with an allosteric mechanism of antagonism for [Sar1Ile8]Ang II. The data are also consistent with a mechanism wherein agonist and antagonist ligands occupy different binding modes at the same receptor site and induce long-term conformational changes in the receptor which are idiosyncratic with respect to the nature of the ligand. An emerging relationship between the actions of angiotensin peptides and non-peptide mimetics of angiotensin is presented.     

Specific cytotoxicity of 16 alpha-[125I]iodoestradiol for estrogen receptor-containing breast cancer cells

We attempted to induce specific killing of estrogen receptor-containing breast cancer cells using estradiol coupled to a high specific activity gamma emitting isotope of iodine. MCF-7 human breast cancer cells were incubated with 16 alpha-[125I]iodoestradiol alone ([125I]E2), or with 16 alpha-[125I]iodoestradiol plus 100-fold excess 17 beta-estradiol (E2), and then viably frozen. After 8 weeks, the [125I]E2 exposed cells and the [125 I]E2 plus E2 exposed cells were 10% and 77% of controls, respectively. When the breast cancer cell line MDA-MB-231 which does not contain estrogen receptors was used, the rate of cell death was similar to the competed MCF-7 cells. When specific cytotoxicity was compared using a cloning technique, nearly a 5 log reduction in surviving cell fraction was seen with [125I]E2, as compared to identical cells treated with [125I]E2 plus competitor. This technique shows promise for selecting a population of cells with defects in their estrogen receptor and in studying subcellular hormone interactions.     

Offspring of xenogeneically-reconstituted scid/scid mice are capable of a primary xenogeneic immune response to DNP-KLH

The distribution of [125I]SRIF-28 ([Leu8,D-Trp22,125I-Tyr25]somatostatin-28), [125I]204-090 ([Tyr3]octreotide) and [125I]CGP 23996 (c[Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) labelled recognition sites was studied by autoradiography in rat brain at embryonic day 18 (E 18) and postnatal day 5 (P 5). These results were compared with mRNA expression of somatostatin receptors SSTR1-5 (named sst1-5 now) as studied by in situ hybridization. [125I]SRIF-28, [125I]204-090 and [125I]CGP 23996 binding displayed different although partially overlapping distributions, and showed an increase between E 18 and P 5, which was less marked for [125I]204-090 binding. -125I-204-090 binding and sst2 receptor mRNA were similarly distributed, whereas [125I]CGP 23996 binding did not correlate with any single somatostatin receptor mRNA. The data suggest that most SRIF receptor subtypes in rat brain are present before birth, but evolve differently.     

Photoaffinity labeling of D1 and D5 dopamine receptors

Although human D1 and D5 dopamine receptors are encoded by distinct genes and share only 50% sequence homology at the amino acid level, their pharmacological properties are identical. Using a selective D1 receptor photoaffinity radioligand, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrahyd ro-1H-3-benzazepine ([125I]MAB), we have further probed the molecular properties of these receptors in transfected GH4C1 rat pituitary cells. Under reversible, non-covalent binding conditions, [125I]MAB bound to both the D1 and the D5 receptors with identical affinities, dopaminergic selectivity and stereospecificity. Upon photoactivation of the bound [125I]MAB, the label was incorporated into a approximately 64,000 mol. wt protein corresponding to the D1 dopamine receptor. However, there was no specific photoincorporation of the ligand observed in D5 receptors. The lack of [125I]MAB photolabeling of D5 receptors was independent of the cell line chosen, since similar results were obtained using other transfected cells. The data suggest that although both D1 and D5 receptors share structurally similar binding sites, the protein domains around the sites are different. Thus, although there are currently no specific compounds which bind preferentially to D1 or D5 receptors, these receptors can be distinguished from one another by the inability of [125I]MAB to photolabel D5, but not D1, receptors. Such selective targeting of a specific receptor may be useful in understanding the functional importance and/or interaction between closely related members of the same receptor family when co-expressed in the same cell.     

Autoradiographic distribution and physiological regulation of 2-[125I]iodomelatonin binding in rat epididymis

Autoradiographic study was conducted to localize 2-[125I]iodomelatonin binding in the rat epididymis. In the peripubertal (6 weeks old), postpubertal (8 weeks old) and adult (3 months old) rats, intense specific 2-[125I]iodomelatonin labelling of the corpus epididymidis was observed. The intensity of 2-[125I]iodomelatonin binding in the distal epididymal segment was significantly decreased in orchidectomized rats but the effect could be reversed with testosterone replacement. The intensity of 2-[125I]iodomelatonin binding in the distal rat epididymal segment did not show any diurnal rhythmicity when mid-light period and mid-dark period levels were compared, and was unaffected by constant lighting. Our data suggest androgen-dependent expression of 2-[125I]iodomelatonin binding sites, independent of light-induced changes in circulating melatonin, in the rat corpus epididymidis. A novel role of melatonin and its receptor in the regulation of the functions of rat corpus epididymidis is strongly implicated.     

Margatoxin binds to a homomultimer of K(V)1.3 channels in Jurkat cells. Comparison with K(V)1.3 expressed in CHO cells

Voltage-gated potassium (K(V)) channels play key roles in setting the resting potential and in the activation cascade of human peripheral T lymphocytes. Margatoxin (MgTX), a 39-amino acid peptide from Centruroides margaritatus, is a potent inhibitor of lymphocyte K(V) channels. The binding of monoiodotyrosinyl margatoxin ([125I]MgTX) to plasma membranes prepared from either Jurkat cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, K(V)1.3, has been used to investigate the properties of lymphocyte K(V) channels. These data were compared with [125I]MgTX binding to heterotetrameric K(V) channels in rat brain synaptic plasma membranes [Knaus, H. G., et al. (1995) Biochemistry 34, 13627-13634]. The affinity for [125I]MgTX is 100-200 fM in either Jurkat or CHO/K(V)1.3 membranes, and the receptor density is 20-120 fmol/mg in Jurkat membranes or 1000 fmol/mg in CHO/K(V)1.3 membranes. In contrast to rat brain, [125I]MgTX binding to Jurkat and CHO/K(V)1.3 membranes exhibits an absolute requirement for K+, with no potentiation of binding by Na+. K(V)1.3 was the only K(V)1 series channel present in either CHO/K(V)1.3 or Jurkat plasma membranes as determined by immunoprecipitation of [125I]MgTX binding or by Western blot analyses using sequence-specific antibodies prepared against members of the K(V)1 family. The relative potencies of a series of peptidyl K(V) channel inhibitors was essentially the same for inhibition of [125I]MgTX binding to Jurkat, CHO, or rat brain membranes and for blocking 86Rb+ efflux from the CHO/K(V)1.3 cells, except that alpha-dendrotoxin was more potent at blocking binding to rat brain membranes than in the other assays. The characteristics of [125I]MgTX binding, the antibody profiles, and the effects of the peptidyl K(V) inhibitors all indicate that the [125I]MgTX receptor in Jurkat lymphocytes is comprised of a homomultimer of K(V)1.3, unlike the heteromultimeric arrangement of the receptor in rat brain.     

Synthesis and radiolabeling of (S)-4-amino-5-iodo-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide, the active enantiomer of [125I]iodozacopride, and re-evaluation of its 5-HT3 receptor affinity

We report an improved synthesis of unlabeled (S)-iodozacopride, the radiolabeling of (S)-[125I]iodozacopride via deschloro-(S)-zacopride, and a re-evaluation of its affinity for the 5-HT3 receptor. Unlabeled (S)-iodozacopride was prepared in seven steps from 4-aminosalicylic acid via alkaline hydrolysis of its 4-acetamide derivative. Catalytic hydrogenation of (S)-iodozacopride gave deschloro-(S)-zacopride, identical to that obtained from (S)-3-amino-quinuclidine and 4-amino-2-methoxybenzoic acid via its corresponding 1-imidazole derivative. Radioiodination to produce (S)-[125I]iodozacopride was accomplished by treatment of deschloro-(S)-zacopride with 5 mCi sodium 125iodide and chloramine-T in hydrochloric acid. Purification of the reaction products using an HPLC system capable of detecting chlorinated side-products revealed a mixture of 2.1 mCi (1.3 nmol) (S)-[125I]iodozacopride and (S)-zacopride (1.5 nmol). Saturation analysis of the binding of the purified (S)-[125I]iodozacopride to whole rat brain homogenates gave an estimated KD of 1.10 +/- 0.07 nM. As anticipated, this is approximately half the KD reported for binding of racemic [125I]iodozacopride, and differs from the previously reported value by an order of magnitude. Analysis of the apparent binding affinity of a 1:1 mixture of (S)-[125I]iodozacopride and (S)-zacopride suggests that the previous result may have been confounded by contamination of the product with unlabeled (S)-zacopride. Competition analysis of the displacement of (S)-[125I]iodozacopride binding by unlabeled (S)-iodozacopride and (S)-zacopride gave Ki values of 0.95 and 0.21 nM, respectively.     

A membrane protein associated with the prolactin receptor. Studies with a photoactivatable human growth hormone derivative

Prolactin receptor from rat liver (PRL-R, 42 kDa) was cross-linked to a radiolabeled azidophenacyl derivative of human growth hormone ([125I]AP-hGH) to yield a 63 kDa adduct. In addition, a protein of Mr 50-52 K was detected as a 73 kDa complex. Microsomes incubated with either (a) increasing amounts of [125I]AP-hGH, or (b) a fixed amount of photoprobe and increasing concentrations of unlabeled hGH, showed that the 73/63 kDa band intensity ratio remains constant (0.71-0.77). Once transferred onto nitrocellulose membranes, only the 42 kDa protein is able to bind [125I]AP-hGH or [125I]hGH. Two anti-PRL-R monoclonal antibodies fail to cross-react with proteins of Mr 50-52 K. In membranes solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a significantly lower amount of the 73 kDa complex is detected. Thus, the 50-52 kDa protein appears to be structurally unrelated to, but is presumably associated with the PRL-R. The 73 kDa complex is also detected under low membrane fluidity conditions (1 degree C), indicating that PRL-R associates to this 50-52 kDa protein prior to hormone binding. Perfusion of rat liver with [125I]AP-hGH shows that this associated protein accompanies the receptor along its intracellular pathway.     

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.     

Binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane to serotonin and dopamine transporters: different ionic requirements for substrate and 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binding

The iodinated cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane (beta-[125I]CIT) binds with high affinity to the platelet plasma membrane serotonin transporter, as previously reported for dopamine transporters from rat brain [Eur. J. Pharmacol. 194:133-134 (1991)]. Unlabeled beta-CIT also inhibits serotonin transport by platelet membrane vesicles. In both rat striatal membranes and platelet plasma membranes, beta-[125I]CIT binding was found to be pH dependent, with a pKa of 6.4-6.9, and did not require the presence of Cl-. Na+ dramatically stimulated beta-[125I]CIT binding to both serotonin and dopamine transporters, although a small fraction of beta-[125I]CIT binding to the serotonin transporter was observed in the absence of Na+. The substrates serotonin and dopamine competed with beta-[125I]CIT for binding to their respective transporters. However, substrate affinity was enhanced by Cl-, whereas beta-[125I]CIT binding affinity was not. [3H]Imipramine binding to the platelet serotonin transporter and [3H]GBR-12935 binding to the dopamine transporter were not inhibited by decreasing the pH from 8 to 6.5. Likewise, the ability of serotonin to compete with [3H]imipramine binding and that of dopamine to inhibit [3H]GBR-12935 binding were equal at pH 6.5 or 8. Thus, beta-[125I]CIT binding to biogenic amine transporters is distinct from serotonin or dopamine binding by virtue of its inhibition by H+ and its insensitivity to Cl-.     

An approach for assaying benzodiazepine receptor binding with radioiodinated ligand: 125I-labeled diazepam derivative

[125I]2'-Iododiazepam (IDZ) was prepared and its application in a benzodiazepine receptor binding assay was studied. [125I]2'-IDZ binds to the rat cortical membrane with a high affinity (Kd, 0.66 nM). Various benzodiazepines showed competition with [125I]2'-IDZ for the binding sites in the rat cortical membrane, and the specificity of its binding correlated well with that of [3H]diazepam (r = 0.992, p < 0.001). These findings suggested that [125I]2'-IDZ binds to the same sites as [3H]diazepam and indicated that [125I]2'-IDZ can be used in a benzodiazepine receptor assay.     

Eel ventricular natriuretic peptide: isolation of a low molecular size form and characterization of plasma form by homologous radioimmunoassay

In order to determine the relation between myocardial fibrosis and (1) angiotensin converting enzyme (ACE) binding density, (2) receptor binding of ACE-related peptides, angiotensin II (AngII) and bradykinin (BK), and (3) the regulation of myocardial ACE by circulating Ang II, we used in vitro quantitative autoradiography to localize and assess ACE ([125I]351A), AngII receptor (125I[Sar1, IIe8]AngII), and BK receptor ([125I]Tyr8) binding densities in the rat myocardium. The experimental groups were (1) AngII (9 micrograms/h subcutaneously) or aldosterone (0.75 microgram/h subcutaneously in uninephrectomized rats on a high-sodium diet) administered by implanted minipump to chronically increase or decrease circulating AngII, respectively, (2) unoperated, untreated age- and sex-matched control rats, and (3) age- and sex-matched uninephrectomized control rats on a high-sodium diet. In the same heart studied at 2, 4, and 6 weeks of hormone treatment, serial sections were assessed for myocardial (hematoxylin and eosin) and fibrillar collagen (picrosirius red) morphology. The authors found that (1) marked ACE binding was coincident with sites of fibrous tissue that appeared in both ventricles as either a perivascular fibrosis of intramyocardial coronary arterioles or microscopic scarring in response to AngII or aldosterone infusion, (2) ACE binding was independent of circulating AngII, (3) BK, not AngII, receptor binding density was markedly increased at fibrous tissue sites, and (4) high-density ACE and BK receptor binding were anatomically coincident. Thus, in these experimental models, ACE is related to fibrous tissue formation where it may use BK, not angiotensin I, as a substrate, and its binding density is independent of circulating AngII.     

The distribution of tachykinin binding sites in the brain of an electric fish (Apteronotus leptorhynchus)

We mapped the distribution of tachykinin binding sites utilizing quantitative autoradiography of iodinated substance P and eledoisin as prototypic ligands for neurokinin-1 (NK1) and neurokinin-3 (NK3) receptors, respectively. The two ligands produced highly heterogenous and quantitatively different patterns of specific binding, suggesting that they revealed different tachykinin receptor subtypes. Although [125I]substance P and [125I]eledoisin binding were correlated in most brain regions, the binding of substance P was usually denser. [125I]substance P binding and substance P-like immunoreactivity were reasonably correlated in most brain areas, although discrepancies were found in some nuclei. Dense [125I]substance P binding was found in most areas of the subpallium and in parts of the pallium related to the olfactory system, as well as in the glomerular layer of the olfactory bulb. Moderate to dense binding of both ligands was observed in preoptic area, hypothalamus, habenula, parts of the thalamus and preglomerular complex. Especially noteworthy was the presence of [125I] substance P binding in the diencephalic prepacemaker nucleus, a region involved in the control of electroncommuncatory behavior. Substance P-like immunoreactivity is sexually dimorphic in certain diencephalic nuclei, including the prepacemaker nucleus (Weld and Maler, 1992); no obvious difference was seen between [125I]substance P or [125I]eledoisin binding in the brains of male versus female fish. In the mesencephalon striking laminar patterns of binding were seen in the torus semicircularis dorsalis and the optic tectum. Dense binding was also noted in the raphé nuclei, the locus ceruleus and the sensory nucleus of the vagus. Although binding of substance P in the electrosensory lateral line lobe and nucleus preeminentialis was light, it was distributed in a discrete fashion, suggesting a role of substance P in electrosensory processing.     

Status of somatostatin receptor messenger RNAs and binding sites in rat brain during kindling epileptogenesis

In situ hybridization histochemistry with somatostatin sst1-sst5 receptor messenger RNA-selective oligoprobes and quantitative receptor autoradiographic binding studies using [125I]Tyr3-octreotide, [Leu2,D-Trp22,125I-Tyr25]somatostatin-28 and [125I]CGP 23996 ([125I]c[Asn-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) were performed to determine the level of expression of somatostatin receptor messenger RNA and receptor binding sites in the hippocampal formation, limbic system and cerebral cortex of adult rats electrically kindled in the dorsal hippocampus. In control rats (implanted with electrodes but not electrically stimulated), the somatostatin-1 receptor-selective [125I]Tyr3-octreotide and the non-subtype-selective [Leu3,D-Trp22,125I-Tyr25]somatostatin-28 preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the molecular layer of the dentate gyrus, the subiculum and presubiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. The non-subtype-selective radioligand [125I]CGP 23996 (in 5 mM Mg2+ buffer) preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the subiculum and the basolateral nucleus of the amygdala. Under conditions where primarily somatostatin-2 receptors were labelled, [125I]CGP 23996 (in 120 mM Na+ buffer) showed strong binding in the strata oriens and radiatum of the CA1 subfield of the hippocampus and the frontal cortex, whereas the dentate gyrus, subiculum and amygdala showed only weak signals. During and after kindling, no significant differences were observed between the ipsi- and contralateral sides of the hippocampus. A significant decrease (about 40%) of somatostatin receptor binding sites was observed in the molecular layer of the dentate gyrus with all radioligands (except [125I]CGP 23996 in Na+ buffer, which did not label this area) at stage 2 (pre-convulsive stage) and one week, but not one month, after stage 5 (generalized motor seizures). In contrast to somatostatin receptor binding, no alterations of the messenger RNA levels for sst1-sst5 receptors were found either at stage 2 or at stage 5. Similarly, no changes in receptor binding or messenger RNA levels were observed in the brain of rats which experienced a single afterdischarge. The present study shows a significant and selective decrease of somatostatin-1 receptor binding sites in the dentate gyrus of kindled rats. This is part of the plastic changes induced by kindling and may contribute to the increased sensitivity for the induction of generalized seizures during kindling.     

Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study

In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin > SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [Sar9]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,Me-Leu9,Nle10]NKA(4-10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [125I]BHSP prefers SP and ranakinin. This toad "NK-1-like receptor" differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.     

Pharmacological characterization of a simple behavioral response mediated selectively by central adenosine A1 receptors, using in vivo and in vitro techniques

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1-32) ([125I]sCT(1-32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1-36) amide ([125I]chPTHrP(1-36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1-32) (EC50 0.05 nM) and 3-fold by chPTHrP(1-36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1-32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1-32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1-36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.     

Multivalent cations depress ligand affinity of insulin-like growth factor-binding proteins-3 and -5 on human GM-10 fibroblast cell surfaces

The effect of multivalent cations on [125I]-IGF binding to cell-associated IGFBPs was investigated using human fibroblasts. The major cell-associated binding site for [125I]-IGF-I is IGFBP-3 and for [125I]-IGF-II are IGFBP-3 and IGFBP-5. Lanthanum and chromium did not affect either [125I]-IGF-I or [125I]-IGF-II binding to cell-associated IGFBPs. By contrast, zinc (Zn2+), gold (Au3+), and cadmium (Cd2+) depressed binding of both ligands. Ligand binding resulted in nonlinear Scatchard plots. Assuming a pre-existent asymmetric model with high- (K[aHi]) and low- (K[aLo]) affinity sites, Zn2+ lowered both K(aHi) and K(aLo). Au3+ eliminated K(aHi). Assuming that the nonlinear plots were caused by ligand-induced negative cooperativity, Zn2+ and Cd2+ lowered both Ke and Kf (affinity of unoccupied and saturated IGFBPs, respectively). Au3+ eliminated Ke and reduced Kf. Zn2+ was active at serum levels in lowering IGF binding. Zinc, gold, and cadmium bind to similar regions within proteins (a zinc-binding motif) indicating similar mechanisms of action. A zinc-binding motif is present in the IGFBPs, but not in the IGFs. We demonstrate for the first time that the trace nutrient zinc and related multivalent cations decrease IGF binding to fibroblast-associated IGFBPs by lowering the affinity of the IGF-IGFBP interaction.