In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.     

The Dopamine Transporter Is Absent in Parkinsonian Putamen and Reduced in the Caudate Nucleus

The neuronal dopamine transporter/uptake site can be covalently labeled with the photoaffinity probe 1-(2-[bis-(4-fluorophenyl) methoxy]ethyl)-4-[2-(4-azido-3-[125I]iodophenyl)ethyl]piperazine [( 125I]FAPP) and visualized following sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Upon photolysis, [125I]FAPP specifically incorporated into a polypeptide of apparent Mr = 62,000 in membranes from both the putamen and the caudate nucleus of control, Alzheimer's, schizophrenia, and Huntington's diseased brain, and following complete deglycosylation, migrated as an Mr approximately 48,000 polypeptide. In parkinsonian postmortem putamen, however, there was no detectable photoincorporation of [125I]FAPP into the ligand binding subunit of the dopamine transporter. [125I]FAPP did specifically label the Mr 62,000 polypeptide of parkinsonian caudate, although with efficiencies of 20-50% of control. The asymmetrical loss of the dopamine transporter in Parkinson's diseased striatum was confirmed in reversible receptor binding experiments using [3H]GBR-12935 (3H-labeled 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)piperazine). In parkinsonian putamen, mazindol competitively inhibited the binding of [3H]GBR-12935 with an estimated affinity (Ki approximately 2,000 nM) 10 times lower than in controls (Ki approximately 30 nM), while the affinity of maxindol for [3H]GBR-12935 binding in the caudate was equal to that seen with controls (Ki approximately 50 nM). The proportion of [3H]GBR-12935 binding sites recognized by mazindol with high affinity in Parkinson's diseased caudate was, however, reduced by 50-80%.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro characterization of radioiodinated (+)-2-[4-(4-iodophenyl) piperidino]cyclohexanol [(+)-pIV] as a sigma-1 receptor ligand

We investigated the binding characteristics of a (+)-enantiomer of radioiodinated 2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[125I]pIV], radioiodinated at the para-position of the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (+)-pIV (Ki=1.30 nM) had more than 10 times higher affinity to the sigma-1 (sigma-1) receptor than (+)-pentazocine (Ki=19.9 nM) or haloperidol (Ki=13.5 nM) known as sigma ligands. Also, the binding affinity of (+)-pIV for the sigma-1 receptor (Ki=1.30 nM), was about 16 times higher than the sigma-2 (sigma-2) receptor (Ki=20.4 nM). (+)-pIV (Ki=1260 nM) had a much lower affinity for VAChT than (-)-vesamicol (Ki=13.0 nM) or (-)-pIV (Ki=412 nM). (+)-[125I]pIV had low affinity for the dopamine, serotonin, adrenaline, and acetylcholine receptors. Furthermore, in a saturation binding study, (+)-[125I]pIV exhibited a K) of 6.96 nM with a Bmax of 799 fmol/mg of protein. These results showed that (+)-pIV binds to the sigma-1 receptor with greater affinity than sigma receptor ligands such as (+)-pentazocine or haloperidol, and that radioiodinated (+)-pIV is suitable as radiotracer for sigma-1 receptor studies in vitro.     

Synthesis and applications of an aldehyde-containing analogue of SCH-23390

SCH-23390 is a high-affinity antagonist selective for D1 dopamine receptors (Ki = 2.5 nM). It does not contain a functional group that can be conveniently coupled to commercially available resins for affinity chromatography or to prepare photolabels for photoaffinity labeling of receptors. To construct an affinity resin for purification of dopamine D1 receptors, an aldehyde analogue of SCH-23390, (+/-)-7-chloro-8-hydroxy-1-(4'-formylphenyl)-3-methyl-2,3,4,5-tetrahydro -1H- 3-benzazepine (ASCH), was synthesized. 8-Methoxy-1-(4'-bromophenyl)-SCH-23390 was lithiated, formylated, and O-demethylated to form the aldehyde. NMR and IR analyses were performed to characterize the product. Assays were performed with the radioligand [125I]SCH-23982 to define the biological activity of the aldehyde. ASCH displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 7.1 nM. ASCH has been coupled through the aldehyde group on the phenyl ring to diaminodipropylamine-agarose for affinity chromatography. After solubilization of caudate membranes in 1% digitonin, the affinity resin retained binding sites for [125I]SCH-23982 that were eluted with 10 mM SCH-23390. The aldehyde was also covalently coupled to biotin hydrazide for fluorescence labeling of dopamine D1 receptors. The biotin-conjugated aldehyde of SCH-23390 displaced [125I]SCH-23982 binding from caudate membranes with a Ki value of 9.3 nM.     

Hormone-like activity of a synthetic decapeptide with the adrenocorticotropin-like sequence of human immunoglobulin G1

The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ?[125I]ACTH-(13-24)?, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.     

Angiotensin-(1-7) binds at the type 1 angiotensin II receptors in rat renal cortex.

Significant angiotensin (Ang) (1-7) production occurs in kidney and effects on renal function have been observed. The present study was undertaken to investigate binding characteristics of the heptapeptide to Ang II receptors present in rat renal cortex. [125I]-Ang II binding to rat glomeruli membranes was analyzed in the presence of increasing concentrations of Ang II, Ang-(1-7), DUP 753 and PD 123319. Linearity of the Scatchard plot of the [125I]-Ang II specific binding to rat glomeruli membranes indicated a single population of receptors, with a Kd value of 0.7 +/- 0.1 nM and a Bmax of 198 +/- 0.04 fmol/mg protein. DUP 753, an specific AT1 receptor antagonist, totally displaced the specific binding of [125I]-radiolabelled hormone with a Ki of 15.8 +/- 0.9 nM, while no changes were observed in the presence of the selective AT2 receptor antagonist, PD 123319. The specific [125I]-Ang II binding to rat glomerular membranes was displaced by Ang-(1-7) with high affinity (Ki = 8.0 +/- 3.2 nM). We conclude that radioligand binding assays in the presence of selective Ang II antagonists DUP 753 and PD 123319 suggest the unique presence of AT1, receptors in rat glomeruli and a possible role in the control of the biological renal effects of Ang-(1-7).     

Photoaffinity labeling of D-2 dopamine binding subunits from rat striatum, anterior pituitary and olfactory bulb with a new probe, [3H]azidosulpride

A novel photoaffinity probe [3H] azidosulpride has been developed for biochemical studies of D-2 dopamine receptors. This ligand binds to the receptors with high affinity (Kd = 3.1 +/- 0.2 nM) and, upon photoactivation, about 20% of the radioactivity bound to membranes becomes covalently incorporated. More than 90% of this irreversible binding is protectable by dopaminergic agents including D-2 selective compounds, whereas D-1 selective and non-dopaminergic compounds are ineffective. Analysis by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals a single band at Mr = 85 kDa for labeled receptors in striatum, anterior pituitary or olfactory bulb, where pharmacologically distinct binding sites have been previously detected.     

125I](+/-)FISCH: a new CNS D-1 dopamine receptor imaging ligand

Radiolabeling and in vitro and in vivo evaluation of an iodinated benzazepine: [125I] FISCH 7-Chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5- tetrahydro-1H-3-benzazepine, as a potential imaging agent for CNS D-1 dopamine receptors in animals, were investigated. After an iv injection, this benzazepine derivative showed good brain uptake in rats (2.70, 1.28, 0.48 %dose/whole brain at 2, 15 and 60 min, respectively). The striatum/cerebellum ratio was 2.50 at 60 min after the injection. The regional distribution in rat brain, as measured by ex vivo autoradiography, displayed highest uptake in the regions of the striatal complex and the substantia nigra, regions known to have a high concentration of D-1 dopamine receptors. Furthermore, this localized regional cerebral distribution was blocked by pretreatment with SCH-23390, a selective D-1 dopamine receptor antagonist. The in vitro binding affinity of this agent in rat striatum tissue preparation displayed a Kd of 1.43 +/- 0.15 nM. Competition data (in vitro) showed the following rank order of potency: SCH-23390 greater than (+/-)IBZP much greater than apomorphine greater than WB 4101 greater than ketanserin approximately spiperone. The preliminary data suggest that this analog of SCH-23390 shows similar selectivity for the CNS D-1 receptor.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Presence of dopamine D-2 receptors in human tumoral cell lines

[125I] Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, [125I] iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.     

Binding of endomorphin-2 to mu-opioid receptors in experimental mouse mammary adenocarcinoma.

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) binds with high affinity and selectivity to the mu-opioid receptor. In the present study, [125I]endomorphin-2 has been used to characterize mu-opioid-binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [125I]endomorphin-2 (1 nM) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K(d) = 18.79 +/- 1.13 nM, B(max) = 635 +/- 24 fmol/mg protein) and the other shows low affinity and higher capacity (K(d) = 7.67 +/- 0.81 microM, B(max) = 157 +/- 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [125I]endomorphin-2 binding site was [d-1-Nal3]morphiceptin > endomorphin-2 > [d-Phe3]morphiceptin > morphiceptin > [d-1-Nal3]endomorphin-2, indicating binding of these peptides to mu-opioid receptors. The uptake of 131I-labeled peptides administered intraperitoneally to tumor-bearing mice was also investigated. The highest accumulation in the tumor was observed for [d-1-Nal3)morphiceptin, which reached the value of 8.19 +/- 1.14% dose/g tissue.     

[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D-1 and D-2 Receptor Sites

[3H]Fluphenazine was used to label both D-1 and D-2 dopamine receptors in mouse striatal membranes. The D-1 and D-2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH-23390 (D-1 selective) and spiperone (D-2 selective). Saturation analyses of these two sites yielded a D-1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D-2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D-2 site was slightly greater than for the D-1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine-specific (apomorphine-sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.     

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide: a ligand for in vitro and in vivo studies of serotonin receptors

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide ([125I]-EIL) has been evaluated as a ligand for in vitro and in vivo studies of cerebral serotonin 5-HT2 receptors. [125I]-EIL exhibited high affinity (KD = 209 pM) for 5-HT2 receptors with a high degree of specific binding (80-95%) in membranes from rat prefrontal cortex. The regional distribution of [125I]-EIL binding in vivo to seven areas of mouse brain correlated significantly (Rs = 0.93) with known densities of 5-HT2 receptors. In vivo specificity, defined by tissue to cerebellum radioactivity ratios, reached a maximum for frontal cortex at 6 hr (21.2) and persisted through 16 hr (8.8). Ketanserin, a 5-HT2 receptor antagonist, fully inhibited binding in a dose dependent fashion in all brain regions except cerebellum. By contrast, blockers for dopamine D2, alpha- or beta-adrenergic receptors did not significantly inhibit radioligand binding in any region. [125I]-EIL selectively labels 5-HT2 receptors in vivo with the highest specificity of any serotonergic ligand reported to date, indicating that [123I]-EIL should prove applicable to single photon emission computed tomography studies in living brain.     

The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.     

Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney

Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.7 +/- 0.3 and 2.3 +/- 0.2 nM, respectively. In competition binding experiments, the solubilized fraction exhibited the same pharmacological profile as the membranes. Chemical crosslinking of [125I]vasopressin to the solubilized fraction followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated a 62-kDa band which was specifically labeled with [125I]vasopressin. Vasopressin binding sites from the solubilized fractions were resolved by gel filtration and ultracentrifugation on a sucrose gradient. In addition, agonist high affinity binding to V2 receptors and its sensitivity to guanine nucleotides were preserved even after solubilization in the absence of prebound agonist prior to solubilization. Addition of guanine nucleotides such as GTP gamma S decreased the specific binding of [3H]arginine vasopressin to these solubilized fractions in a dose-dependent manner, suggesting the solubilization of a V2 receptor-G protein complex. [32P]ADP ribosylation of the solubilized fraction by cholera and pertussis toxins revealed specifically labeled proteins with molecular weights of 42,000-43,000 and 39,000-41,000, respectively, on sodium dodecyl sulfate polyacrylamide gels. Furthermore [35S]GTP gamma S binding to these solubilized fractions was enhanced by vasopressin, confirming that a significant proportion of the vasopressin receptors must be closely coupled to G proteins even when these receptors are solubilized in the absence of agonist. These results are in contrast with those reported for beta, alpha 2 adrenergic and D2 dopaminergic receptor systems, but in agreement with D1 dopaminergic and A1 adenosine receptors. The molecular mechanism responsible for this difference remains to be determined.     

Measles virus modulates human T-cell somatostatin receptors and their coupling to adenylyl cyclase.

The possible role of immunomodulatory peptide somatostatin (SRIF) in measles virus (MV)-induced immunopathology was addressed by analysis of SRIF receptors and their coupling to adenylyl cyclase in mitogen-stimulated Jurkat T cells and human peripheral blood mononuclear cells (PBMC). SRIF-specific receptors were assayed in semipurified membrane preparations by using SRIF14 containing iodinated tyrosine at the first position in the amino acid chain ([125I]Tyr1) as a radioligand. A determination of receptor number by saturation of radioligand binding at equilibrium showed that in Jurkat cells, MV infection led to a dramatic decrease in the total receptor number. The virus-associated disappearance of one (Ki2 = 12 +/- 4 nM [mean +/- standard error of the mean [SEM]]; n = 4) of two somatostatin binding sites identified in control Jurkat cells (Ki1 = 78 +/- 3 pM and Ki2 = 12 +/- 4 nM [mean +/- SEM]; n = 4) was also observed. Almost identical results were obtained for phytohemagglutinin-activated human PBMC. In the absence of MV infection, two somatostatin binding sites were present (Ki1 = 111 +/- 31 pM and Ki2 = 17 +/- 2 nM [mean +/- SEM]; n = 2), whereas in MV-infected cells, only the high-affinity (Ki1 = 48 +/- 15 pM [mean +/- SEM]; n = 2) binding site remained. In addition, MV infection reinforced the inhibitory effects of SRIF on adenylyl cyclase activity, since maximal inhibition at 1 microM peptide was 11% +/- 4% in control cells versus 25% +/- 3% (P < 0.05) in infected Jurkat cells. Moreover, MV infection severely impaired the capacity of adenylyl cyclase to be activated directly (by forskolin) or indirectly (via Gs protein-coupled vasoactive intestinal peptide receptor). An assessment of [methyl-3H]thymidine incorporation showed that SRIF increased proliferative responses to mitogens only in control cells, not in MV-infected cells. Altogether, our data emphasize that MV-associated alteration of SRIF transduction appears to be related to the loss of SRIF-dependent increase of mitogen-induced proliferation.     

Characterization of a new radioiodinated probe for the alpha2C adrenoceptor in the mouse brain

[125I]17alpha-hydroxy-20alpha-yohimban-16beta-(N-4-p6 hydroxyphenethyl)carboxamide or [125I]rauwolscine-OHPC, a new radioiodinated probe derived from rauwolscine was synthesized and its binding characteristics investigated on sections of the mouse caudate putamen. [125I]rauwolscine-OHPC binding was saturable and revealed interaction with a single class of binding sites (KD= 0.171 nM, Bmax = 3082 pCi/mg of tissue). The kinetically derived affinity was in close agreement with the affinity evaluated by saturation experiments: k(-1)/k(+1)(0.0403 min(-1)/114 10(6) M(-1) min(-1))=0.35 nM. Competition studies revealed interaction with one single class of binding sites for each of the twelve compounds tested. The rank of potency suggested an interaction with alpha2 adrenoceptors (atipamezole > or = RX 821002 > yohimbine > (-)epinephrine). Moreover, the good affinity of [125I] rauwolscine-OHPC binding sites for spiroxatrine, yohimbine, WB 4101, the relatively good affinity for prazosin (Ki =37.4 nM) and the affinity ratio prazosin/oxymetazoline (37.4/43.4=0.86) were consistent with an alpha2C selective labelling of [125I]rauwolscine-OHPC. The distribution of [125I]rauwolscine-OHPC binding sites in mouse brain was characterized by autoradiography. The density of binding sites was high in the islands of Calleja, accumbens nucleus, caudate putamen and olfactory tubercles, moderate in the hippocampus, amygdala and anterodorsal nucleus of the thalamus. These findings demonstrated that [125I]rauwolscine-OHPC is a useful radioiodinated probe to label alpha2C adrenoceptors in mouse brain.     

[3H]N-Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.     

125I-Spiperone: a novel ligand for D2 dopamine receptors

125I-Spiperone binds with high affinity (KD 0.3 nM) to a single specific site (Bmax 34 pmol/g wet weight) in homogenates of rat corpus striatum. Specific binding is about 40-60 percent of total binding and is displaced stereo-specifically by butaclamol and clopenthixol. Neuroleptic drugs of various classes are potent inhibitors of 125I-spiperone binding (Ki's 1-10 nM). Selective dopamine antagonists such as sulpiride (Ki 50 nM) and dopamine agonists such as apomorphine (Ki 200 nM) are also potent inhibitors. The drug specificity of 125I-spiperone binding correlates well with that of 3H-spiperone binding, providing good evidence that 125I-spiperone labels D2 dopamine receptors in striatal membranes. 125I-Spiperone, with its high specific activity (2200 Ci/mmol) may prove to be a useful ligand in studies examining D2 dopamine receptors in soluble preparations and by autoradiography. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.