Iptakalim as a human nicotinic acetylcholine receptor antagonist

Nicotinic acetylcholine receptors (nAChRs) play many critical roles in nervous system function and have been implicated in a variety of diseases. Drugs acting at nAChRs, perhaps in nAChR subtype-selective manners, can be used to dissect receptor function and perhaps as medications. In the present study, we used patch-clamp whole-cell recording and pharmacological manipulations to evaluate effects of iptakalim hydrochloride (Ipt), which is a drug reported to act as an ATP-sensitive potassium (K(ATP)) channel opener, on selected human nAChRs heterologously expressed in the native nAChR-null SH-EP1 human epithelial cell line. Ipt reduced both peak and steady-state whole-cell current amplitudes mediated by human alpha4beta2-nAChRs in response to nicotinic agonists. It also accelerated current decay, caused a decline in apparent efficacy of agonists, and acted in voltage- and use-dependent manners at alpha4beta2-nAChRs. These findings and the inability of Ipt to block radiolabeled epibatidine binding to alpha4beta2-nAChRs suggest a noncompetitive mechanism of antagonism. Other studies discount effects of Ipt on nAChR internalization or involvement of K(ATP) channels in Ipt-induced inhibition of alpha4beta2-nAChR function. By comparison, alpha7-nAChRs were less sensitive than alpha4beta2-nAChRs to Ipt acting as an antagonist. Thus, alpha4beta2-nAChRs are among the molecular targets of Ipt, which has utility as a tool in functional characterization and pharmacological profiling of nAChRs.     

Quantitative imaging in live human cells reveals intracellular alpha(1)-adrenoceptor ligand-binding sites

Cellular distribution and binding characteristics of native alpha(1)-adrenoceptors (ARs) were determined in a live, single, human smooth muscle cell (SMC) with confocal laser scanning microscopy and a fluorescent ligand, BODIPY-FL prazosin (QAPB). This allowed single-cell competitive ligand binding and showed that 40% of alpha(1)-AR-binding sites in native cells are intracellular. QAPB had high affinity and acted as a nonselective, competitive antagonist versus [(3)H]prazosin at cloned human alpha(1a)-, alpha(1b)-, and alpha(1d)-AR subtypes on membrane preparations and whole cells. RS100329 had 70-fold selectivity for alpha(1a)-ARs versus alpha(1b)- and alpha(1d)-ARs, validating its use to identify this subtype. In similar cells QAPB-associated fluorescence provided quantitative data analogous and comparable to [(3)H]prazosin binding in whole cells. In human, dissociated, prostatic smooth muscle cells QAPB-associated fluorescence binding exhibited specific high-affinity binding properties (FK(D) = 0.63 +/- 0.02 nM), which was 3- to 4-fold higher compared with recombinant cells (FK(D) = 2. 1-2.3 nM). Internal consistency in the data showed that affinity is greater, in general, in membrane preparations than in cells but also greater in the native prostatic tissues or cells than in equivalent recombinant receptors. Fluorescence revealed binding sites both on the plasmalemmal membrane and on intracellular compartments: at all locations RS100329 inhibited QAPB binding identifying the sites as alpha(1A)-ARs. Quantitative three-dimensional mapping of QAPB-associated fluorescence binding in native human cells showed that 40% of high-affinity-binding sites was in intracellular compartments. This provides a potential new site for physiological agonism and makes intracellular access a potential differentiator of drug action.     

S18616, a highly potent, spiroimidazoline agonist at alpha(2)-adrenoceptors: I. Receptor profile, antinociceptive and hypothermic actions in comparison with dexmedetomidine and clonidine

S18616 ((S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4, 2'-(8'-chloro-1',2',3',4'-tetrahydronaphthalene)]) displayed high affinity at native rat alpha(2)-adrenoceptors (AR)s (pK(i), 9.8), native human (h)alpha(2A)-ARs (9.6), and cloned halpha(2A)- (9.5), halpha(2B)- (9.2), and halpha(2C)- (9.0) ARs. It showed 40-fold lower affinity for halpha(1A)-ARs (8.4) and >/=100-fold lower affinity for rat alpha(1)-ARs (7.1), halpha(1B)-ARs (7.7), halpha(1D)-ARs (7.6), imidazoline(1) (7.4), and imidazoline(2) (7.4) sites and >100-fold lower affinity for all other (>50) sites. At halpha(2A)-ARs, in guanosine-5'-O-(3-[(35)S]thio)triphosphate binding studies, S18616 was a potent (partial) agonist: log effective concentration (pEC(50)), 9.3/maximal effect, 51%. This observation was corroborated employing a halpha(2A)-Gi1alpha fusion protein/GTPase assay (9.0/40%) in which the actions of S18616 were blocked by pertussis toxin. Employing guanosine-5'-O-(3-[(35)S]thio)triphosphate binding assays, S18616 was also a partial agonist at halpha(2C)-ARs (8.2/63%) but a full agonist (8.4/124%) at halpha(2B)-ARs. At halpha(2A)-, halpha(2B)-, and halpha(2C)-ARs, the selective alpha(2)-AR antagonist, atipamezole, abolished the actions of S18616: pK(b) values of 9.1, 9. 1, and 9.4, respectively. As determined by depletion of membrane-bound [(3)H]phosphatidyl inositols, S18616 behaved as a (less potent) agonist (7.8/79%) at halpha(1A)-ARs, an action abolished by prazosin (pK(b), 8.9). Reflecting alpha(2)-AR agonist properties, S18616 potently (>/=1 microg/kg, s.c.) and dose dependently elicited hypothermia and antinociception (nine diverse models) in rodents. These actions were dose dependently inhibited by chemically diverse alpha(2)- versus alpha(1)-AR antagonists, atipamezole, idazoxan, RX821,002, and BRL44418 (a preferential alpha(2A)-AR ligand). In contrast, the actions of S18616 were unaffected by the alpha(1)-AR antagonists, ARC239 and prazosin (which preferentially block alpha(2B/2C)- versus alpha(2A)-ARs). Although the affinity of dexmedetomidine at alpha(2)-ARs was lower than S18616; it displayed a similar receptor and functional profile. Clonidine displayed lower efficacy than S18616, was substantially less potent, and had marked affinity for imidazoline(1) sites and alpha(1)-ARs. In conclusion, S18616 is a novel, selective, and highly potent agonist at alpha(2)-ARs.     

Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. I. A multivariate analysis of the binding profiles of 14 drugs at 21 native and cloned human receptor subtypes

Because little comparative information is available concerning receptor profiles of antiparkinson drugs, affinities of 14 agents were determined at diverse receptors implicated in the etiology and/or treatment of Parkinson's disease: human (h)D(1), hD(2S), hD(2L), hD(3), hD(4), and hD(5) receptors; human 5-hydroxytryptamine (5-HT)(1A), h5-HT(1B), h5-HT(1D), h5-HT(2A), h5-HT(2B), and h5-HT(2C) receptors; halpha(1A)-, halpha(1B)-, halpha(1D)-, halpha(2A)-, halpha(2B)-, halpha(2C)-, rat alpha(2D)-, hbeta(1)-, and hbeta(2)-adrenoceptors (ARs); and native histamine(1) receptors. A correlation matrix (294 pK(i) values) demonstrated substantial "covariance". Correspondingly, principal components analysis revealed that axis 1, which accounted for 76% variance, was associated with the majority of receptor types: drugs displaying overall high versus modest affinities migrated at opposite extremities. Axis 2 (7% of variance) differentiated drugs with high affinity for hD(4) and H(1) receptors versus halpha(1)-AR subtypes. Five percent of variance was attributable to axis 3, which distinguished drugs with marked affinity for hbeta(1)- and hbeta(2)-ARs versus hD(5) and 5-HT(2A) receptors. Hierarchical (cluster) analysis of global homology generated a dendrogram differentiating two major groups possessing low versus high affinity, respectively, for multiple serotonergic and hD(5) receptors. Within the first group, quinpirole, quinerolane, ropinirole, and pramipexole interacted principally with hD(2), hD(3), and hD(4) receptors, whereas piribedil and talipexole recognized dopaminergic receptors and halpha(2)-ARs. Within the second group, lisuride and terguride manifested high affinities for all sites, with roxindole/bromocriptine, cabergoline/pergolide, and 6,7-dihydroxy-N,N-dimethyl-2-ammotetralin (TL99)/apomorphine comprising three additional subclusters of closely related ligands. In conclusion, an innovative multivariate analysis revealed marked heterogeneity in binding profiles of antiparkinson agents. Actions at sites other than hD(2) receptors likely participate in their (contrasting) functional profiles.     

Alpha-conotoxin Arenatus IB[V11L,V16D] [corrected] is a potent and selective antagonist at rat and human native alpha7 nicotinic acetylcholine receptors

A recently developed alpha-conotoxin, alpha-conotoxin Arenatus IB-[V11L,V16D] (alpha-CtxArIB[V11L,V16D]) [corrected], is a potent and selective competitive antagonist at rat recombinant alpha7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. alpha7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional alpha7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. alpha-CtxArIB[V11L,V16D] specifically inhibited alpha7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate alpha7 nAChRs were unaffected. Human alpha7 nAChRs were also sensitive to alpha-CtxArIB[V11L, V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human alpha7 nAChRs were inhibited by alpha-CtxArIB[V11L,V16D] (IC(50), 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat alpha7 nAChRs in PC12 cells. alpha-CtxArIB[V11L,V16D] inhibited human native alpha7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain alpha7 nAChRs contribute to dopamine release from striatal minces; alpha-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that alpha-CtxArIB[V11L,V16D] selectively inhibits human and rat native alpha7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating alpha7 nAChR functions.     

The comparative pharmacology and up-regulation of rat neuronal nicotinic receptor subtype binding sites stably expressed in transfected mammalian cells

We stably transfected human embryonic kidney cells (HEK 293 cells) with genes encoding rat neuronal nicotinic receptor alpha2, alpha3, or alpha4 subunits in combination with the beta2 or beta4 subunit to generate six cell lines that express defined subunit combinations that represent potential subtypes of rat neuronal nicotinic acetylcholine receptors (nAChRs). These cell lines were designated KXalpha2beta2, KXalpha2beta4, KXalpha3beta2, KXalpha3beta4, KXalpha4beta2, and KXalpha4beta4. The Kd values of [3H](+/-)epibatidine ([3H]EB) binding to membranes from these six cell lines ranged from approximately 0.02 to 0.3 nM. The pharmacological profiles of the agonist binding sites of these putative nAChR subtypes were examined in competition studies in which unlabeled nicotinic ligands, including 10 agonists and two antagonists, competed against [3H]EB. Most nicotinic ligands examined had higher affinity for the receptor subtypes containing the beta2 subunit compared with those containing the beta4 subunit. An excellent correlation (r > 0.99) of the binding affinities of the 10 agonists was observed between receptors from KXalpha4beta2 cells and from rat forebrain tissue, in which [3H]EB binding represents predominantly alpha4beta2 nAChRs. More important, the affinities (Ki values) for the two tissues were nearly identical. The densities of the binding sites of all six cell lines were increased after a 5-day exposure to (-)-nicotine or the quaternary amine agonist carbachol. These data indicate that these cell lines expressing nAChR subunit combinations should be useful models for investigating pharmacological properties and regulation of the binding sites of potential nAChR subtypes, as well as for studying the properties of nicotinic compounds.     

Antiparkinsonian agent piribedil displays antagonist properties at native, rat, and cloned, human alpha(2)-adrenoceptors: cellular and functional characterization

Compared with cloned, human (h)D(2) receptors (pK(i) = 6.9), the antiparkinsonian agent piribedil showed comparable affinity for halpha(2A)- (7.1) and halpha(2C)- (7.2) adrenoceptors (ARs), whereas its affinity for halpha(2B)-ARs was less marked (6.5). At halpha(2A)- and halpha(2C)-ARs, piribedil antagonized induction of [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding by norepinephrine (NE) with pK(b) values of 6.5 and 6.9, respectively. Furthermore, Schild analysis of the actions of piribedil at halpha(2A)-ARs indicated competitive antagonism, yielding a pA(2) of 6.5. At a porcine alpha(2A)-AR-Gi1alpha-Cys351C (wild-type) fusion protein, piribedil competitively abolished (pA(2) = 6.5) GTPase activity induced by epinephrine. However, at a alpha(2A)-AR-Gi1alpha-Cys351I (mutant) fusion protein of amplified sensitivity, although still acting as a competitive antagonist (pA(2) = 6.2) of epinephrine, piribedil itself manifested weak partial agonist properties. Similarly, piribedil weakly induced mitogen-activated protein kinase phosphorylation via wild-type halpha(2A)-ARs, although attenuating its phosphorylation by NE. As demonstrated by functional [(35)S]GTPgammaS autoradiography in rats, piribedil antagonized activation by NE of alpha(2)-ARs in cortex, amygdala, and septum. Antagonist properties were also expressed in a dose-dependent enhancement of the firing rate of adrenergic neurons in locus ceruleus (0.125-4.0 mg/kg i.v.). Furthermore, piribedil (2.5-4.0 mg/kg s.c.) accelerated hippocampal NE synthesis, elevated dialysis levels of NE in hippocampus and frontal cortex, and blocked hypnotic-sedative properties of the alpha(2)-AR agonist xylazine. Finally, piribedil showed only modest affinity for rat alpha(1)-ARs (5.9) and weakly antagonized NE-induced activation of phospholipase C via halpha(1A)-ARs (pK(b) = 5.6). In conclusion, piribedil displays essentially antagonist properties at cloned, human and cerebral, rat alpha(2)-ARs. Blockade of alpha(2)-ARs may, thus, contribute to its clinical antiparkinsonian profile.     

Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. II. Agonist and antagonist properties at subtypes of dopamine D(2)-like receptor and alpha(1)/alpha(2)-adrenoceptor

The accompanying multivariate analysis of the binding profiles of antiparkinson agents revealed contrasting patterns of affinities at diverse classes of monoaminergic receptor. Herein, we characterized efficacies at human (h)D(2SHORT(S)), hD(2LONG(L)), hD(3), and hD(4.4) receptors and at halpha(2A)-, halpha(2B)-, halpha(2C)-, and halpha(1A)-adrenoceptors (ARs). As determined by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding, no ligand displayed "full" efficacy relative to dopamine (100%) at all "D(2)-like" sites. However, at hD(2S) receptors quinpirole, pramipexole, ropinirole, quinerolane, pergolide, and cabergoline were as efficacious as dopamine (E(max)100%); TL99, talipexole, and apomorphine were highly efficacious (79-92%); piribedil, lisuride, bromocriptine, and terguride showed intermediate efficacy (40-55%); and roxindole displayed low efficacy (11%). For all drugs, efficacies were lower at hD(2L) receptors, with terguride and roxindole acting as antagonists. At hD(3) receptors, efficacies ranged from 33% (roxindole) to 94% (TL99), whereas, for hD(4) receptors, highest efficacies (approximately 70%) were seen for quinerolane, quinpirole, and TL99, whereas piribedil and terguride behaved as antagonists and bromocriptine was inactive. Although efficacies at hD(2S) versus hD(2L) sites were highly correlated (r = 0.79), they correlated only modestly to hD(3)/hD(4) sites (r = 0.44-0.59). In [(35)S]GTPgammaS studies of halpha(2A)-ARs, TL99 (108%), pramipexole (52%), talipexole (51%), pergolide (31%), apomorphine (16%), and quinerolane (11%) were agonists and ropinirole and roxindole were inactive, whereas piribedil and other agents were antagonists. Similar findings were obtained at halpha(2B)- and halpha(2C)-ARs. Using [(3)H]phosphatidylinositol depletion, roxindole, bromocriptine, lisuride, and terguride displayed potent antagonist properties at halpha(1A)-ARs. In conclusion, antiparkinson agents display diverse agonist and antagonist properties at multiple subtypes of D(2)-like receptor and alpha(1)/alpha(2)-AR, actions, which likely contribute to their contrasting functional profiles.     

Characterization of epibatidine binding to medial habenula: potential role in analgesia

The objective of the present study was to characterize a recently described binding site in the habenula, which has high affinity for [(3)H]epibatidine and low affinity for nicotine and acetylcholine. We report that the extension of this binding area in coronal and horizontal sections corresponds to the anatomical extension of the medial habenula. The affinity (K(D)) of the medial habenula receptors for [(3)H]epibatidine was estimated to be 0.5 nM using an autoradiographic saturation assay, whereas the affinity of the binding site for nicotine and acetylcholine was estimated to be 5 and 8 microM, respectively. The receptor density (B(max)) in the medial habenula was estimated to be about 1100 fmol/mg wet weight using [(3)H]epibatidine. The subunit composition of the "epibatidine receptor" was investigated by the ability of different compounds with affinity to various subtypes of nicotinic receptors to displace [(3)H]epibatidine bound to the receptor. The results suggest that the receptor contains alpha3 subunits but that it is unlikely to be an alpha3beta4 nicotinic receptor. Systemic administration of epibatidine has analgesic effects in rats. Here we report that 2 x 1 microl of 10 nM epibatidine, resulting in a 2 x 10-fmol dose, administered directly to the medial habenula by bilateral stereotactic injection had an analgesic effect measured in the hot-plate test. This dose of epibatidine increased hot-plate latency significantly, whereas 2 x 2 fmol of epibatidine or 2 x 10 fmol of nicotine were without effect. This leads us to suggest that the medial habenular epibatidine binding site might be a valuable target for the development of non-opiate analgesics.     

3H]A-585539 [(1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane], a novel high-affinity alpha7 neuronal nicotinic receptor agonist: radioligand binding characterization to rat and human brain

Receptor binding was characterized for [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane ([(3)H]A-585539), a selective high-affinity alpha7 nicotinic acetylcholine receptor (nAChR) agonist with rapid kinetics, low nonspecific binding, and high specific activity. At 4 degrees C, the association was monophasic and rapid (t((1/2)) = 8.0 min); dissociation was slower (t((1/2)) = 64.2 min). The K(d) in rat brain at 4 degrees C was 0.063 nM, whereas at 22 and 37 degrees C, the K(d) values were 0.188 and 0.95 nM, respectively. In contrast, the B(max) (34 fmol/mg protein) was unaffected by temperature. In human cortex, [(3)H]A-585539 bound with a K(d) of 0.066 nM and a B(max) of 5.8 fmol/mg protein at 4 degrees C, whereas under similar conditions, specific [(3)H]methyllycaconitine ([(3)H]MLA) binding was not measurable. A number of agonist and antagonist nAChR ligands displaced binding to rat brain membranes with rank order of affinity similar to that for [(3)H]MLA, and in general, a 5 to 10-fold higher affinity was observed for [(3)H]A-585539 binding. There was also a good correlation of K(i) values between [(3)H]A-585539 binding to rat brain and human cortex. The use of a alpha7/5-hydroxytryptamine type-3 chimera revealed that the N-terminal domain of alpha7 nAChR was sufficient to faithfully reproduce the pharmacology of [(3)H]A-585539 binding. Autoradiographic studies comparing [(3)H]A-585539 and [(125)I]alpha-bungarotoxin revealed a similar pattern of labeling in the rat. In summary, [(3)H]A-585539 was shown to have excellent binding characteristics in rat and human brain and represents the first high-affinity alpha7 agonist radioligand with utility in the characterization of this important nAChR subtype that is targeted toward ameliorating cognitive deficits underlying neuropsychiatric and neurodegenerative disorders.     

Effects of prolonged nicotinic ligand exposure on function of heterologously expressed,human alpha4beta2- and alpha4beta4-nicotinic acetylcholine receptors

Effects of prolonged nicotinic ligand exposure on the function of human alpha4beta2- and alpha4beta4-nicotinic acetylcholine receptor (nAChR) subtypes were studied using receptors heterologously expressed in SH-EP1 human epithelial cells. Magnitudes of acute, nAChR-mediated, specific 86Rb+ efflux responses to 1 mM carbamylcholine were reduced after pretreatment with specific nAChR ligands in effects that depended on pretreatment drug dose, duration of drug pretreatment, and duration of drug-free recovery. Fifty percent inhibition of alpha4beta2-nAChR function following 5 min of recovery occurred after 1 min of pretreatment with 1 mM nicotine but also after 1-h pretreatment at 10 nM nicotine. Seventy-five percent loss in function persisted 1 h after drug removal following 15 min or more of exposure to 1 mM nicotine. However, functional recovery was nearly complete after 1 h in drug-free medium following 1 min to 24 h pretreatment with 0.1 to 1 microM nicotine, i.e., in the range of smoker plasma nicotine levels. alpha4beta4-nAChR was similarly sensitive to persistent inactivation by prolonged nicotine exposure. Carbamylcholine exhibited slightly lower persistent inactivation potency than nicotine at both alpha4beta2- and alpha4beta4-nAChR. The nAChR antagonist, mecamylamine, exhibited persistent inactivation potency and efficacy similar to nicotine at alpha4beta2-nAChR but had a reduced effect on alpha4beta4-nAChR. These studies illustrate persistent inactivation of human alpha4beta2- or alpha4beta4-nAChR induced by prolonged exposure to nicotine and show that other ligands induce nAChR persistent inactivation in a subtype-specific manner.     

Characterization of opioid-binding sites in zebrafish brain

The pharmacological profile of opioid-binding sites in zebrafish brain homogenates has been studied using radiolabeled binding techniques. The nonselective antagonist [(3)H]diprenorphine binds with high affinity (K(D) = 0.27 +/- 0.08 nM and a B(max) = 212 +/- 14.3 fmol/mg protein), displaying two different binding sites with affinities of K(D1) = 0.08 +/- 0.02 nM and K(D2) = 17.8 +/- 9.18 nM. The nonselective agonist [(3)H]bremazocine also binds with high affinity to zebrafish brain membranes but only displays one single binding site with a K(D) = 1.1 +/- 0.09 nM and a B(max) = 705 +/- 19.3 fmol/mg protein. Competition binding assays using [(3)H]diprenorphine and several unlabeled ligands were performed. The synthetic selective agonists for mammalian opioid receptors DPDPE ([DPen(2),D-Pen(5)]-enkephalin), DAMGO ([D-Ala(2),NMe-Phe(4),Gly(5)-ol]-enkephalin), and U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide] failed to effectively displace [(3)H]diprenorphine binding, whereas nonselective ligands and the endogenous opioid peptides such as dynorphin A showed good affinities in the nanomolar range, although several of the endogenous peptides only displaced approximately 50% of the specifically bound [(3)H]diprenorphine. Our results provide evidence that, although the selective synthetic compounds for mammalian receptors do not fully recognize the opioid-binding sites in zebrafish brain, the activity of the endogenous zebrafish opioid system might not significantly differ from that displayed by the mammalian opioid system. Hence, the study of zebrafish opioid activity may contribute to an understanding of endogenous opioid systems in higher vertebrates.     

Synthesis and characterization of 125I-alpha-conotoxin ArIB[V11L;V16A], a selective alpha7 nicotinic acetylcholine receptor antagonist

The alpha7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-alpha-bungarotoxin is commonly used to identify alpha7 nAChRs specifically. However, alpha-bungarotoxin also interacts potently with alpha1* and alpha9alpha10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the alpha7 subtype. [3H]Methyllycaconitine is also frequently used as an alpha7-selective antagonist, but it has significant affinity for alpha6* and alpha9alpha10 nAChR subtypes. In this study, we have developed a highly alpha7-selective alpha-conotoxin radioligand by iodination of a naturally occurring histidine. Both mono- and diiodo derivatives were generated and purified (specific activities were 2200 and 4400 Ci mmol(-1), respectively). The properties of the mono- and diiodo derivatives were very similar to each other, but the diiodo was less stable. For monoidodo peptide, saturation binding to mouse hippocampal membranes demonstrated a K(d) value of 1.15 +/- 0.13 nM, similar to that of 125I-alpha-bungarotoxin in the same preparations (0.52 +/- 0.16 nM). Association and dissociation kinetics were relatively rapid (k(obs) for association at 1 nM was 0.027 +/- 0.007 min(-1); k(off) = 0.020 +/- 0.001 min(-1)). Selectivity was confirmed with autoradiography using alpha7-null mutant tissue: specific binding was abolished in all regions of alpha7(-/-) brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-alpha-conotoxin ArIB[V11L; V16A] should prove useful where alpha7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on alpha7 nAChRs, something that is impossible with 125I-alpha-bungarotoxin.     

Identification of RANTES receptors on human monocytic cells: competition for binding and desensitization by homologous chemotactic cytokines

RANTES (regulated on activation, normal T expressed and secreted) is a member of the chemotactic cytokine (chemokine) beta subfamily. High affinity receptors for RANTES have been identified on a human monocytic leukemia cell line THP-1, which responded to RANTES in chemotaxis and calcium mobilization assays. Steady-state binding data analyses revealed approximately 700 binding sites/cell on THP-1 cells with a Kd value of 400 pM, comparable to that expressed on human peripheral blood monocytes. The RANTES binding to monocytic cells was competed for by monocyte chemotactic and activating factor (MCAF) and macrophage inflammatory protein 1 (MIP-1) alpha, two other chemokine beta cytokines. Although MCAF and MIP-1 alpha competed for RANTES binding to monocytes with apparent lower affinity (with estimated Kd of 6 and 1.6, nM respectively) both of these cytokines effectively desensitized the calcium mobilization induced by RANTES. The chemotactic response of THP- 1 cells to RANTES was also markedly inhibited by preincubation with MCAF or MIP-1 alpha. In contrast, RANTES did not desensitize the THP-1 calcium mobilization and chemotaxis in response to MCAF or MIP-1 alpha. These results, together with our previous observations that RANTES did not compete for MCAF or MIP-1 alpha binding on monocytic cells, indicate the expression of promiscuous receptors on monocytes that recognize one or more cytokines within the chemokine beta family.