Biomedical subjects

A E Jacobson

Publications and source records attributed to A E Jacobson.

At least 109 records · Page 6Linked to original sources

PubMed1988

Evidence for a delta-class opioid receptor on cells of the immune system.

Opioid receptors have been reported to be present on cells of the immune system. Both opioid receptor-ligand interactions which are reversible by naloxone (classical) and those interactions which are irreversible by naloxone (nonclassical) appear to alter various immunologic parameters through the ligand-receptor interactions. In an effort to characterize opioid receptors (which operate with classical interactions) on cells of the immune system, we employed the delta-class opioid receptor-specific ligand, cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT). We herein report the specific labeling of a protein with a molecular weight of 70,000 Da under reducing conditions (58,000 Da under nonreducing conditions) from both B and T cell-enriched murine splenocytes and peripheral blood lymphocytes. Similar results are demonstrated by human peripheral blood lymphocytes and plastic-adherent (monocyte-enriched) leukocytes.

Animals↗

PubMed1988

Mu-, delta- and kappa-opioid receptor-mediated inhibition of neurotransmitter release and adenylate cyclase activity in rat brain slices: studies with fentanyl isothiocyanate.

We investigated the effects of [D-Ala2,D-Leu5]enkephalin (DADLE). [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) (0.01-1 microM) and bremazocine (0.001-0.3 microM) on the electrically evoked release of radiolabelled neurotransmitters and on the dopamine (DA)-stimulated cyclic AMP efflux from superfused rat brain slices. The differential inhibitory effects of these agonists on the evoked neurotransmitter release indicate that the opioid receptors mediating presynaptic inhibition of [3H]noradrenaline (NA, cortex), [14C]acetylcholine (ACh, striatum) and [3H]DA (striatum) release represent mu, delta and kappa receptors, respectively. In agreement with this classification, preincubation (60 min) of the slices with the delta-opioid receptor-selective irreversible ligand, fentanyl isothiocyanate (FIT, 0.01-1 microM), antagonized the inhibitory effects of DADLE and DPDPE on striatal [14C]ACh release only. On the other hand, the D-1 DA receptor-stimulated cyclic AMP efflux from striatal slices appeared to be inhibited by activation of mu as well as of delta receptors. In this case, the reversible mu antagonist, naloxone (0.1 microM), fully antagonized the inhibitory effect of the mu agonist, DAGO, without changing the effect of the delta agonist DPDPE but was ineffective as an antagonist in slices pretreated with FIT (1 microM). The inhibitory effect of DAGO on the electrically evoked [3H]NA release was antagonized by naloxone whether the receptors were irreversibly blocked by FIT or not. These data not only further support the existence of independent presynaptic mu-, delta- and kappa-opioid receptors in rat brain but also evidence strongly that mu and delta receptors mediating the inhibition of DA-sensitive adenylate cyclase could share a common binding site (for naloxone and FIT) and, therefore, may represent constituents of a functional opioid receptor complex.

Acetylcholine↗

PubMed1988

Differential coupling of mu-competitive and mu-noncompetitive delta opiate receptors to guanine nucleotide binding proteins in rat brain membranes.

The effects of fentanyl isothiocyanate (FIT) and pertussis toxin on the binding of [3H]D-Ala2, D-Leu5-enkephalin ([3H]DADLE) to rat brain membranes were compared. The site of action of pertussis toxin was confirmed by the labeling of a 41,000 dalton protein in the presence of [alpha-32P]NAD. Both reagents produced inhibition of [3H]DADLE binding when binding was assayed in 10 mM Tris-HCl buffer alone. FIT inhibited binding 91% whereas pertussis toxin treatment resulted in 27% inhibition. However, when binding was assayed in 10 mM Tris-HCl containing SMG (100 mM NaCl, 3 mM manganese acetate, and 2 microM guanosine triphosphate), inhibition due to both reagents was attenuated markedly: 66% for FIT and 5% for toxin. In addition, both reagents markedly potentiated enhancement of binding by SMG. Thus, the effects of FIT and pertussis toxin on [3H]DADLE binding were qualitatively similar. These results suggest that FIT and pertussis toxin affect binding of [3H]DADLE to the same population of delta receptors. This was further supported by the observation that treatment of membranes with FIT prior to pertussis toxin treatment blocked the effect of toxin on [3H]DADLE binding. FIT selectively eliminates the SMG-insensitive, mu-competitive [3H]DADLE binding site [Rothman et al., Neuropeptides 4, 201 (1984); Rothman et al., Molec. Pharmac. 27, 399 (1985)]. These results indicate that this site is coupled to G protein substrates for pertussis toxin and that it mediates the inhibitory effects of delta ligands on adenylate cyclase. The FIT-insensitive, SMG-sensitive mu-noncompetitive [3H]DADLE site appears not to be coupled to G protein substrates for pertussis toxin and may mediate some other biochemical effects of delta ligands.

Adenylate Cyclase Toxin↗

PubMed1988

Chronic administration of morphine and naltrexone up-regulate[3H][D-Ala2,D-leu5]enkephalin binding sites by different mechanisms.

Previous studies have demonstrated that chronic administration of morphine up-regulated the lower affinity binding site for [3H][D-ala2,D-leu5]enkephalin, without producing a detectable alteration in the higher affinity binding site for [3H][D-ala2,D-leu5]enkephalin (Rothman et al., Eur. J. Pharmac. 124: 113-119, 1986). The experiments reported in this paper tested the hypothesis that chronic administration of morphine and naltrexone up-regulated the binding sites for [3H][D-ala2,D-leu5]enkephalin by different mechanisms. Rats were given either morphine or naltrexone chronically. Chronic administration of morphine up-regulated the lower affinity site, while chronic administration of naltrexone up-regulated both the higher and lower affinity binding sites for [3H][D-ala2,D-leu5]enkephalin. Unlike the lower affinity binding site for [3H][D-ala2,D-leu5]enkephalin present in membranes prepared from rats treated with placebo pellets, the lower affinity binding sites which were up-regulated by naltrexone and morphine were partially (naltrexone) or completely (morphine) labile to preincubation for 60 min at 25 degrees C in 50 mM Tris-HCl, pH 7.4, containing 0.4 M NaCl. These data suggest that chronic administration of morphine and naltrexone up-regulate binding sites for [3H][D-ala2,D-leu5]enkephalin through different mechanisms, and that the lower affinity binding sites for [3H][D-ala2, D-leu5]enkephalin which are up-regulated by chronic administration of morphine and naltrexone might differ biochemically from the lower affinity binding sites present in membranes treated with placebo.

Animals↗

PubMed1988

Metaphit prevents locomotor activation induced by various psychostimulants and interferes with the dopaminergic system in mice.

Metaphit, an isothiocyanate analog of phencyclidine and a proposed phencyclidine receptor acylator, inactivated the carrier involved in the neuronal uptake of dopamine in in vitro experiments with preparations of the striatum in the mouse. In ex vivo experiments 2 and 24 hr after the intravenous administration of metaphit, no changes were observed either in the binding of [3H]cocaine to striatal membranes or in the uptake of [3H]dopamine into synaptosomes or slices. In in vivo experiments 24 hr after pretreatment with metaphit, selective labelling of uptake sites for dopamine in the striatum of the mouse with [3H]GBR 12935 was unaffected. In these in vivo experiments, however, metaphit antagonized the locomotor stimulation induced by blockers of the uptake of dopamine (methylphenidate, mazindol, cocaine, GBR 12909) but not that induced by drugs that affect locomotion by other mechanisms (amphetamine, phencyclidine). Twenty-four hours after treatment with metaphit there was an increase in homovanillic acid in all regions of the brain studied (striatum, olfactory tubercle, cerebral cortex). There was no effect of metaphit on the disappearance rate of 3,4-dihydroxyphenylacetic acid and homovanillic acid from the striatum during the inhibition of monoamine oxidase with pargyline. If the increase in homovanillic acid reflected a greater rate of dopamine catabolism in metaphit-treated mice, it could explain the lack of locomotor stimulation of blockers uptake of the dopamine in these animals, resulting from a rapid breakdown of extracellularly accumulated dopamine.

Amphetamine↗

PubMed1988

Tritiated-6-beta-fluoro-6-desoxy-oxymorphone ([3H]FOXY): a new ligand and photoaffinity probe for the mu opioid receptors.

6-Beta-fluoro-6-desoxy-oxymorphone (FOXY) is a fluorinated derivative of oxymorphone originally developed as a potential PET scanning ligand. Preliminary work (Rothman et al., Neuropeptides 4: 311-317, 1984) demonstrated that [3H]FOXY selectively labeled mu opioid binding sites with low levels of nonspecific binding. In this study the opiate receptor subtypes labeled by [3H]FOXY and [3H]D-ala2-MePhe4, Gly-ol5-enkephalin ([3H] DAGO) were compared using site directed acylating agents and binding surface analysis. Although the data indicated that both ligands selectively label mu opiate receptors, other experiments demonstrated that [3H]DAGO and [3H]FOXY labeled mu binding sites differently. Additional experiments demonstrated that [3H]FOXY can be used as a high yield photoaffinity label for the mu opiate receptor subtype.

Affinity Labels↗

PubMed1988

A brief study of the selectivity of norbinaltorphimine, (-)-cyclofoxy, and (+)-cyclofoxy among opioid receptor subtypes in vitro.

Norbinaltorphimine (nor-BNI) is a bifunctional reagent developed as a selective antagonist of the kappa opioid receptor. In this paper we examined the in vitro selectivity of nor-BNI, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY), and the enantiomer of cycloFOXY, among opioid receptor subtypes. Nor BNI exhibited the highest affinity for kappa binding sites labeled by 3H-U69593 (Ki = 1.8nM), and was 27- to 29-fold less potent at mu and delta binding sites. In contrast, cycloFOXY had the highest affinity for mu binding sites (Ki = 2.62 nM), and bound to kappa and delta binding sites with Ki's of 9.3 nM and 89 nM, respectively. The enantiomer of cycloFOXY, did not inhibit binding even at concentrations greater than 10 microM, validating in part the use of 18F-labeled (+)-cycloFOXY to estimate "non-specific binding" in positron emission tomography. Additionally, we report that (S,S)-U50 488 and (R.R)-U50 488 bind to kappa binding sites labeled by 3H-U69 593 with Ki's of 0.89 nM and 299 nM, respectively.

Animals↗

PubMed1988

Interaction of enantiomeric pairs of opiates with phencyclidine binding sites in rat brain: identification of (+) pentazocine as a ligand potentially suitable for imaging sigma binding sites using positron emission tomography.

Some unnatural opiates, which do not interact with classical opiate receptors, interact with phencyclidine (PCP) receptors. Among their many pharmacological actions, drugs which bind to the PCP receptor antagonize the actions of glutamic acid mediated via the NMDA excitatory amino acid receptor, leading to their potential use as anti-ischemic and anticonvulsant agents. Despite an enormous effort, identification of a PCP receptor antagonist, which would be useful for research and therapeutics, has not yet been reported. Chemical modification of unnatural opiates as a means to produce a PCP antagonist, or PCP agonists with properties different than PCP, has not been fully explored. Towards this end, we determined the equilibrium dissociation constants of eight enantiomeric pairs of opiates for the rat brain PCP receptor.

Animals↗

PubMed1988

Synthesis, absolute configuration, and molecular modeling study of etoxadrol, a potent phencyclidine-like agonist.

Etoxadrol (2a), one of the eight possible optical isomers of 2-ethyl-2-phenyl-4-(2-piperidyl)-1,3-dioxolane, was synthesized from (S,S)-1-(2-piperidyl)-1,2-ethanediol, which was obtained from cleavage of dexoxadrol (1a, (S,S)-2,2-diphenyl-4-(2-piperidyl)-1,3-dioxolane). The absolute configuration of etoxadrol hydrochloride, a phencyclidine-like compound biologically, was determined to be 2S, 4S, and 6S at its three chiral centers by single-crystal X-ray analysis. Epietoxadrol (2b), epimeric with etoxadrol at the C-2 center, was also obtained from the synthesis. This much less potent enantiomer has the 2R,4S,6S configuration. The affinity of etoxadrol to the phencyclidine binding site was found to be comparable to that of phencyclidine itself and was 35 times more potent than its epimer, epietoxadrol. Three diastereomeric mixtures were prepared that had low affinity for the phencyclidine site. In studies of the discriminative stimulus properties of these compounds, it was found that only etoxadrol substituted for the phencyclidine stimulus. With use of computer-assisted molecular modeling techniques, a hypothetical phencyclidine binding site model has been developed that, unlike our former hypothesis based on Dreiding models, correctly predicts the higher affinity of etoxadrol and the lesser affinity of epietoxadrol for the phencyclidine site.

Animals↗

PubMed1988

Synthesis, configuration, and evaluation of two conformationally restrained analogues of phencyclidine.

Brown oxidation of cis-bicyclo[3.1.0]hexan-3-ol afforded bicyclo[3.1.0]hexan-3-one in 98% yield. Treatment of this ketone with either phenyllithium or phenylamagnesium bromide in ether at room temperature followed by solvolysis of the resulting alcohol in a mixture of trifluoroacetic acid, sodium azide, and chloroform gave a mixture of cis- and trans-3-azido-3-phenylbicyclo[3.1.0]hexanes. LAH reduction of this crude mixture of azides afforded a 1:3.5 mixture of cis- and trans-3-phenyl-3-bicyclo[3.1.0]hexylamine, respectively, in 51% overall yield from the alcohol. Separation of the mixture of amines by column chromatography followed by cyclization of each by heating at 60 degrees C in DMF solution with 1 equiv of 1,5-dibromopentane furnished the two conformationally restrained analogues of phencyclidine (PCP), cis- and trans-3-phenyl-3-piperidinylbicyclo[3.1.0]hexane (1 and 2, respectively), in high yield. Configurations were assigned on the basis of an X-ray crystallographic analysis of the cis isomer (1). Bond lengths and angles are similar to those found in PCP and its derivatives. Binding to PCP receptors and sigma sites as well as behavioral effects of 1 and 2 in rats was determined relative to PCP. In displacement of specifically bound [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine) from PCP receptors, 1 and 2 were nearly equipotent and about one-seventh as potent as PCP. These compounds were about one-fifth as potent as PCP in displacing [3H]-(+)-SKF 10,047 from its binding site. Calculation of the ED50 values of 1 and 2 for stereotyped behavior and ataxia indicated that they were about equipotent, and 2-3-fold less active than PCP.

Animals↗

PubMed1988

Synthesis, pharmacological action, and receptor binding affinity of the enantiomeric 1-(1-phenyl-3-methylcyclohexyl)piperidines.

The cis and trans enantiomers of the 1-(1-methylcyclohexyl)piperidines were prepared from either 3(R)- or 3(S)-methylcyclohexanone through the Bruylents reaction or a modified azide route, respectively. Separation of the intermediate amines 5 and 6 was achieved through chromatography or selective crystallization of a fumarate salt. The cis isomer 2b had about one-third of the affinity of phencyclidine for the PCP receptor. The other isomers were less potent. There was a 40-fold difference between the binding affinity of the cis enantiomers 2a and 2b and a fourfold difference between the affinities of the trans enantiomers 1a and 1b. None of the compounds antagonized the stereotypy induced by phencyclidine in the rotorod assay in mice, after intraperitoneal introduction.

Animals↗

PubMed1988

beta-FNA binds irreversibly to the opiate receptor complex: in vivo and in vitro evidence.

beta-Funaltrexamine (beta-FNA) is an alkylating derivative of naltrexone. Considerable data support its use as an irreversible mu receptor antagonist. However, pretreatment of rats with beta-FNA attenuates the ability of delta antagonists and naloxone to reverse delta receptor-mediated physiological effects, suggesting that physically adjacent mu and delta receptors interact in vivo. The purpose of this study was to determine which opiate receptor subtype is altered by i.c.v. injections of beta-FNA, as well as by in vitro incubations with beta-FNA, and then to examine the hypothesis that pretreatment of rats with beta-FNA increases the IC50 for naloxone at the altered binding site. The results demonstrate that beta-FNA alters the conformation of the opiate receptor complex, as evidenced by a decrease in the Bmax of the lower affinity [3H]D-Ala2-D-Leu5-enkephalin binding site and a doubling of the naloxone IC50 for displacing [3H]D-Ala3-D-Leu5-enkephalin from this site. [3H]D-Ala2-MePhe4,Gly-ol5-enkephalin binding sites were not detectably altered by i.c.v. injections of beta-FNA. These data collectively support the concept of coupling among opioid receptor subtypes.

Animals↗

PubMed1987

Phencyclidine (PCP)-like inhibition of N-methyl-D-aspartate-evoked striatal acetylcholine release, H-TCP binding and synaptosomal dopamine uptake by metaphit, a proposed PCP receptor acylator.

The phencyclidine (PCP) receptor acylator, metaphit, has been reported to act as a PCP antagonist. Recent electrophysiological and behavioral assessments of metaphit action have revealed, however, that this compound can also act as a PCP-like agonist. The present study examined the effects of metaphit on the inhibition of N-methyl-D-aspartate (NMDA)-induced 3H-acetylcholine (ACh) release, 3H-TCP binding and synaptosomal 3H-dopamine (DA) uptake in the rat striatum. Preincubation of striatal slices for 10 min in the presence of metaphit, followed by a prolonged washout, produced a concentration-dependent inhibition of the ACh release evoked by 300 microM NMDA. At high concentrations, preincubation with PCP also resulted in inhibition of this measure. However, this could be reduced by extending the washout period, a procedure which had no effect on the inhibition produced by metaphit. At 10 microM, metaphit resulted in a 53% reduction in NMDA-evoked ACh release while PCP had no effect under identical conditions. Preincubation of slices in 10 microM PCP and metaphit reduced the metaphit inhibition by 62%. The effects of PCP and metaphit, alone or in combination, on NMDA-induced ACh release were paralleled by a loss of 3H-TCP binding sites in striatal tissue incubated under identical conditions suggesting that metaphit exerts long-lasting agonist-like actions on PCP receptors coupled to NMDA receptors. Although these results do not explain the ability of metaphit to antagonize PCP effects in other assays, we did observe that preincubation of striatal synaptosomes with metaphit also resulted in an irreversible inhibition of 3H-DA uptake. These data are discussed in relation to the interaction of metaphit with PCP receptors in various systems.

Acetylcholine↗

PubMed1987

Immunoaffinity-purified opiate receptor specifically binds the delta-class opiate receptor ligand, cis-(+)-3-methylfentanylisothiocyanate, SUPERFIT.

Using an antibody generated against the opiate receptor on NG108-15 cells, we recently purified the putative receptor from this hybrid cell line. We herein report that the purified receptor complex specifically binds tritiated cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT), with the predominant binding associated with a 58 kDa polypeptide chain. Consistent with these findings is the in situ labeling of a 58 kDa protein with [3H]SUPERFIT on NG108-15 cells.

Animals↗

PubMed1987

The mu-opioid receptor in the 7315c tumor cell.

Experiments were carried out to show that the 7315c tumor cell possesses mu-, but apparently not delta- or kappa-opioid receptors. The binding data for [3H]Tyr-D-Ala-Gly-Me-Phe-Gly-ol ([3H]DAGO) indicate the presence of one class of high affinity site (Kd = 1.5 +/- 0.3 nM, Bmax = 50 fmol/mg). An irreversible alkylating agent, 2-(p-ethoxybenzyl) 1-diethylaminoethyl-5-isothiocyanobenzimidazole isothiocyanate (BIT), with selectivity for mu- over delta-opioid receptors, completely blocked [3H]Tyr-D-Ala-Gly-Phe-Met-NH2 ([3H]DALAMID) binding to 7315c cell membranes. Another irreversible alkylating agent, fentanyl isothiocyanate (FIT) with selectivity for delta- over mu-opioid receptors, had no effect on [3H]DALAMID binding. Since [3H]DALAMID binds equally well to mu- and delta-opioid receptors, these results indicate the presence of mu- but not delta-opioid receptors on 7315c cells. The Ki of U50488, a kappa selective ligand, for [3H]ethylketocyclazocine ([3H]EKC) binding sites was 400 +/- 100 nM, suggesting the absence of kappa-opioid receptors on the 7315c tumor cell. These results are consistent with the presence of mu-opioid receptors in 7315c tumor cells.

Cell Membrane↗

PubMed1987

Synthesis and absolute configuration of optically pure enantiomers of a kappa-opioid receptor selective agonist.

The enantiomers of U50,488, ligands highly selective for kappa-opioid receptors, have been prepared by a refined procedure and their optical purity demonstrated. The absolute configuration of (+)-trans-2-pyrrolidinyl-N-methylcyclohexylamine, a chemically versatile intermediate for synthesis of analogs of kappa-opioid receptor ligands with defined chirality, has been determined to be 1S,2S by X-ray crystallographic analysis. This intermediate has been used to synthesize the optically pure U50,488 enantiomers with known absolute configuration.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1987

Chronic morphine upregulates a mu-opiate binding site labeled by [3H]cycloFOXY: a novel opiate antagonist suitable for positron emission tomography.

CycloFOXY (17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta- fluoromorphinan) is a novel opiate antagonist synthesized as a ligand suitable for in vivo visualization of opiate receptors using positron emission transaxial tomography. In this paper we report that [3H]cycloFOXY labels two distinct opiate binding sites in rat brain membranes, tentatively identified as mu and kappa. Furthermore, chronic administration of morphine results in a selective up-regulation of the mu binding site. The implications of this finding for models of the opioid receptors and the mechanism of the sodium effect are discussed.

Alkylating Agents↗

PubMed1987

Metaphit, a proposed phencyclidine (PCP) antagonist, prevents PCP-induced locomotor behavior through mechanisms unrelated to specific blockade of PCP receptors.

Metaphit, a derivative of phencyclidine (PCP) which irreversibly binds to a population of PCP receptor sites in rat brain, has been considered as a potentially specific PCP antagonist. In whole animal studies, however, metaphit has been shown to have either antagonist or PCP-like actions depending upon the animal species used and the experimental variable evaluated. In the present study, the ability of PCP to produce hyperactivity was assessed in rats following pretreatment with intravenous, intraventricular or intracerebral injections of metaphit. Whereas, intravenous and intraventricular metaphit pretreatment failed to alter the locomotor stimulatory effects of PCP, direct injections of metaphit into the nucleus accumbens resulted in a dose-dependent reduction of PCP-induced hyperactivity. This result also was mimicked by intra-accumbens injections of equimolar concentrations of PCP, but not by the local anesthetic procaine. Additionally, intra-accumbens metaphit prevented d-amphetamine-induced hyperactivity and also depleted significantly accumbens dopamine content; effects not seen after intracerebral PCP administration. These results suggest that metaphit produces a functional antagonism of PCP-induced locomotor activity through presynaptic mechanisms unrelated to specific blockade of PCP receptors.

Animals↗