Biomedical subjects

K C Rice

Publications and source records attributed to K C Rice.

At least 271 records · Page 15Linked to original sources

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

LY164929: a highly selective ligand for the lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding site.

Most radiolabeled ligands used to label opiate receptors bind to multiple binding sites. Subtype-selective ligands make possible the labeling of single sites by virtue of their ability to "block" binding of the radiolabeled ligand to selected subtypes. This study compares the selectivity of several ligands for the higher and lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding sites. The results demonstrated that while morphine and D-ala2-MePhe4,Gly-ol5-enkephalin were 80- and 256-fold selective for the lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding site, LY164929 was 1,986-fold selective. Additional experiments indicated that whereas morphine was a noncompetitive inhibitor at the lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding site, LY164929 was a competitive inhibitor, suggesting that this peptide might exhibit different properties in vivo than other mu-like ligands.

Animals↗

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↗

PubMed1988

Purification, properties and polyclonal antibodies for the particulate, low Km cAMP phosphodiesterase from bovine adipose tissue.

The particulate, cGMP- and cilostamide-inhibited "low Km" cAMP phosphodiesterase was purified greater than 100,000-fold in good yield (approximately 20%) from bovine omental fat by a procedure similar to that utilized for purification of an analogous enzyme from rat epididymal adipose tissue; ten-fold more enzyme protein (20-30 micrograms) could be prepared from bovine omentum (25 kg) than from rats (approximately 900 fat pads). Kinetic parameters (all similar to those for the rat enzyme) were: for cAMP, Km and Vmax = 0.3 microM and 2.5 mumol/min/mg, respectively; for cGMP, 0.8 microM and 1.6 mumol/min/mg. For inhibition of cAMP hydrolysis, IC50 values were: for cGMP = 0.6 microM and for OPC 3911, milrinone, CI 930, 0.1-2.0 microM. The purified enzyme, the activity of which eluted from Sephadex G-200 with Mr(app) = 110,000 and was associated with a single protein band during non-denaturing electrophoresis, exhibited on SDS-PAGE silverstained bands of 62 (probably a 61-63 doublet) and 77 kDa, perhaps due to proteolytic nicking. On Western blots, each of the polypeptides cross-reacted with a monoclonal antibody toward the bovine cardiac low Km cAMP and polyclonal rabbit antibody generated toward the purified bovine omental phosphodiesterase. Rabbit anti-phosphodiesterase IgG, which inhibited bovine and rat phosphodiesterase enzymatic activity, did not cross-react with purified bovine retina cGMP-binding or bovine cGMP-stimulated phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗

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↗

PubMed1987

Purification of the putative hormone-sensitive cyclic AMP phosphodiesterase from rat adipose tissue using a derivative of cilostamide as a novel affinity ligand.

A "low Km" cAMP phosphodiesterase with properties of a peripheral membrane protein accounts for approximately 90% of total cAMP phosphodiesterase activity in particulate (100,000 X g) fractions from rat fat cells. Incubation of fat cells with insulin for 10 min increased particulate (but not soluble) cAMP phosphodiesterase activity, with a maximum increase (approximately 100%) at 1 nM insulin. Most of the increase in activity was retained after solubilization (with non-ionic detergent and NaBr) and partial purification (approximately 20-fold) on DEAE-Sephacel. The solubilized enzyme from adipose tissue was purified approximately 65,000-fold to apparent homogeneity (yield approximately 20%) by chromatography on DEAE-Sephacel and Sephadex G-200 and affinity chromatography on aminoethyl agarose conjugated with the N-(2-isothiocyanato)ethyl derivative of the phosphodiesterase inhibitor cilostamide (OPC 3689). A 63,800 +/- 200-Da polypeptide (accounting for greater than 90% of the protein eluted from the affinity column) was identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (with or without reduction). Enzyme activity was associated with the single protein band after electrophoresis under nondenaturing conditions. On gel permeation, Mr(app) was 100,000-110,000, suggesting that the holoenzyme is a dimer. A pI of 4.9-5.0 was estimated by isoelectric focusing. At 30 degrees C, the purified enzyme hydrolyzed both cAMP and cGMP with normal Michaelis-Menten kinetics; the pH optimum was 7.5. The Km(app) for cAMP was 0.38 microM and Vmax, 8.5 mumol/min/mg; for cGMP, Km(app) was 0.28 microM and Vmax, 2.0 mumol/min/mg. cGMP competitively inhibited cAMP hydrolysis with a Ki of approximately 0.15 microM. The enzyme was also inhibited by several OPC derivatives and "cardiotonic" drugs, but not by RO 20-1724. It was very sensitive to inhibition by agents which covalently modify protein sulfhydryls, but not by diisopropyl fluorophosphate. The activation by insulin and other findings indicate that the purified enzyme, which seems to belong to a subtype of low Km cAMP phosphodiesterases that is specifically and potently inhibited by cGMP, cilostamide, other OPC derivatives, and certain cardiotonic drugs, is likely to account for the hormone-sensitive particulate low Km cAMP phosphodiesterase activity of rat adipocytes.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1987

The pattern of [3H]cyclofoxy retention in rat brain after in vivo injection corresponds to the in vitro opiate receptor distribution.

Cyclofoxy, a fluorinated analog of naltrexone, has been designed specifically to permit in vivo labeling of opiate receptors in experimental animals and ultimately, humans. Recently, using positron emission tomography (PET), 3-[18F]acetylcyclofoxy was shown to accumulate in opiate receptor-rich brain regions of a live baboon and to be stereospecifically displaced by injections of (-)-naloxone but not (+)-naloxone. Autoradiographic evidence is presented here that the unacetylated compound, [3H]cyclofoxy, labels a population of opiate receptors in brain after in vivo injections that is virtually identical to that labeled by [3H]naloxone. The in vivo binding patterns of [3H]cyclofoxy in brain were similar to those obtained following incubation of slide-mounted brain sections in vitro. Intravenous injections of [3H]cyclofoxy to rats yielded high (greater than 4:1) striatal and thalamic to cerebellar binding ratios in brain homogenates, supernatants and in 24 micron-thick brain sections 60 min after 30 mu Ci per animal (spec. act. = 16.4 Ci/mmol). Autoradiographs revealed the typical opiate antagonist binding profile with marked retention of [3H]cyclofoxy in the striatal patches, subcallosal streak, medial habenula and central thalamus with little retention of label in cerebellum. [3H]Cyclofoxy binding was reversible since the radiolabeled drug disappeared from brain tissue within 2 h after injections to rats, or could be removed from brain slices in vitro by washing slide-mounted tissue sections for 45 min. In addition, after in vitro washing the same brain sections again bound [3H]cyclofoxy or [3H]naloxone in the same pattern. When pre-washed brain sections were incubated with [3H]cyclofoxy in the presence of unlabeled naloxone, [3H]cyclofoxy binding was reduced to background levels. These data show that [3H]cyclofoxy labels-sensitive opiate receptors in vivo and in vitro. The present results combined with evidence that cyclofoxy demonstrates a low level of toxicity in animals suggest that cyclofoxy is an excellent tool with which to study the physiological role of opiate receptors in living animals using in vivo autoradiography, and in humans using PET.

Animals↗