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G R Uhl

Publications and source records attributed to G R Uhl.

At least 19 recordsLinked to original sources
PubMed2000

Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization.

Determining which domains and amino acid residues of the mu opioid receptor are phosphorylated is critical for understanding the mechanism of mu opioid receptor phosphorylation. The role of the C-terminus of the receptor was investigated by examining the C-terminally truncated or point-mutated mu opioid receptors in receptor phosphorylation and desensitization. Both wild-type and mutated receptors were stably expressed in Chinese hamster ovary (CHO) cells. The receptor expression was confirmed by receptor radioligand binding and immunoblottting. After exposure to 5 microM of DAMGO, phosphorylation of the C-terminally truncated receptor and the mutant receptor T394A was reduced to 40 and 10% of that of the wild-type receptor, respectively. Mutation effects on agonist-induced desensitization were studied using adenylyl cyclase inhibition assays. The C-terminally truncated receptor and mutant receptor T394A both showed complete loss of DAMGO-induced desensitization, while the mutant T/S-7A receptor only lost part of its ability to desensitize. Taken together, these results suggest that the C-terminus of the mu opioid receptor participates in receptor phosphorylation and desensitization with threonine 394, a crucial residue for both features. DAMGO-induced mu opioid receptor phosphorylation and desensitization are associated and appear to involve both the mu opioid receptor C-terminus and other domains of the receptor.

Adenylyl Cyclases↗
PubMed2000

delta-Opioid receptor agonists produce antinociception and [35S]GTPgammaS binding in mu receptor knockout mice.

We examined the effects of [D-Pen(2),D-Pen(5)]enkephalin (DPDPE), [D-Ala(2),Glu(4)]deltorphin (DELT), and (+)-4-[(alphaR)-alpha((2S, 5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N, N-diethylbenzamide (SNC80) on [35S]GTPgammaS binding in brain membranes prepared from micro-opioid receptor knockout (-/-) mice. The potency and maximal response (E(max)) of these agonists were unchanged compared to control mice. In contrast, while the potency of [D-Pen(2),pCl-Phe(4),D-Pen(5)]enkephalin (pCl-DPDPE) was not significantly different, the E(max) was reduced as compared to controls. In the tail-flick test, intracerebroventricular (i.c.v.) or intrathecal (i.th.) DELT produced antinociceptive effects in -/- mice with potency that did not differ significantly from controls. In contrast, the antinociceptive potency of i.c.v. and i.th. DPDPE was displaced to the right by 4- and 9-fold in -/- compared to control mice, respectively. Reduced DPDPE antinociceptive potency in -/- mice, taken together with reduced DPDPE- and pCl-DPDPE- stimulated G protein activity in membranes prepared from -/- mice, demonstrate that these agonists require mu-opioid receptors for full activity. However, because DELT mediated G protein activation and antinociception were both comparable between -/- and wild type mice, we conclude that the mu-opioid receptor is not a critical component of delta-opioid receptor function.

Analgesics, Opioid↗
PubMed2000

Dopamine transporter proline mutations influence dopamine uptake, cocaine analog recognition, and expression.

Analyses of mutation effects can aid in understanding how large proteins act. The dopamine transporter (DAT) mediates complex actions in recognizing cocaine and in recognizing and translocating dopamine, sodium, and chloride. DAT proline residues, especially those in transmembrane (TM) domains, are good candidates for involvement in these DAT actions. We now report production of mutants substituting alanine and/or glycine residues for 16 prolines located in or near putative DAT TM domains. We examine effects of these modifications on DAT expression, dopamine uptake, and cocaine analog binding. Mutants in prolines located in five DAT TM domains and four connecting loops alter apparent DAT membrane targeting. Five mutations decrease dopamine affinities more than threefold without significantly decreasing cocaine analog affinities. One decreases cocaine analog affinity without decreasing dopamine affinity. Two mutations decrease affinities for both dopamine and cocaine analog. P101 is especially implicated in dopamine uptake. Alanine substitution for this proline yields dopamine V(max) values of less than 3% of wild-type values despite dopamine affinities more than fourfold higher than wild-type and normal Na(+) and Cl(-) dependence. These DAT proline mutants identify DAT regions likely for dopamine translocation and for recognition of dopamine and cocaine.

Amino Acid Sequence↗
PubMed2000

Dopamine transporter transmembrane domain polar mutants: DeltaG and DeltaDeltaG values implicate regions important for transporter functions.

Polar residues in dopamine transporter (DAT) transmembrane domains (TMs) are likely to act individually and even interactively in recognizing cocaine and dopamine. We initially evaluated the effects of alanine substitution mutants that remove the polar side chains from residues in each of the 12 putative DAT TMs on the recognition of dopamine and the cocaine analog CFT. Eleven combination mutants with multiple substitutions in DAT TMs 4, 5, 7, or 11 were then selected as candidates for more detailed evaluation based on mutation effects on dopamine and cocaine analog affinities. An evaluation of Gibbs free energy changes displayed by single and combined TM mutants (DeltaG(o) and DeltaDeltaG(o)(int)) reveals three categories of potential interactions among mutants: 1) independent, noncooperative interactions (five influenced CFT and two influenced dopamine affinities), 2) synergistic influences (two for CFT and four for dopamine), and 3) complementation of influences on CFT recognition (four mutants) or on dopamine affinity (five). Combined mutations in TMs 4 and 5 yield the largest DeltaDeltaG(o)(int) values for dopamine uptake. TMs 4 and 11 mutants provide the largest DeltaDeltaG(o)(int) for CFT binding. Interactions between residues lying in DAT TMs 4 and 5 support current DAT structural models that suggest the juxtaposition of these two TMs. These data also support contributions of TM 4 and 11 residues to a polar pocket important for cocaine recognition. These candidate interactive DAT polar domains provide larger target sites for compounds that could modulate specific DAT functions than those provided by single mutations alone.

Alanine↗
PubMed1999

Cocaine reward and MPTP toxicity: alteration by regional variant dopamine transporter overexpression.

Polygenic factors play important roles in animal models of substance abuse and susceptibility to dopaminergic neurodegeneration. Genetic factors are also likely to contribute to the etiology of human drug abuse disorders, and may alter human vulnerabilities to Parkinsonian neurodegeneration. The dopamine transporter (DAT; SLC6A3) is densely expressed by the dopaminergic midbrain neurons that play central roles in drug reward and is believed to be a primary site of action for cocaine reward. This transporter is necessary for the action of selective dopaminergic neurotoxins, and is uniquely expressed on neurons that are the primary targets of Parkinsonian neurodegeneration. To study possible influences of variant DAT expression on these processes, we have constructed transgenic mice (THDAT) in which tyrosine hydroxylase (TH) promoter sequences drive expression of a rat DAT cDNA variant, increase striatal DAT expression by 20-30%, and provide modest alterations in striatal levels of dopamine and its metabolites. THDAT mice habituate more rapidly to a novel environment than wildtype littermates. These animals display enhanced reward conferred by cocaine, as measured by conditioned place preference. However, locomotor responses to cocaine administration are similar to those of wildtype mice, except at high cocaine doses. THDAT mice display more than 50% greater losses of dopaminergic neurons following a course of MPTP treatment than do wildtype control mice. These results document a model for allelic variation at a gene locus that can exert significant effects in murine models of human substance abuse vulnerability and dopaminergic neurodegeneration.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1999

Heterozygous VMAT2 knockout mice display prolonged QT intervals: possible contributions to sudden death.

Heterozygous knockout (KO) mice with half of wild-type levels of expression of the vesicular monoamine transporter (VMAT2) can suddenly die in midlife. To seek mechanisms for this sudden death, we have examined electrocardiogram (ECG) data telemetered from freely-moving heterozygote and wild-type littermate mice. Many ECG parameters were indistinguishable in mice of these two strains. However, heterozygous mice displayed prolonged QT intervals. These findings provide likely contributions to differences in vulnerability to lethal arrhythmias in these animals, and a candidate gene for contributions to human interindividual differences in vulnerability to cardiac arrhythmias.

Animals↗
PubMed1999

Cholinergic axon terminals in the ventral tegmental area target a subpopulation of neurons expressing low levels of the dopamine transporter.

Cholinergic activation of dopaminergic neurons in the ventral tegmental area (VTA) is thought to play a major role in cognitive functions and reward. These dopaminergic neurons differentially project to cortical and limbic forebrain regions, where their terminals differ in levels of expression of the plasmalemmal dopamine transporter (DAT). This transporter selectively identifies dopaminergic neurons, whereas the vesicular acetylcholine transporter (VAchT) is present only in the neurons that store and release acetylcholine. We examined immunogold labeling for DAT and immunoperoxidase localization of VAchT antipeptide antisera in single sections of the rat VTA to determine whether dopaminergic somata and dendrites in this region differ in their levels of expression of DAT and/or input from cholinergic terminals. VAchT immunoreactivity was prominently localized to membranes of small synaptic vesicles in unmyelinated axons and axon terminals. VAchT-immunoreactive terminals formed almost exclusively asymmetric synapses with dendrites. Of 159 dendrites that were identified as cholinergic targets, 35% contained plasmalemmal DAT, and 65% were without detectable DAT immunoreactivity. The DAT-immunoreactive dendrites postsynaptic to VAchT-labeled terminals contained less than half the density of gold particles as seen in other dendrites receiving input only from unlabeled terminals. These results suggest selective targeting of cholinergic afferents in the VTA to non-dopaminergic neurons and a subpopulation of dopaminergic neurons that have a limited capacity for plasmalemmal reuptake of dopamine, a characteristic of those that project to the frontal cortex.

Acetylcholine↗
PubMed1999

The mu opiate receptor as a candidate gene for pain: polymorphisms, variations in expression, nociception, and opiate responses.

There are differences between human individuals and between mouse strains in levels of mu opiate receptor (muOR) expression, responses to painful stimuli, and responses to opiate drugs. One of the best candidates for contributing to these differences is variation at the muOR gene locus. Support for this idea comes from analyses of the human and murine muOR genes. Assessments of individual differences in human muOR expression add further support. Studies with mice, including knockout-transgenic, quantitative trait locus, and strain-comparison studies, also strongly support the possibility that muOR gene alleles would be strong candidates for contributing to individual differences in human nociception and opiate drug responses. This paper reviews current analyses of the murine and human muOR genes, their important variants, and correlations between these variants and opiate influences on pain.

Animals↗
PubMed1999

Visceral chemical nociception in mice lacking mu-opioid receptors: effects of morphine, SNC80 and U-50,488.

Writhing responses to intraperitoneal acetic acid administration and their modulation by mu-, kappa- and delta-opioid receptor agonists were compared in wild-type and mu-opioid receptor knockout mice. Unpretreated homozygous knockout mice displayed less writhing than wild-type mice. U-50,488 [trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl]-benze neacetamide]) reduced writhing responses in wild-type and knockouts. Morphine and SNC80 [(+)-4-[9-alpha-R)-alpha-(2S,5RO-4-allyl-2,5-dimethyl-1-piperaziny l)-3-methoxybenzyl]-N,N-diethylbenzamide] were effective in wild-type mice but ineffective in knockouts. Mu-opioid receptors appear to play important roles in responses to this visceral nociceptive stimulus and its modulation by mu- and delta-opioid receptor agonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1999

The mu-opioid receptor gene-dose dependent reductions in G-protein activation in the pons/medulla and antinociception induced by endomorphins in mu-opioid receptor knockout mice.

There appear to be different relationships between mu-opioid receptor densities and the acute and neuroadaptive mu-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating mu-opioid receptor knockout mice allows studies of mu-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the mu-opioid receptor gene. We now report that the binding of mu-opioid receptor ligand, [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous mu-opioid receptor knockout mice and eliminated in homozygous mu-opioid receptor knockout mice. The endogenous mu-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [35S]guanosine-5'-o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous mu-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The mu-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of mu-opioid agonists. These data support the notion that there are limited physiological mu-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

Analgesics, Opioid↗
PubMed1999

Absence of G-protein activation by mu-opioid receptor agonists in the spinal cord of mu-opioid receptor knockout mice.

1. The ability of mu-opioid receptor agonists to activate G-proteins in the spinal cord of mu-opioid receptor knockout mice was examined by monitoring the binding to membranes of the non-hydrolyzable analogue of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). 2. In the receptor binding study, Scatchard analysis of [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin ([3H]DAMGO; mu-opioid receptor ligand) binding revealed that the heterozygous mu-knockout mice displayed approximately 40% reduction in the number of mu-receptors as compared to the wild-type mice. The homozygous mu-knockout mice showed no detectable mu-binding sites. 3. The newly isolated mu-opioid peptides endomorphin-1 and -2, the synthetic selective mu-opioid receptor agonist DAMGO and the prototype of mu-opioid receptor agonist morphine each produced concentration-dependent increases in [35S]GTPgammaS binding in wild-type mice. This stimulation was reduced by 55-70% of the wild-type level in heterozygous, and virtually eliminated in homozygous knockout mice. 4. No differences in the [35S]GTPgammaS binding stimulated by specific delta1- ([D-Pen2,5]enkephalin), delta2-([D-Ala2]deltorphin II) or kappa1-(U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes. 5. The data clearly indicate that mu-opioid receptor gene products play a key role in G-protein activation by endomorphins, DAMGO and morphine in the mouse spinal cord. They support the idea that mu-opioid receptor densities could be rate-limiting steps in the G-protein activation by mu-opioid receptor agonists in the spinal cord. These thus indicate a limited physiological mu-receptor reserve. Furthermore, little change in delta1-, delta2- or kappa1-opioid receptor-G-protein complex appears to accompany mu-opioid receptor gene deletions in this region.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1999

Dopamine transporter: transmembrane phenylalanine mutations can selectively influence dopamine uptake and cocaine analog recognition.

Cocaine blocks the normal role of the dopamine transporter (DAT) in terminating dopamine signaling through molecular interactions that are only partially understood. Cocaine analog structure-activity studies have suggested roles for both cationic and aromatic interactions among DAT, dopamine, and cocaine. We hypothesized that phenylalanine residues lying in putative DAT transmembrane (TM) domains were good candidates to contribute to aromatic and/or cationic interactions among DAT, dopamine, and cocaine. To test this idea, we characterized the influences of alanine substitution for each of 29 phenylalanine residues lying in or near a putative DAT TM domain. Cells express 22 mutants at near wild-type levels, manifest by DAT immunohistochemistry and binding of the radiolabeled cocaine analog [(3)H](-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane (CFT). Seven mutants fail to express at normal levels. Four mutations selectively reduce cocaine analog affinities. Alanine substitutions at Phe(76), Phe(98), Phe(390), and Phe(361) located in TM domains 1 and 2, the fourth extracellular loop near TM 4 and in TM 7, displayed normal affinities for dopamine but 3- to 8-fold reductions in affinities for CFT. One TM 3 mutation, F(155)A, selectively decreased dopamine affinity to less than 3% of wild-type levels while reducing CFT affinity less than 3-fold. In a current DAT structural model, each of the residues at which alanine substitution selectively reduces cocaine analog or dopamine affinities faces a central transporter cavity, whereas mutations that influence expression levels are more likely to lie at potential helix/helix interfaces. Specific, overlapping sets of phenylalanine residues contribute selectively to DAT recognition of dopamine and cocaine.

Alanine↗
PubMed1998

Consensus phosphorylation sites of human GABA(c)/GABArho receptors are not critical for inhibition by protein kinase C activation.

The mechanism of inhibition of human GABA(C)/GABArho receptors by protein kinase C (PKC) activation was investigated in Xenopus oocytes. Phorbol 12-myristate 13 acetate (PMA), a potent PKC activator, at 25 nM inhibited the currents through GABArho2 receptors, which have one consensus phosphorylation site by PKC in the predicted intracellular loops. The time-courses and amplitudes of inhibition were not significantly different from those occurring through GABArho1 receptors, which have six such sites. The inhibitory effect of PMA was also observed after removing each consensus phosphorylation site in both GABArho1 and rho2 receptors by site-directed mutagenesis. These results suggest that phosphorylation of consensus sites in the intracellular loops is not involved in the inhibition of human GABA(C)/GABArho receptors by PKC activation.

Animals↗
PubMed1998

MPP+ toxicity and plasma membrane dopamine transporter: study using cell lines expressing the wild-type and mutant rat dopamine transporters.

The Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) causes specific cell death in dopaminergic neurons after accumulation by the dopamine transporter (DAT). COS cells, a non-neuronal cell line insensitive to high doses of MPP+, becomes sensitive to MPP+ when transfected with the rat DAT cDNA. We analyzed the bi-directional transport of MPP+ and its toxicity in several cell lines expressing wild or mutant DATs. Cell death in COS cells expressing wild DAT by exposure to MPP+ was concentration-dependent and cocaine-reversible. Increased wild DAT expression caused higher sensitivities to the toxin in HeLa cells. Although several mutant DATs demonstrated greater transport activity than the wild-type, they displayed similar or lower sensitivity to MPP+ toxicity. Reverse transport of preloaded [3H]MPP+ through DAT was facilitated in COS cells expressing certain mutant DATs, which consistently displayed less sensitivity to MPP+ toxicity. These results suggest that re-distribution of MPP+ due to influx/efflux turnover through the transporter is a key factor in MPP+ toxicity.

1-Methyl-4-phenylpyridinium↗
PubMed1998

No heroin or morphine 6beta-glucuronide analgesia in mu-opioid receptor knockout mice.

Recent reports suggest that heroin and its metabolite morphine 6beta-glucuronide can produce analgesia independent of the morphine-preferring mu-opioid receptor. We have tested heroin and morphine 6beta-glucuronide analgesia in wild-type, homozygous and heterozygous mu-opioid receptor knockout mice. Homozygotes display no heroin or morphine 6beta-glucuronide analgesia. Heterozygous mice with one mu-opioid receptor gene copy reveal reduced heroin and morphine 6beta-glucuronide analgesia. The mu-opioid receptor-dependence of heroin and morphine 6beta-glucuronide fails to support a requirement for a heroin-specific opiate receptor subtype.

Analgesia↗
PubMed1998

Cocaine reward models: conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice.

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.

Animals↗
PubMed1998

Studies of the biogenic amine transporters. VII. Characterization of a novel cocaine binding site identified with [125I]RTI-55 in membranes prepared from human, monkey and guinea pig caudate.

[125I]RTI-55 is a cocaine analog with high affinity for dopamine (DA) and serotonin (5-HT) transporters. Quantitative ligand binding studies revealed a novel high affinity [125I]RTI-55 binding site assayed under 5-HT transporter (SERT) conditions which has low affinity for almost all classic biogenic amine transporter ligands, including high affinity 5-HT transporter inhibitors such as paroxetine, but which retains high affinity for cocaine analogs. This site, termed SERT(site2) for its detection under 5-HT transporter conditions (not for an association with the SERT) occurs in monkey caudate, human caudate, and guinea pig caudate membranes, but not in rat caudate membranes. SERT(site2) is distinguished from the DA transporter (DAT) and SERT by several criteria, including a distinct ligand-selectivity profile, the inability to detect SERT(site2) in cells stably expressing the cloned human DAT, and insensitivity to irreversible ligands which inhibit [125I]RTI-55 binding to the DAT and SERT. Perhaps the most striking finding about SERT(site2) is that a wide range of representative antidepressant agents have very low affinity for SERT(site2). The affinity of cocaine for this site is not very different from the concentration cocaine achieves in the brain at pharmacological doses. Viewed collectively with the observation that ligands with high affinity for SERT(site2) are mostly cocaine analogs, these data lead us to speculate that actions of cocaine which differ from those of classic biogenic amine uptake inhibitors may be mediated in part via SERT(site2).

Animals↗