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G R Uhl

Publications and source records attributed to G R Uhl.

At least 37 records · Page 2Linked to original sources
PubMed2000

Stress-induced analgesia in mu-opioid receptor knockout mice reveals normal function of the delta-opioid receptor system.

Stress-induced analgesia (SIA) was examined in wildtype and mu-opioid receptor knockout mice. We used thermal paw withdrawal (TPW) latency following a continuous 3-min swim in 20 degrees C water, and found a significant increase in TPW latency in both wild-type and knockout mice. Pre-treatment prior to the swim with naltrindole, a selective delta-opioid receptor antagonist, blocked the increase in TPW latency in knockout mice. These results demonstrate an intact delta-receptor-mediated function of a physiologically-released endogenous agonist in the mu-opioid receptor knockout mouse. The present findings are in contrast with previous reports that analgesia induced by exogenous delta agonists is reduced in the knockout mice.

Analgesia↗
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

Human dopamine transporter gene: coding region conservation among normal, Tourette's disorder, alcohol dependence and attention-deficit hyperactivity disorder populations.

The dopamine transporter (DAT) provides major regulation of the synaptic levels of dopamine and is a principal target of psychostimulant drugs. Associations between DAT gene polymorphisms and human disorders with possible links to dopaminergic neurotransmission, including attention-deficit/hyperactivity disorder (ADHD) and consequences of cocaine and alcohol administration, have been reported. We now report approximately 60000 bp of genomic sequence containing the entire DAT gene. This sequence was used to amplify each of the 15 DAT gene exons and several introns and analyze these amplification products by single-stranded sequence conformation (SSCP) and/or direct sequencing. These results define silent allelic single nucleotide sequence variants in DAT gene exons 2, 6, 9 and 15. Rare conservative mutations are identified in amino acids encoded by DAT exons 2 and 8. Analyses of the common nucleotide variants and the previously reported VNTR in the non-coding region of exon 15 define the pattern of linkage disequilibrium across the DAT locus. These comprehensive analyses, however, fail to identify any common protein coding DAT sequence variant in more than 150 unrelated individuals free of neuropsychiatric disease, 109 individuals meeting City of Hope criteria for Tourette's syndrome, 64 individuals with DSM-IV diagnoses of ethanol dependence, or 15 individuals with ADHD. These data are consistent with substantial evolutionary conservation of the DAT protein sequence. They suggest that gene variants that alter levels of DAT expression provide the best current candidate mechanism for reported associations between DAT gene markers, ADHD and other more tentatively associated neuropsychiatric disorders.

Adolescent↗
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

The VMAT2 gene in mice and humans: amphetamine responses, locomotion, cardiac arrhythmias, aging, and vulnerability to dopaminergic toxins.

Monoamine compartmentalization in monoaminergic neurons uses serial action of the plasma membrane and vesicular monoamine (VAMT2) transporters. We can now define the sequences of the genes encoding these transporters in mice and humans, examine influences of deletions of this gene and alteration in its expression levels in transgenic mice, and identify sequence polymorphisms in the human VMAT2 gene. Examination of VMAT2 variants can provide potential insights into roles for allelic variants at these loci in variant drug responses and in diseases linked to monoaminergic systems, including substance abuse and Parkinson's disease.

Aging↗
PubMed2000

Dopamine transporter tryptophan mutants highlight candidate dopamine- and cocaine-selective domains.

Cocaine blocks the normal role of the dopamine transporter (DAT) in terminating dopamine signaling and in restricting its spatial spread through molecular interactions that remain largely obscure. Cocaine analog structure-activity studies suggest roles for cationic and hydrophobic interactions between DAT, dopamine, cocaine, and the sodium and chloride ions whose gradients power uptake processes. Tryptophan residues lying in putative DAT transmembrane domains could contribute to both aromatic and cationic interactions between DAT and dopamine or cocaine. We thus produced mutant DATs with alanine substitutions for tryptophans lying in or near putative DAT transmembrane domains. We have focused analyses on mutations that exert selective influences on affinities for dopamine or the cocaine analog CFT [(-)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane]. Substitutions W162A, W255A, and W310A reduced dopamine uptake affinities. 5W266A, 12W555A, and 12W561A each reduced dopamine superficial recognition affinities by more than 3-fold and all retained affinity for CFT. W406A, W496A and W523A each reduced CFT affinity, and W84A increased CFT affinity. None of these four mutations decreased dopamine uptake affinity. These data, current provisional DAT structural models, and results from parallel studies of other mutants identify candidate dopamine-selective DAT domains for transmembrane dopamine permeation and regions in which mutations selectively lower CFT affinities. Tryptophan residues may contribute more extensively to these selective domains than other previously studied DAT amino acids. These sites provide tempting targets for selective blockers of cocaine recognition by DAT.

Alanine↗
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↗