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Biomedical subjects

W R Roeske

Publications and source records attributed to W R Roeske.

At least 19 recordsLinked to original sources
PubMed2000

The role of the G protein gamma(2) subunit in opioid antinociception in mice.

We examined the role of the gamma(2) subunit of G proteins (Ggamma(2)) in the antinociception produced by c[D-Pen(2), D-Pen(5)]enkephalin (DPDPE) in mice. DPDPE produced 84.0+/-9.0% antinociception in vehicle-treated mice. After intracerebroventricular (i.c.v.) treatment with an antisense phosphorothioate oligodeoxynucleotide to the Ggamma(2) subunit, DPDPE-mediated antinociception decreased to 24.4+/-7.4%. The mismatch phosphorothioate oligodeoxynucleotide-treated mice showed 65.1+/-10.3% antinociception, while the missense phosphorothioate oligodeoxynucleotide-treated mice showed 76.4+/-23.6% antinociception by DPDPE. The reduction of analgesia in antisense phosphorothioate oligodeoxynucleotide-treated mice was significant in comparison with vehicle-treated (P<0.001), mismatch phosphorothioate oligodeoxynucleotide-treated (P<0.01) and missense phosphorothioate oligodeoxynucleotide-treated (P<0.05) mice. These results suggest that the G protein gamma(2) subunit is involved in the transduction pathway leading to antinociception by DPDPE.

Analgesics, Opioid↗
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

Differential down-regulation of the human delta-opioid receptor by SNC80 and [D-Pen(2),D-Pen(5)]enkephalin.

We examined the contribution of the human delta-opioid receptor carboxyl terminal tail to (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)- and cyclic[D-Pen(2),D-Pen(5)]enkephalin (DPDPE)-mediated receptor down-regulation. Both SNC80 and DPDPE mediated down-regulation of an epitope tagged human delta-opioid receptor. Truncation of the human delta-opioid receptor after Gly(338) blocked DPDPE-mediated down-regulation. However, SNC80 mediated significant down-regulation of the truncated receptor. These findings suggest that SNC80-mediated down-regulation involves receptor domains in addition to the carboxyl terminal tail.

Benzamides↗
PubMed2000

Coupling of human delta-opioid receptor to retinal rod transducin in Chinese hamster ovary cells.

Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G(ialpha2), G(ialpha3), and G(oalpha) in both cell lines. G(ialpha1) and G(alphaz) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(talpha1)) in CHO, and the mouse cone transducin (G(talpha2)) in B82 cells. The presence of the transducin alpha-subunit proteins in CHO and B82 cells was confirmed by immunoprecipitation with specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod transducin mutant was transfected into a CHO cell line stably expressing the human delta-opioid receptor (hDOR/CHO). (+)-4-[(alphaR)-alpha-((2S,2R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[(35)S]thio)triphosphate binding by 293 +/- 36% after Ptx pretreatment in the mutant cell line with an EC(50) value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells.

Animals↗
PubMed1999

(2S,3R)TMT-L-Tic-OH is a potent inverse agonist at the human delta-opioid receptor.

We examined the pharmacologic effect of beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinone-3- carboxylic acid ((2S,3R)TMT-L-Tic-OH) on G protein activation in membranes prepared from Chinese Hamster Ovary cells transfected with cDNA of the human delta-opioid receptor. (2S,3R)TMT-L-Tic-OH inhibited G protein activation to 58% of basal with an EC50 of 0.72 nM as determined by [35S]GTPgammaS binding. These findings suggest that (2S,3R)TMT-L-Tic-OH is a highly potent inverse agonist at the human delta-opioid receptor.

Animals↗
PubMed1999

Phosphorylation of adenylyl cyclase VI upon chronic delta-opioid receptor stimulation.

An immunoprecipitation method was used to measure [32P]phosphate incorporation into the adenylyl cyclase VI protein in Chinese Hamster Ovary (CHO) cells stably expressing the human delta-opioid receptor. Chronic SNC 80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N ,N-diethyl-benzamide) 1 microM, 24 h) treatment increased the incorporation of [32P] into a 200 kDa protein band 2.5-fold after gel electrophoresis. The increase in phosphorylation of adenylyl cyclase VI was antagonized by naltrindole (1 microM) and the immunoprecipitation was prevented by the saturation of the antibody with the blocking peptide.

Adenylyl Cyclases↗
PubMed1998

Identification of adenylyl cyclase isoenzymes in CHO and B82 cells.

The identification of adenylyl cyclase isoenzymes in mammalian host cells is important for the interpretation of data obtained from cell lines heterologously expressing G-protein coupled receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify adenylyl cyclase cDNAs from Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. The isolated fragments were identified by restriction analyses and by sequencing. We found mRNAs for adenylyl cyclases VI and VII in CHO and adenylyl cyclases IX and VII in B82 cells.

Adenylyl Cyclases↗
PubMed1998

Endomorphin-1 and endomorphin-2 are partial agonists at the human mu-opioid receptor.

Recently two tetrapeptide ligands that bind preferentially to the mu-opioid receptor were identified and named endomorphin-1 and endomorphin-2. We examined the ability of these peptides to stimulate G protein activation in human mu-opioid receptor transfected B82 fibroblasts as measured by [35S]GTPgammaS binding to cell membranes. Both endomorphin-1 and -2 act as partial agonists in this assay system compared with the mu-selective agonist [D-Ala2,N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). In addition, endomorphins demonstrate efficacy similar to morphine. These findings demonstrate that endomorphin peptides have similar activity at the mu-opioid receptor as morphine and suggest that these peptides have the potential to modulate neuronal activity in vivo.

Analgesics, Opioid↗
PubMed1998

AM630 is an inverse agonist at the human cannabinoid CB1 receptor.

The present investigation examines WIN 55,212-2 and AM630 at the cloned human cannabinoid CB1 receptor stably expressed in Chinese hamster ovary (CHO) cells. The effect of various concentrations of WIN 55,212-2 and AM630 on basal [35S]GTPgammaS binding to cell membranes was determined. WIN 55,212-2 (100 microM) stimulated basal [35S]GTPgammaS binding 77.9% with an EC50 value of 0.36 microM. Conversely, AM630 (100 microM) inhibited basal [35S]GTPgammaS binding by 20.9% with an EC50 value of 0.90 microM. These results show that WIN 55,212-2 is an agonist and AM630 is an inverse agonist in this system.

Animals↗
PubMed1998

The efficacy of delta-opioid receptor-selective drugs.

Delta-opioid receptor-selective drugs may provide an alternative to mu-opioid-selective drugs currently used for the relief of pain. To develop improved delta-opioid receptor-selective drugs, better measures of drug activity are necessary. In this review we suggest that efficacy calculations provide a superior measure of drug activity as compared to dissociation constants and drug potencies in functional assays. Efficacy, as discussed in this review, is defined as a quantitative measurement of the ability of a drug to stimulate second messenger systems or measurable functional responses in cells or tissues under standard conditions. Efficacy values will allow medicinal chemists to understand the contributions of both the coupling efficiency and dissociation constant to drug potencies in the development of new delta-opioid receptor-selective drugs.

Analgesics↗
PubMed1998

Structural correlates for down-regulation of m1 and m2 muscarinic receptor subtypes.

Three chimeric receptors stably expressed in murine fibroblast (B82) cells were used to examine how different parts of the rat muscarinic m1 and m2 receptors contribute to the down-regulation process. The MCH7 chimeric m2 receptor contained a fragment between VIth TM and C-terminal end derived from the m1 receptor. The MCH3 and MCH5 receptors have exchanged N-terminal and third intracellular loop regions of the MCH7 receptor. Fibroblast cells stably expressing individual muscarinic wild type (m1, m2) or chimeric (MCH3, MCH5, or MCH7) receptors were treated with plain medium (control) or medium containing carbachol for 24 h. Receptor density changes were measured by [3H](-)1-N-methyl-3-quinuclidinyl benzilate ([3H](-)MQNB) saturation binding studies. There was a significant loss of receptor density, different for each receptor studied, following carbachol treatment relative to control cells. We related this loss of [3H](-)MQNB binding to the number of amino acids derived from m1 or m2 receptors for each constructed chimera and to the affinity of carbachol to the receptors studied. We demonstrate that: 1) the region from the VIth TMD to the end of C-terminal controls the extent of m1 and m2 receptor down-regulation; 2) the overall receptor conformation and the interaction between intracellular portions of the receptor influence the extent of receptor down-regulation; and 3) resistance to down-regulation by carbachol correlates with the affinity of carbachol to the muscarinic receptor construct.

Animals↗
PubMed1998

Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line.

The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with Kd values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and Bmax values of 97 +/- 4 and 338 +/- 16 fmol/10(6) cells, respectively. Inhibition of [3H](-)-N-methyl-3-quinuclidinyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihidro-6H-pyrido-[2,3-b](1, 4)-benzodiazepine-6-one > hexahydro-sila-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin-stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 microM (0.2-2.8) and 1.1 microM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2-B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms.

Amino Acid Sequence↗
PubMed1997

Relative efficacies of cannabinoid CB1 receptor agonists in the mouse brain.

We measured (-)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohe xyl]-phenol (CP 55,940)-, (-)11-OH-delta8-tetrahydrocannabinol-dimethylheptyl (HU-210)-, anandamide- and delta9-tetrahydrocannabinol-stimulated G protein activation in mouse brain using the [35S]GTPgammaS functional assay. The Ki values for these drugs were determined by agonist competition binding with the cannabinoid CB1 receptor antagonist [3H]N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride ([3H]SR141716A). This information was used to calculate the efficacy for drug stimulation of G protein activity. The rank order of efficacy was CP 55,940 > HU-210 > anandamide > delta9-tetrahydrocannabinol with the latter two drugs being partial agonists. Since efficacy values relate receptor occupancy to functional responses, we believe efficacy values are a better measure of drug-mediated functional responses compared with measurements of drug potency.

Animals↗
PubMed1997

SR141716A is an inverse agonist at the human cannabinoid CB1 receptor.

The effects of R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4- benzoxazin-yl]-(1-napthalenyl)methanone mesylate (WIN 55,212-2) and N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazo le-carboxamide (SR141716A) on guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes isolated from human cannabinoid CB1 receptor-transfected Chinese hamster ovary (CHO) cells were examined. WIN 55,212-2 stimulated [35S]GTPgammaS binding 76.3% above basal levels whereas SR141716A produced a 22.3% decrease in basal [35S]GTPgammaS binding. These findings demonstrate that WIN 55,212-2 is an agonist and SR141716A is an inverse agonist in this system.

Analgesics↗
PubMed1997

Relative efficacies of delta-opioid receptor agonists at the cloned human delta-opioid receptor.

The present study was conducted to determine the relative efficacies of the selective delta-opioid receptor agonists SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPgammaS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human delta-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/Emax)(A'/A + 1) X 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of (-)-TAN67.

Animals↗
PubMed1997

delta 9-Tetrahydrocannabinol is a partial agonist of cannabinoid receptors in mouse brain.

We measured the ability of the cannabinoid agonists delta 9-tetrahydrocannabinol and R(+)-[2,3,-dihydro-5-methyl-3- [(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-napht halenyl) methanone mesylate (WIN 55,212-2) to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in mouse brain membranes. delta 9-Tetrahydrocannabinol stimulated [35S]GTP gamma S binding by about 25% as compared to WIN 55,212-2. This is the first report demonstrating that delta 9-tetrahydrocannabinol acts as a partial agonist in stimulating [35S]GTP gamma S binding in the mouse brain.

Animals↗
PubMed1997

AM630 antagonism of cannabinoid-stimulated [35S]GTP gamma S binding in the mouse brain.

This research was designed to determine the action of the novel aminoalkylindole AM630 (6-iodo-pravadoline) at the cannabinoid receptor by studying its interaction with the cannabinoid receptor agonist WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-y]-(1-naphthalenyl)methanone mesylate) on guanosine-5'-O-(3-[35S]thio) triphosphate ([35S]GTP gamma S) binding in mouse brain. WIN 55,212-2 stimulated [35S]GTP gamma S binding, while AM630 had no effect. AM630 antagonized WIN 55,212-2-2induced [35S]GTP gamma S binding and shifted the WIN 55,212-dose-response curve to the right. These results clearly demonstrate that AM630 exerts cannabinoid receptor antagonist properties in the brain.

Analgesics↗