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P G Strange

Publications and source records attributed to P G Strange.

At least 19 recordsLinked to original sources
PubMed1998

Pharmacological characterisation of the D2 dopamine receptor expressed in the yeast Schizosaccharomyces pombe.

The rat D2(long) dopamine receptor has been expressed in the fission yeast Schizosaccharomyces pombe at levels of about 1 pmol/mg of protein. The recombinant receptor, analysed in ligand binding experiments, exhibits properties typical of a D2 dopamine receptor and the affinities of antagonists agree with values obtained for the receptor expressed in mammalian systems although the affinities of some antagonists are lower. Substituted benzamide antagonists show lower affinities in the absence of sodium ions whereas clozapine and classical antagonists mostly show higher affinities. Agonist binding is insensitive to the effects of GTP indicating lack of a stable interaction with G-proteins.

Animals↗
PubMed1998

Agonist action at D2(long) dopamine receptors: ligand binding and functional assays.

1. The activities of a range of agonists at D2(long) dopamine receptors expressed in CHO cells have been determined in ligand binding and in a functional assay, the stimulation of [35S]-GTPgammaS binding. 2. For several agonists (apomorphine, dopamine, pergolide, quinpirole, NPA, ropinirole, talipexole) binding in the absence of added guanine nucleotides was best described in terms of interaction at higher and lower affinity states, whereas for other agonists (bromocriptine, DHEC, lisuride, 3-PPP) a one binding site model was a good description of the data. In the presence of GTP (100 microM) all agonist binding data were best described by a one site model. 3. All of the agonists tested increased [35S]-GTPgammaS binding above the basal level and the maximal effects and potencies of the agonists in this test were different. There was no clear relation between the ability of an agonist to stabilize the formation of the ternary complex of agonist/receptor/G-protein and the maximal activity of the agonist or the amplification factor (ratio of dissociation constant for binding to receptor to EC50 in functional assay). 4. A comparison was made between the profiles of the D2(short) and D2(long) receptor isoforms in these assays.

Animals↗
PubMed1998

Activation of microtubule-associated protein kinase (Erk) and p70 S6 kinase by D2 dopamine receptors.

The ability of human and rat D2(short) and D2(long) dopamine receptors to activate microtubule-associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose-dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D2 dopamine receptor. Activation of Erk and of p70 S6 kinase via the D2 dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the Gi or Go family. Inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3-kinase-dependent and -independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3-kinase.

Animals↗
PubMed1997

Evidence that antipsychotic drugs are inverse agonists at D2 dopamine receptors.

1. The effects of a number of D2-like dopamine receptor antagonists have been determined on forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary (CHO) cells expressing the human D2short dopamine receptor (CHO-D2S cells). 2. Dopamine inhibited the effect of forskolin (as expected for a D2 receptor). However, all of the antagonists tested, apart from UH232 and (-)-butaclamol, were able to increase cyclic AMP accumulation above the forskolin control level. (+)-Butaclamol elicited a similar stimulation of forskolin-stimulated cyclic AMP accumulation in a CHO cell line expressing human D2long dopamine receptors whereas it exhibited no stimulating effect on forskolin-stimulated cyclic AMP accumulation in untransfected CHO-K1 cells. 3. There was a strong correlation between the EC50 values of these compounds for potentiation of cyclic AMP accumulation and their Ki values from radioligand binding experiments in CHO-D2S cells. 4. The effects of both (+)-butaclamol and dopamine in CHO-D2S cells were inhibited by pre-treatment with pertussis toxin indicating a role for Gi/Go proteins. 5. UH232 did not significantly affect forskolin-stimulated cyclic AMP accumulation but this substance was able to inhibit the effects of both dopamine and (+)-butaclamol in a concentration-dependent manner. Thus the effects of (+)-butaclamol on forskolin-stimulated cyclic AMP accumulation are mediated directly via the D2 receptor rather than by reversal of the effects of an endogenous agonist. 6. These data suggest that the D2 dopamine receptor antagonists tested here, many of which are used clinically as antipsychotic drugs, are in fact inverse agonists at human D2 dopamine receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1997

Agonist action at D2(short) dopamine receptors determined in ligand binding and functional assays.

Mechanisms of agonist action at the G protein-coupled D2(short) dopamine receptor expressed in Chinese hamster ovary cells have been investigated. Agonist binding was assayed in the presence and absence of GTP (100 microM). Data in the absence of GTP were fitted best by a two-site model (apomorphine, dopamine, 10,11-dihydroxy-N-n-propylnorapomorphine hydrochloride, and quinpirole) or a one-site model [bromocriptine, dihydroergocristine, and (-)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride], whereas in the presence of GTP a one-site model was the best fit for all compounds. Agonist binding parameters were used to provide a measure of the ability of the agonist to stabilise the ternary complex of agonist/receptor/G protein. Agonist stimulation of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding for a range of agonist concentrations was measured and the EC50 and maximal effects determined. The initial rates of [35S]GTPgammaS binding induced by maximally stimulating agonist concentrations were also recorded. Simultaneous inhibition of agonist-stimulated [35S]GTPgammaS binding and receptor occupancy by spiperone was determined. Agonist inhibition of forskolin-stimulated cyclic AMP accumulation was determined for a range of agonist concentrations and the EC50 and maximal inhibition recorded. The data on the maximal agonist responses showed that it was possible to detect a spectrum of agonist efficacy (partial and full agonism) in both functional assays. The data on the apparent potencies of agonists to elicit the functional responses showed that different extents of amplification of response were seen for different agonists in both assays. The maximal activity data have been compared with the stabilisation of the agonist/receptor/G protein ternary complex as measured in binding assays.

Animals↗
PubMed1996

D2-like dopamine receptors are not detectable on human peripheral blood lymphocytes.

The binding of [3H]-nemonapride to human peripheral blood lymphocytes (PBL) was studied using various competing ligands specific for D2-like dopamine receptors. There is no detectable stereoselectivity for the stereoisomers of butaclamol, and competitions with haloperidol and sulpiride also show no evidence of specific binding to D2-like dopamine receptors. RT-PCR of RNA from human lymphocytes showed that there is no detectable D2 mRNA (even with nested PCR). D3 mRNA was, however, detectable by RT-PCR, but only at low levels that could not be detected by Northern blots of PBL total RNA.

Adult↗
PubMed1996

The energetics of ligand binding at catecholamine receptors.

One of the key events in the actions of agonists and antagonists is their binding to receptors. Understanding this event is of interest in terms of understanding receptor function but it also has immense practical relevance for the design of drugs. If the ligand-binding process could be understood in detail, including the nature of the interactions made between ligand and receptor, then this could help in the design of more-selective drugs. The interaction of a ligand with its receptor is clearly of importance in determining the specificity of ligand action but ligand-receptor interaction also initiates the processes of signalling that are exhibited in the efficacy of ligand action. Here Philip Strange considers these events for catecholamine receptors, concentrating mostly on dopamine receptors; where necessary the discussion is widened to include other receptor systems.

Drug Design↗
PubMed1996

Investigation of the role of conserved serine residues in the long form of the rat D2 dopamine receptor using site-directed mutagenesis.

Three serine residues (Ser193, Ser194, Ser197) in the fifth transmembrane-spanning region of the D2 dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand-binding experiments with [3H] spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 microM). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist-binding curves in the absence of GTP, when higher and lower affinity agonist-binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala193 and Ala194 mutations, but the Ala197 mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.

Alanine↗
PubMed1996

Pharmacological analysis of dopamine stimulation of [35S]-GTP gamma S binding via human D2short and D2long dopamine receptors expressed in recombinant cells.

1. The activation of G-proteins by agonist-occupied D2 or D3 dopamine receptors in membranes from recombinant cells expressing the cloned receptors has been analysed by a [35S]-guanosine 5'-[gamma-thio] triphosphate ([35S]-GTP gamma S) binding assay. 2. The rate of [35S]-GTP gamma S binding was increased by dopamine in a dose-dependent manner in membranes from CHO cells stably expressing either the D2short or D2long dopamine receptor. 3. The dopamine-induced stimulation of [35S]-GTP gamma S binding could be inhibited by a range of antagonists. Affinities for antagonists derived from the inhibition of the dopamine stimulation of [35S]-GTP gamma S binding correlated very well with affinities derived from radioligand binding studies. 4. When the maximum [35S]-GTP gamma S binding responses stimulated by dopamine acting at different receptor subtypes were compared, there was a tendency for the stimulation via the D2short receptor to be greater than via the D2long receptor and for the stimulation via the D3 dopamine receptor to be less than for either D2 receptor. These differences in maximal response were also seen when the inhibitory effects of dopamine on adenylyl cyclase via the three receptor subtypes were compared. 5. The stimulation of [35S]-GTP gamma S binding by dopamine in membranes from recombinant cells therefore provides an excellent system for studying the molecular nature of agonism and the receptor/G-protein interactions for these receptors.

Adenylyl Cyclases↗
PubMed1996

Regulation of D2 dopamine receptors by amiloride and amiloride analogs.

Allosteric regulation of rat D2 dopamine receptors by amiloride and amiloride analogs has been studied by investigating their ability to accelerate the dissociation of [3H]spiperone from the receptors expressed in Ltk- cells. The amiloride analogs were more potent and produced a greater maximal effect on the rate of [3H]spiperone dissociation than did amiloride. [3H]Spiperone dissociation was biphasic and could be resolved into contributions from fast and slow rates in the absence and presence of the modulators. Methylisobutylamiloride accelerated both the fast and slow rates of dissociation and modulated the proportions of the two rates. The association of [3H]spiperone in the absence of modulators was also biphasic. The combination of the two sets of association and dissociation rate constants gave very similar equilibrium dissociation constants, and this was confirmed in equilibrium binding experiments that were consistent with a single binding site. It is proposed that there are two binding states for [3H]spiperone that can be distinguished kinetically but not in equilibrium binding experiments. The proportions of these states are differentially modulated with the use of sodium ions and magnesium ions, whereas GTP has no significant effect. Allosteric regulation of [3H]spiperone binding by methylisobutylamiloride could also be observed in saturation and inhibition binding experiments. These effects can be accounted for in a model in which the modulator binds to the competitive site of the receptor and to an allosteric site on the receptor from which it exerts negatively cooperative effects on [3H]spiperone binding to the competitive site and positively cooperative effects on the binding of the modulator to the competitive site.

Amiloride↗
PubMed1995

pH dependence of ligand binding to D2 dopamine receptors.

The binding of a range of ligands to D2 dopamine receptors in bovine caudate nucleus and recombinant CHO cells expressing the receptor has been determined at different pH values between 4.5 and 8.5. The maximum number of D2 dopamine receptor binding sites in each tissue was not affected by the change in pH, but the affinity of ligands for binding to the receptors was decreased as the pH was decreased. For classical dopamine antagonists, e.g. spiperone and haloperidol, the data on pH dependence of the dissociation constant for receptor binding indicated that the protonation of a single ionizing group on the receptor (pKa approximately 6) influenced the binding process. For antagonists of the substituted benzamide class, the data indicated that the protonation of two ionizing groups (pKa between 6 and 7) influenced the ligand binding process. These ionizing residues may correspond to Asp 114 for the classical antagonists and Asp 114 and Asp 80 for the substituted benzamide antagonists. Further evidence for the participation of carboxyl residues in the ligand binding process was obtained from the inhibition by N,N'-dicyclohexylcarbodiimide of the binding of [3H]spiperone and [3H]YM 09151-2 to D2 receptors in the recombinant CHO cells.

Animals↗