PHARMACOLOGICAL ASPECTS OF R-(+)-7-OH-DPAT, A PUTATIVE DOPAMINE D-3 RECEPTOR-LIGAND

The R-(+)-isomer of 7-hydroxy-2-(N,N-di-n-propylamino)tetralin (7-OH-DPAT) bound with a more than 200-fold higher affinity to cloned human dopamine D-3 receptors (K-i=0.57 nM) than to dopamine D-2 receptors; the corresponding S-(-)-enantiomer had considerably less affinity for both dopamine receptor subtypes, indicating that the known enantiomer selectivity of 7-OH-DPAT for the 'classical' dopamine D-2 receptor subtype extends to the recently discovered dopamine D-3 receptor subtype. In rats R-(+)-7-OH-DPAT dose dependently (10-1000 nmol/kg) decreased dopamine release and induced yawning, while sniffing behaviour occurred at the highest dose tested (1000 nmol/kg). The possibility that the inhibition of dopamine release and the elicitation of yawning are mediated by dopamine D-3 receptors is considered

Affinity for dopamine D-2, D-3, and D-4 receptors of 2-aminotetralins. Relevance of D-2 agonist binding for determination of receptor subtype selectivity

A series of 2-aminotetralins, substituted with a methoxy or a hydroxy group on the 5- or 7-position, and with varying N-alkyl or N-arylalkyl substituents, were prepared and evaluated in binding assays for human dopamine (DA) D-2, D-3, and D-4 receptors. Some members of this series were prepared in former studies, but were never tested in vitro with single receptor subtypes, and these were examined again. None of the tested 2-aminotetralins showed high affinity for the dopamine D-4 receptor. However, a number of the 2-aminotetralins showed high affinity for both the D-2 and the D-3 DA receptors, as exemplified by compounds 11-15 and 21-26, while some had a reasonable selectivity for the DA D-3 receptors. The affinities of the 2-aminotetralins for the D-2L receptor depended on the type of radioligand (agonist or antagonist) used. The agonist affinity data, obtained by using the agonist ligand [H-3]N-0437, are thought to be more relevant for calculating DA receptor subtype selectivity

Selection for intrabody solubility in mammalian cells using GFP fusions

International audienceEctopically expressed intracellular recombinant antibodies, or intrabodies, are powerful tools to visualize proteins and study their function in fixed or living cells. However, many intrabodies are insoluble and aggregate in the reducing environment of the cytosol. To solve this problem, we describe an approach based on GFP-tagged intrabodies. In this protocol, the GFP is used both as a folding-reporter to select correctly folded intrabodies and as a fluorescent tag to localize the scFv and its associated antigen in eukaryotic cells. Starting from a scFv gene cloned in a retroviral vector, we describe retrovirus production, cell line transduction, and soluble intrabody characterization by microscopy and FACS analysis

Neurochemical and functional characterization of the preferentially selective dopamine D3 agonist PD 128907

The present study determined the biochemical and pharmacological effects of PD 128907 [R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-6]-1,4-oxazin-9-ol], a dopamine (DA) receptor agonist that shows a preference for the human D3 receptor. In transfected Chinese hamster ovary cells (CHO K1), PD 128907 displaced [H-3]spiperone in a biphasic fashion which fit best to a two-site model, generating K-i values of 20 and 6964 nM for the high- and low-affinity sites for the D2L receptors and 1.43 and 413 nM for the corresponding sites for the D3 receptors. Addition of sodium and the GTP analog Gpp(NH)p to both the D2L and D3 caused a modest reduction in the affinity of the compound suggestive of an agonist type action. In agonist binding ([H-3]N-0437), PD 128907 exhibited an 18-fold selectivity for D3 versus D2L, a selectivity similar to that found with antagonist binding to the high-affinity sites. PD 128907 exhibited only weak affinity for D4.2 receptors (K-i = 169 nM). No significant affinity for a variety of other receptors was observed. PD 128907 stimulated cell division (measured by [H-3]thymidine uptake) in CHO p-5 cells transfected with either D2L or D3 receptors exhibiting about a 6.3-fold greater potency in activating D3 as compared to D2L receptors. In vivo the compound was active in reducing DA synthesis both in normal and gamma-butyrolactone (GEL) treated rats; in the GEL model, the decrease was greater in the higher D3-expressing mesolimbic region as compared with striatum which has a lower expression of D3 receptors. PD 128907 decreased DA release (as measured by brain microdialysis) both in rat striatum, nucleus accumbens and medial frontal cortex, as well as in monkey putamen. Behaviorally PD 128907 decreased spontaneous locomotor activity (LMA) in rats at low doses, whereas at higher doses stimulatory effects were observed. PD 128907 at high doses reversed the reserpine-induced decrease in LMA and induced stereotypy in combination with the D1 agonist SKF 38393 indicating postsynaptic DA agonist actions. It is unclear which of the subtypes of DA receptors might be mediating the pharmacological effects of PD 128907. However, the present findings indicating that PD 128907 shows a preference for DA D3 over D2L and D4.2 receptors indicates that its action at low doses may be due to interaction with D3 receptors and at higher doses, with both D2 and D3 receptors.</p

Neurochemical and functional characterization of the preferentially selective dopamine D3 agonist PD 128907

The present study determined the biochemical and pharmacological effects of PD 128907 [R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-6]-1,4-oxazin-9-ol], a dopamine (DA) receptor agonist that shows a preference for the human D3 receptor. In transfected Chinese hamster ovary cells (CHO K1), PD 128907 displaced [H-3]spiperone in a biphasic fashion which fit best to a two-site model, generating K-i values of 20 and 6964 nM for the high- and low-affinity sites for the D2L receptors and 1.43 and 413 nM for the corresponding sites for the D3 receptors. Addition of sodium and the GTP analog Gpp(NH)p to both the D2L and D3 caused a modest reduction in the affinity of the compound suggestive of an agonist type action. In agonist binding ([H-3]N-0437), PD 128907 exhibited an 18-fold selectivity for D3 versus D2L, a selectivity similar to that found with antagonist binding to the high-affinity sites. PD 128907 exhibited only weak affinity for D4.2 receptors (K-i = 169 nM). No significant affinity for a variety of other receptors was observed. PD 128907 stimulated cell division (measured by [H-3]thymidine uptake) in CHO p-5 cells transfected with either D2L or D3 receptors exhibiting about a 6.3-fold greater potency in activating D3 as compared to D2L receptors. In vivo the compound was active in reducing DA synthesis both in normal and gamma-butyrolactone (GEL) treated rats; in the GEL model, the decrease was greater in the higher D3-expressing mesolimbic region as compared with striatum which has a lower expression of D3 receptors. PD 128907 decreased DA release (as measured by brain microdialysis) both in rat striatum, nucleus accumbens and medial frontal cortex, as well as in monkey putamen. Behaviorally PD 128907 decreased spontaneous locomotor activity (LMA) in rats at low doses, whereas at higher doses stimulatory effects were observed. PD 128907 at high doses reversed the reserpine-induced decrease in LMA and induced stereotypy in combination with the D1 agonist SKF 38393 indicating postsynaptic DA agonist actions. It is unclear which of the subtypes of DA receptors might be mediating the pharmacological effects of PD 128907. However, the present findings indicating that PD 128907 shows a preference for DA D3 over D2L and D4.2 receptors indicates that its action at low doses may be due to interaction with D3 receptors and at higher doses, with both D2 and D3 receptors