Sigmaplot 13 0 Crack Cocaine
Agathe Thies
Pre-session intraperitoneal (i.p.) injection with (+)-pentazocine dose-dependently shifted the cocaine self-administration dose-effect curve leftward, without affecting maximum response rate (Fig. 1B). The lowest dose of (+)-pentazocine (1.0 mg/kg) was inactive, whereas doses of 3.2 and 10 mg/kg produced leftward shifts that approximated 3- and 10-fold, respectively. These changes were obtained without appreciable effects on the temporal patterns of responding (Fig. 1A, bottom record). The major difference in the performances after (+)-pentazocine or vehicle pretreatment were that the highest response rates were obtained at lower doses of cocaine after (+)-pentazocine. Two-way repeated measures analysis of variance (ANOVA) indicated a significant effect on response rate (F4,60 = 5.60; p = 0.003), with drug pretreatment dose and component (EXT and cocaine dose) as factors. Post-hoc Tukey comparisons indicated that response rates maintained at 0.32 mg/kg/injection of cocaine were significantly decreased by 3.2 and 10 mg/kg of (+)-pentazocine (q = 5.78, 6.25, respectively; p values
The relation between cocaine self-administration and DAT function suggests that potentiation of cocaine self-administration by σ1R agonists may involve modulation of DAT by these drugs. Because cocaine inhibits the DAT by direct binding, we tested the effects of σ1R ligands on the binding of [3H]"type":"entrez-protein","attrs":"text":"WIN35428","term_id":"2516415317","term_text":"WIN35428"WIN35428, a radiolabeled cocaine analog, in rat brain tissues with a high density of DAT. Freshly harvested rat striatal slices were incubated with σ1R agonists, washed multiple times to remove residual drugs, then homogenized to measure binding the of [3H]"type":"entrez-protein","attrs":"text":"WIN35428","term_id":"2516415317","term_text":"WIN35428"WIN35428. Specific binding was adjusted for variations of protein concentrations among treatment groups. After preincubation with 10 μm (+)-pentazocine or PRE-084, Bmax values of [3H]"type":"entrez-protein","attrs":"text":"WIN35428","term_id":"2516415317","term_text":"WIN35428"WIN35428 binding in striatal homogenates were significantly increased (mean S.E.: 187 30% and 166 15% of vehicle, respectively; Fig. 2, A and B), whereas Ki values were not substantially changed.
We further examined the effects of σ1R agonists on DAT function in HEK293 cells co-transfected with DAT and σ1R. Because cocaine preferentially binds to DAT in the outward-facing conformation (7), we reasoned that potential changes of DAT conformation and cocaine binding might be more easily unmasked under conditions of low extracellular Na+, in which the conformational equilibrium of DAT is shifted toward the inward-facing state. We found that [3H]"type":"entrez-protein","attrs":"text":"WIN35428","term_id":"2516415317","term_text":"WIN35428"WIN35428 binding was correlated with increasing extracellular Na+ in a dose-dependent manner in these cells, and that although binding at 50 mm Na+ was reduced to half that in normal Na+ (150 mm), it still could be measured reliably (data not shown). Thus, cells were incubated with σ1R ligands and subsequently assayed for DAT binding or substrate uptake in a buffer with 50 mm NaCl and 100 mmN-methyl-d-glucamine. Similar to results obtained using striatal tissues, there was a significant increase of [3H]"type":"entrez-protein","attrs":"text":"WIN35428","term_id":"2516415317","term_text":"WIN35428"WIN35428 binding Bmax in these cells pretreated with (+)-pentazocine or PRE-084 (130 7% or 125 6% of vehicle, respectively; Fig. 2, C and D) without substantial changes in Ki values. Similar effects of σ1R agonists on DAT binding were observed in another cell line expressing HA-tagged DAT and Myc-tagged σ1R (data not shown). Additionally, there was a significant increase of [3H]DA uptake Vmax values in cells preincubated with (+)-pentazocine or PRE-084 (133 8% or 145 12% of vehicle, respectively; Fig. 2, E and F) without substantial changes of Km values.
We previously studied conformational changes of DAT using cysteine accessibility assays. Upon cocaine binding, the conformation of DAT was changed so that several residues exhibited altered accessibility, including cysteine 306 (Cys-306) and threonine 316 (Thr-316) on TM6a (Fig. 4A), a key domain forming a part of the extracellular vestibule in the outward-facing conformation of DAT (26). To understand why preincubation with σ1R agonists increased cocaine binding (Fig. 2), we examined whether σ1R drugs could modulate the DAT conformation. In HEK293 cells co-transfected with wild-type DAT and Myc-tagged σ1R, exposure of 1 μm cocaine substantially increased the accessibility of Cys-306 (253 29%, mean S.E., compared with vehicle), as probed by maleimide-PEG2-biotin, a sulfhydryl-specific, membrane-impermeant reagent. Preincubation with PRE-084 increased the cocaine-induced effects in a dose-dependent manner, with 10 μm PRE-084 producing a significant enhancement to 364 36% of vehicle (Fig. 4B). Similarly, 10 μm (+)-pentazocine enhanced cocaine-induced changes in Cys-306 accessibility from 273 20% to 349 29% of vehicle treatment (Fig. 4C). However, neither PRE-084 nor (+)-pentazocine pretreatment significantly altered Cys-306 accessibility in the absence of cocaine (Fig. 4, B and C). Moreover, neither σ1R agonists changed the cell-surface expression levels of DAT, as measured by cell-surface biotinylation using sulfo-NHS-SS-biotin, a membrane-impermeant probe that selectively reacts with primary amine moieties in proteins (Fig. 4D).
Pretreatment with 2 μm CM304, a novel antagonist with subnanomolar affinity and a high selectivity for σ1R (27, 28), effectively blocked the potentiating effects of 10 μm PRE-084 (Fig. 4E), verifying that these effects were mediated specifically through σ1R. CM304 alone did not alter cocaine-induced changes in Cys-306 accessibility on DAT.
We then probed the cysteine accessibility at a position in closer proximity to the inhibitor-binding pocket of DAT, using a substituted cysteine construct T316C/C306A in which Thr-316 and Cys-306 were mutated to cysteine and alanine, respectively. In cells co-transfected with T316C/C306A DAT and Myc-tagged σ1R, cocaine binding significantly increased T316C thiol side-chain reactivity toward maleimide-PEG2-biotin to 387 22% of vehicle (Fig. 4F). The effect of cocaine was significantly enhanced after preincubation with 20 μm (+)-pentazocine or PRE-084 to 487 26% or 548 49%, respectively (Fig. 4F). As in Fig. 4, B and C, neither PRE-084 nor (+)-pentazocine altered T316C accessibility in the absence of cocaine (Fig. 4F).
Together, these biochemical data indicated that pre-exposure to σ1R agonists enhanced cocaine-induced conformational changes of DAT, suggesting that the interaction with σ1R likely shifted the conformational equilibrium of DAT toward the outward-facing state which preferentially binds cocaine.
Hypothesis schematic summarizing the modulation of DAT conformation and cocaine binding by σ1R. Binding of σ1R agonists facilitates dissociation of σ1R multimers to monomers, which then dynamically interact with DAT to promote an outward-facing conformation of DAT, thus enhancing cocaine binding and potentiating cocaine's behavioral response.
The transport cycle of DAT involves coordinated movement of key domains that enables DAT to switch between the outward-facing and inward-facing conformations. Pharmacological studies (26, 29) and recent atomic structures of Drosophila DAT (7) have confirmed that cocaine binds to DAT in the outward-facing conformation. Based on biochemical, pharmacological and behavioral data presented in this study, we speculate that interaction with σ1R shifts the DAT conformation toward an outward-facing state in which cocaine binding is facilitated, although detailed molecular mechanisms involved in such modulation requires further elucidation. Our cysteine accessibility assays were optimized to examine cocaine-induced conformational changes of DAT at Cys-306 and T316C, but they did not detect significant effects of σ1R agonists in the absence of cocaine (Fig. 4). It is possible that such modulation may involve conformational changes of other domains on DAT. Because cocaine binding to DAT is promoted by DAT-σ1R interaction, modulatory effects by σ1R agonists were eventually unmasked as potentiation of cysteine accessibility changes at Cys-306 or T316C. In other words, conformational changes of DAT induced by cocaine binding are apparently enhanced after σ1R agonists treatment.
The σ1R agonist (+)-pentazocine dose-dependently shifted the cocaine self-administration dose-effect curve to the left (Fig. 1). A similar effect was obtained previously with other σ1R agonists, PRE-084 and DTG (22). Additionally, the effects of the σ1R agonists on cocaine self-administration were similar to the effects of DA uptake inhibitors, such as methylphenidate or "type":"entrez-protein","attrs":"text":"WIN35428","term_id":"2516415317","term_text":"WIN35428"WIN35428 (22), suggesting that σ1R agonists may act through DAT to potentiate the reinforcing effects of cocaine. In contrast, σ1R antagonists at doses effective in blocking σ1R agonist effects had no effects on cocaine self-administration when administered alone, suggesting a crucial modulatory role of σ1R on the DA signaling that is critically involved in cocaine self-administration. In the present study we identified a novel modulatory mechanism of σ1R on DAT, the key element initiating DA signaling due to cocaine administration. Data presented here suggest that the interaction with σ1R likely stabilizes a conformation of DAT, which is preferable for cocaine binding. This correlates with the observations that lower doses of cocaine were sufficient to produce reinforcing effects, i.e. a leftward shift of cocaine dose response.
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