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Studies of radiotracer uptake were performed in monolayers grown at the bottom of well plates. At the end of incubation 60 min , the medium was quickly removed and the monolayer was washed 3 times with chilled phosphate-buffered saline PBS.
Cells were then treated with 0. When the cells had detached from the bottom of the well within 5 min , 1 mL of DMEM was added to stop the proteolytic action.
Radioactivity in the cell suspension 1. C6 cells were grown and harvested as described; 2 wells from a well plate were pooled. After each washing step, a cell pellet was collected by short centrifugation 5 min, 2, rpm; Hettich tabletop centrifuge.
Data were plotted using CellQuest software Becton Dickinson ; at least 5, events were analyzed for each sample. C6 cells were grown and harvested as described; 4 wells from a well plate were pooled. A cell pellet was collected by a short centrifugation as above. The pellet was resuspended in 0. Equal volumes of cell lysate 0.
A blank incubation 0. Preliminary experiments indicated that the thymidine kinase assay was linear for incubation times up to 90 min. Therefore, an incubation time of 60 min was chosen in subsequent experiments. The bioluminescence was quantified using a Lumicount microplate luminometer Packard Instruments Co. A reaction blank and a standard curve with 7 concentrations of ATP were run in parallel.
Samples were diluted to ensure that the concentration of ATP was within the range of the calibration curve. The number of viable cells per well decreased as a result of chemotherapy. The 3 cytostatic agents inhibited cellular proliferation with different time courses Fig. Decreases in cell number A and changes in S-phase fraction B after onset of chemotherapy.
The 3 cytostatic agents also had different effects on the fraction of cells in S-phase Fig. During the first 4 h of treatment, all compounds caused a slight increase in this parameter.
However, whereas doxorubicin and 5-fluorouracil caused a significant decrease in the relative numbers of cells in S-phase after 24 h, cisplatin had the opposite effect Fig. A statistically significant increase of tracer binding was already noted within 1 h of treatment with doxorubicin or 5-fluorouracil and within 2 h of incubation with cisplatin.
During 24 h of treatment of cells with doxorubicin, the binding of 18 F-FE-SA per cell did not show any change Fig. However, a significant increase of 18 F-FE-SA binding was observed after 3, 4, and 24 h of treatment with cisplatin and after 24 h of incubation with 5-fluorouracil. During the initial 4 h of treatment with doxorubicin and cisplatin, uptake of 11 C-choline per viable cell remained constant, but a rapid increase within 1 h was induced by 5-fluorouracil Fig.
During treatment of C6 cells with doxorubicin and cisplatin, uptake of 18 F-FDG per cell remained constant throughout the entire study period Fig. After treatment with 5-fluorouracil, 18 F-FDG uptake remained constant during the initial 4 h. The uptake of 11 C-methionine per cell was depressed by chemotherapy, although this was a relatively late effect Fig. During the initial 4 h of treatment with all cytostatic agents, cellular uptake of the amino acid remained constant.
On some curves, error bars are within size of symbols and, therefore, are not visible. A rapid and significant decrease within 1 h was observed after treatment of cells with doxorubicin; after 24 h of treatment 18 F-FLT uptake was almost completely suppressed 1. A less rapid decrease occurred during chemotherapy with cisplatin. Incubation of cells with 5-fluorouracil had a biphasic effect on cellular 18 F-FLT uptake. The activity of cellular thymidine kinase normalized for the number of viable cells showed a similar pattern of changes after 24 h of treatment with cytostatic agents as the fraction of cells in S-phase Fig.
Error bars are within size of symbols. On the basis of the doses used, we expected a reduction in cell numbers after treatment of C6 cells with cytostatic agents. The reduction in the number of viable C6 cells that we observed after 24 h of treatment with cytostatic agents Fig. In our in vitro chemotherapy model, cytostatic agents had different effects on the fraction of cells in S-phase after 24 h. Doxorubicin and 5-fluorouracil decreased the S-phase fraction, in contrast to cisplatin Fig.
In contrast, cisplatin initially blocks C6 cells in the S-phase, resulting in a transient increase in the S-phase fraction. Three responses of tracer uptake to chemotherapy were observed in the current study: Our data indicate that the cellular uptake of these tracers is initially increased Figs.
A study in the literature has indicated that radiolabeled choline is not very suitable for evaluation of cytotoxic effects as the uptake of 3 H-choline and also of 18 F-FDG in prostate tumor cells did not show any decrease after treatment of the cells with 2-methoxyestradiol Our own data indicate that uptake of 11 C-choline in C6 cells is increased after 24 h of antitumor therapy in contrast to that of the proliferation marker 18 F-FLT Figs. A relatively small response of 18 F-FDG uptake to chemotherapy, compared with the response of radiolabeled nucleosides, has often been reported.
The authors concluded that, in their cell line, 18 F-FDG measured a substantially different parameter viable cell number than 3 H-thymidine proliferative rate. These findings are very similar to our own in vitro observations Figs.
However, this correlation did not hold for 11 C-methionine uptake after cisplatin treatment. The increase in the S-phase fraction after cisplatin treatment was not accompanied by an increased cellular uptake of 11 C-methionine compare Figs.
The aberrant behavior of 11 C-methionine uptake after cisplatin treatment can be attributed to the fact that cisplatin inhibits not only cell division but also the transport of amino acids across the tumor cell membrane. This side effect of cisplatin treatment has been noted previously, using 14 C-methionine and L murine leukemia cells 36 or C6 and P tumors grown in rats in vivo 37 , This observation is in accordance with studies in the literature that reported close, or fairly close, correlations between the fraction of tumor cells in S-phase and the cellular uptake of 18 F-FLT 35 , However, we did not observe any correlation between 18 F-FLT uptake and the S-phase fraction after treatment of C6 cells with cisplatin or after 1, 2, 3, or 4 h of incubation with doxorubicin.
Under such conditions, uptake of the nucleoside was reduced in contrast to the S-phase fraction, which remained high compare Figs. The activity of this enzyme is determined not only by the fraction of cells in S-phase and the corresponding level of TK1 expression but also by the availability of the cofactor ATP A bioluminescence assay that we performed indicated that cellular ATP content was decreased after treatment of C6 cells with doxorubicin but not after treatment with cisplatin or 5-fluorouracil Fig.
Thus, the effect of doxorubicin on 18 F-FLT uptake may be related to depletion of the cellular ATP pool in the presence of the cytostatic agent, but the mechanism of the cisplatin effect is as yet unknown. Cisplatin may interfere not only with amino acid transport but also with the transport of nucleosides across the tumor cell membrane The 6 PET tracers that we studied showed different uptake kinetics after therapy.
Uptake of 18 F-FLT and 11 C-methionine showed a decline; these tracers acted as proliferation markers. Accumulation of 18 F-FDG reflected not proliferation rate, but the number of viable cells per well. If rapid increases in the binding of 11 C-SA are also observed in vivo and are related to the final therapy outcome, 11 C-SA may be useful for therapy monitoring and may provide information complementary to 18 F-FLT. Been 1 , Kiichi Ishiwata 2 , Rudi A.
Dierckx 1 and Philip H. Previous Section Next Section. Incubation with Radiotracer Experiments were performed in quadruplicate. Thymidine Kinase Assay C6 cells were grown and harvested as described; 4 wells from a well plate were pooled.
Cell Number The number of viable cells per well decreased as a result of chemotherapy. Fraction of Cells in S-phase The 3 cytostatic agents also had different effects on the fraction of cells in S-phase Fig.
Activity of Cellular Thymidine Kinase The activity of cellular thymidine kinase normalized for the number of viable cells showed a similar pattern of changes after 24 h of treatment with cytostatic agents as the fraction of cells in S-phase Fig. Cell Number On the basis of the doses used, we expected a reduction in cell numbers after treatment of C6 cells with cytostatic agents.
S-Phase Fraction In our in vitro chemotherapy model, cytostatic agents had different effects on the fraction of cells in S-phase after 24 h. Effect of oxybutynin and imidafenacin on central muscarinic receptor occupancy and cognitive function: Implicit Memory in Monkeys: PET studies in nonhuman primate models of cocaine abuse: Duke , Michael A.
Cholinergic modulation of working memory activity in primate prefrontal cortex. References Publications referenced by this paper. Showing of references. Effects of drugs of abuse and cholinergic agents on delayed matching-to-sample responding in the squirrel monkey.
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Most prior work with positron emission tomography (PET) dopamine subtype 2/3 receptor (D2/3R) non-selective antagonist tracers suggests. PET measurement of D2 and S2 receptor binding of 3-N-([2′F]fluoroethyl) spiperone in baboon brain. Authors; Authors and affiliations. Heinz H. Coenen. Somatostatin receptor (SSTR) PET has demonstrated a signif- icant improvement . exception to this may be well-differentiated grade 3 NETs, for which optimal.