Journal: Frontiers in Pharmacology
Article Title: Dopamine internalization via Uptake 2 and stimulation of intracellular D 5 -receptor-dependent calcium mobilization and CDP-diacylglycerol signaling
doi: 10.3389/fphar.2024.1422998
Figure Lengend Snippet: Effects of dopamine (DA) microinjection or bath application on intracellular free calcium concentrations [iCa 2+ ] in hDRD5 receptor-expressing cells. (A) . Human U2-OS osteosarcoma cells natively expressing DRD1 and PMAT, but not DRD5, were pre-transfected with GFP-fluorescent human DRD5 receptor (green GFP-D5 fluorescence). Dopamine and other treatments were microinjected into the cell Soma followed by continuous 0.25 Hz recording of [iCa 2+ ] signal as indicated by Fura-2AM. fluorescence. Pictograms of [iCa 2+ ] from respective representative cells are shown (at least 12 transfected cells were tested with similar outcomes, and average results from 12 cells are shown in part D below). The control cell tested in parallel (top panel) was microinjected with buffered media alone, while the DA injected cell received 10 nM final intracellular concentration of DA. The [iCa 2+ ] signal integrated over a 6 min observation window as well as point observations at 2 and 6 min, are shown. (B) . Effects of co-microinjection of SCH23390 (SCH) 10 nM with dopamine 10 nM (Top panel) or of quinpirole microinjection (up to 10 μM, bottom panel) on [iCa 2+ ] response. SCH23390 blocked the iCa 2+ response to dopamine, while the D 2 agonist quinpirole (Quin) was without effect. (C) . Pictograms from representative experiments testing the effects of bath-applied DA on iCa 2+ mobilization in U2-OS cells. Bath application of 10 nM dopamine had no effect (Top panel), while 10 µM dopamine monitored over 11 min revealed a slowly rising wave of [iCa 2+ ] response that was highest toward the latest 11 min time point observed (Bottom panel). (D) . Quantified observations and temporal patterns of responses for microinjected versus bath applied dopamine. Continually recorded [iCa 2+ ] data resampled at 0.133 min intervals from 12 experiments are shown for microinjected DA 10 nM (Left graph, peak effect = 416%) in contrast with the effects of bath-applied DA 10 nM (Middle graph) and bath-applied DA 10 µM (Right graph). For bath-applied DA 10 μM, the average of the three observations made in the last 12 s of recording (106.2%, N = 12) was compared to the average of the three observations made in the first 12 s (0.2 min) of recording as the baseline (100%, N = 12); the comparison indicated a statistically significant increase in bath-applied dopamine-stimulated [iCa 2+ ] response (paired t -test, p = 0.019). The experimental system could not support extension of observation period past the 11th minute. (E) Expression indications for various dopamine receptors and PMAT in U2-OS cells. While D 1 and D 2 receptors as well as PMAT were expressed, there was no indication of D 5 receptor expression in the U2-OS cells.
Article Snippet: U2-OS cells were transfected with GFP-tagged human D 5 receptor (DRD5) cDNA ( OriGene.com , #RG202502) or pCMV6-AC-mGFP vector ( OriGene.com #PS100040) using Turbofectin-8 transfection reagent ( Origene.com #TF81001) according to manufacturer recommendations.
Techniques: Microinjection, Expressing, Transfection, Fluorescence, Control, Injection, Concentration Assay, Comparison
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![Effects of dopamine (DA) microinjection or bath application on intracellular free calcium concentrations [iCa 2+ ] in hDRD5 receptor-expressing cells. (A) . Human U2-OS osteosarcoma cells natively expressing DRD1 and PMAT, but not <t>DRD5,</t> were pre-transfected with GFP-fluorescent human DRD5 receptor (green GFP-D5 fluorescence). Dopamine and other treatments were microinjected into the cell Soma followed by continuous 0.25 Hz recording of [iCa 2+ ] signal as indicated by Fura-2AM. fluorescence. Pictograms of [iCa 2+ ] from respective representative cells are shown (at least 12 transfected cells were tested with similar outcomes, and average results from 12 cells are shown in part D below). The control cell tested in parallel (top panel) was microinjected with buffered media alone, while the DA injected cell received 10 nM final intracellular concentration of DA. The [iCa 2+ ] signal integrated over a 6 min observation window as well as point observations at 2 and 6 min, are shown. (B) . Effects of co-microinjection of SCH23390 (SCH) 10 nM with dopamine 10 nM (Top panel) or of quinpirole microinjection (up to 10 μM, bottom panel) on [iCa 2+ ] response. SCH23390 blocked the iCa 2+ response to dopamine, while the D 2 agonist quinpirole (Quin) was without effect. (C) . Pictograms from representative experiments testing the effects of bath-applied DA on iCa 2+ mobilization in U2-OS cells. Bath application of 10 nM dopamine had no effect (Top panel), while 10 µM dopamine monitored over 11 min revealed a slowly rising wave of [iCa 2+ ] response that was highest toward the latest 11 min time point observed (Bottom panel). (D) . Quantified observations and temporal patterns of responses for microinjected versus bath applied dopamine. Continually recorded [iCa 2+ ] data resampled at 0.133 min intervals from 12 experiments are shown for microinjected DA 10 nM (Left graph, peak effect = 416%) in contrast with the effects of bath-applied DA 10 nM (Middle graph) and bath-applied DA 10 µM (Right graph). For bath-applied DA 10 μM, the average of the three observations made in the last 12 s of recording (106.2%, N = 12) was compared to the average of the three observations made in the first 12 s (0.2 min) of recording as the baseline (100%, N = 12); the comparison indicated a statistically significant increase in bath-applied dopamine-stimulated [iCa 2+ ] response (paired t -test, p = 0.019). The experimental system could not support extension of observation period past the 11th minute. (E) Expression indications for various dopamine receptors and PMAT in U2-OS cells. While D 1 and D 2 receptors as well as PMAT were expressed, there was no indication of D 5 receptor expression in the U2-OS cells.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3475/pmc11543475/pmc11543475__fphar-15-1422998-g006.jpg)
