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5-Hydroxytryptamine (5-HT) cellular sequestration during chronic exposure delays 5-HT3 receptor resensitization due to its subsequent release.

Hothersall JD, Alexander A, Samson AJ, Moffat C, Bollan KA, Connolly CN - J. Biol. Chem. (2014)

Bottom Line: The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered.Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum.Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization.

View Article: PubMed Central - PubMed

Affiliation: From the Medical Research Institute, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

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Cytosolic accumulation of 5-HT correlates with the loss of receptor binding sites.A, cells expressing 5-HT3A, SERT, or 5-HT3A + SERT were loaded with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Postnuclear cytosolic (TX-114 supernatant at 37 °C) fractions were counted (wash 1). The membrane pellet washed (wash 2 and 3) and remaining membrane pellet fraction (TX-114) were counted. Data represent an average of three experiments performed in triplicate. B, 5-HT3A-expressing cells were incubated with 10 μm 5-[3H]HT with (nonspecific binding (NSB)) or without (total) 10 μm ondansetron (60 min at 37 °C). C, cells expressing 5-HT3A, SERT, or both were incubated with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Cells were permeabilized with digitonin (100 μg/ml) to release cytosolic 5-HT and compared with cell-associated signal. Data represent an average of three experiments performed in triplicate. D, cells expressing 5-HT3A only (3A), 5-HT3A and SERT (3A + SERT cotransfection), or cells expressing either 5-HT3A or SERT and subsequently mixed (3A/SERT combined) were examined for 5-HT (10 μm or 1 mm, 60 min, 37 °C)-induced down-regulation of receptor binding sites ([3H]granisetron (Gran)). UT, untreated.
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Figure 4: Cytosolic accumulation of 5-HT correlates with the loss of receptor binding sites.A, cells expressing 5-HT3A, SERT, or 5-HT3A + SERT were loaded with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Postnuclear cytosolic (TX-114 supernatant at 37 °C) fractions were counted (wash 1). The membrane pellet washed (wash 2 and 3) and remaining membrane pellet fraction (TX-114) were counted. Data represent an average of three experiments performed in triplicate. B, 5-HT3A-expressing cells were incubated with 10 μm 5-[3H]HT with (nonspecific binding (NSB)) or without (total) 10 μm ondansetron (60 min at 37 °C). C, cells expressing 5-HT3A, SERT, or both were incubated with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Cells were permeabilized with digitonin (100 μg/ml) to release cytosolic 5-HT and compared with cell-associated signal. Data represent an average of three experiments performed in triplicate. D, cells expressing 5-HT3A only (3A), 5-HT3A and SERT (3A + SERT cotransfection), or cells expressing either 5-HT3A or SERT and subsequently mixed (3A/SERT combined) were examined for 5-HT (10 μm or 1 mm, 60 min, 37 °C)-induced down-regulation of receptor binding sites ([3H]granisetron (Gran)). UT, untreated.

Mentions: To gain further insight into a potential mechanism of action, we investigated whether transported 5-[3H]HT was associated with cellular membranes or remained soluble within the cytoplasm. Cells were incubated with 5-HT (10 μm, 10% of which was 5-[3H]HT) in Opti-MEM for 60 min at 37 °C, and cells were washed with binding buffer before being solubilized with 1% TX-114 (5 min, 4 °C). Radioactivity associated with the membrane phase and each sequential wash (cytosol) was counted (Fig. 4A). In keeping with previous observations, where cells lacking SERT exhibited little cellular uptake (Fig. 3D) and no loss of function (Fig. 3A) at this concentration of 5-HT, we found no significant intracellular 5-[3H]HT accumulation (Fig. 4A). In SERT-expressing cells (regardless of 5-HT3 receptor expression), robust counts were detected in the first cytosolic fraction (wash 1), with diminishing counts associated with subsequent washes. Minimal 5-[3H]HT remained in the final TX-114 membrane fraction, suggesting that intracellular 5-HT is predominantly freely soluble. Importantly, in cells co-expressing SERT and 5-HT3A receptors, no increased cellular retention or membrane association was observed. Therefore, prolonged receptor association through a putative intracellular binding site is not responsible. Moreover, in our assays, 5-HT (10 μm) does not exhibit prolonged 5-HT3 receptor binding, as no 5-[3H]HT binding (Fig. 4B, total) was observed above the nonspecific binding (as determined by competition with excess ondansetron) after rapid washing in 5-HT3A-transfected cells.


5-Hydroxytryptamine (5-HT) cellular sequestration during chronic exposure delays 5-HT3 receptor resensitization due to its subsequent release.

Hothersall JD, Alexander A, Samson AJ, Moffat C, Bollan KA, Connolly CN - J. Biol. Chem. (2014)

Cytosolic accumulation of 5-HT correlates with the loss of receptor binding sites.A, cells expressing 5-HT3A, SERT, or 5-HT3A + SERT were loaded with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Postnuclear cytosolic (TX-114 supernatant at 37 °C) fractions were counted (wash 1). The membrane pellet washed (wash 2 and 3) and remaining membrane pellet fraction (TX-114) were counted. Data represent an average of three experiments performed in triplicate. B, 5-HT3A-expressing cells were incubated with 10 μm 5-[3H]HT with (nonspecific binding (NSB)) or without (total) 10 μm ondansetron (60 min at 37 °C). C, cells expressing 5-HT3A, SERT, or both were incubated with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Cells were permeabilized with digitonin (100 μg/ml) to release cytosolic 5-HT and compared with cell-associated signal. Data represent an average of three experiments performed in triplicate. D, cells expressing 5-HT3A only (3A), 5-HT3A and SERT (3A + SERT cotransfection), or cells expressing either 5-HT3A or SERT and subsequently mixed (3A/SERT combined) were examined for 5-HT (10 μm or 1 mm, 60 min, 37 °C)-induced down-regulation of receptor binding sites ([3H]granisetron (Gran)). UT, untreated.
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Figure 4: Cytosolic accumulation of 5-HT correlates with the loss of receptor binding sites.A, cells expressing 5-HT3A, SERT, or 5-HT3A + SERT were loaded with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Postnuclear cytosolic (TX-114 supernatant at 37 °C) fractions were counted (wash 1). The membrane pellet washed (wash 2 and 3) and remaining membrane pellet fraction (TX-114) were counted. Data represent an average of three experiments performed in triplicate. B, 5-HT3A-expressing cells were incubated with 10 μm 5-[3H]HT with (nonspecific binding (NSB)) or without (total) 10 μm ondansetron (60 min at 37 °C). C, cells expressing 5-HT3A, SERT, or both were incubated with 10 μm 5-HT (10% 5-[3H]HT) for 60 min at 37 °C. Cells were permeabilized with digitonin (100 μg/ml) to release cytosolic 5-HT and compared with cell-associated signal. Data represent an average of three experiments performed in triplicate. D, cells expressing 5-HT3A only (3A), 5-HT3A and SERT (3A + SERT cotransfection), or cells expressing either 5-HT3A or SERT and subsequently mixed (3A/SERT combined) were examined for 5-HT (10 μm or 1 mm, 60 min, 37 °C)-induced down-regulation of receptor binding sites ([3H]granisetron (Gran)). UT, untreated.
Mentions: To gain further insight into a potential mechanism of action, we investigated whether transported 5-[3H]HT was associated with cellular membranes or remained soluble within the cytoplasm. Cells were incubated with 5-HT (10 μm, 10% of which was 5-[3H]HT) in Opti-MEM for 60 min at 37 °C, and cells were washed with binding buffer before being solubilized with 1% TX-114 (5 min, 4 °C). Radioactivity associated with the membrane phase and each sequential wash (cytosol) was counted (Fig. 4A). In keeping with previous observations, where cells lacking SERT exhibited little cellular uptake (Fig. 3D) and no loss of function (Fig. 3A) at this concentration of 5-HT, we found no significant intracellular 5-[3H]HT accumulation (Fig. 4A). In SERT-expressing cells (regardless of 5-HT3 receptor expression), robust counts were detected in the first cytosolic fraction (wash 1), with diminishing counts associated with subsequent washes. Minimal 5-[3H]HT remained in the final TX-114 membrane fraction, suggesting that intracellular 5-HT is predominantly freely soluble. Importantly, in cells co-expressing SERT and 5-HT3A receptors, no increased cellular retention or membrane association was observed. Therefore, prolonged receptor association through a putative intracellular binding site is not responsible. Moreover, in our assays, 5-HT (10 μm) does not exhibit prolonged 5-HT3 receptor binding, as no 5-[3H]HT binding (Fig. 4B, total) was observed above the nonspecific binding (as determined by competition with excess ondansetron) after rapid washing in 5-HT3A-transfected cells.

Bottom Line: The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered.Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum.Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization.

View Article: PubMed Central - PubMed

Affiliation: From the Medical Research Institute, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

Show MeSH
Related in: MedlinePlus