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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.

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

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5-HT release and its impact on receptor resensitization.A, time course of 5-HT release from COS-7 cells after loading (300 μm 5-HT (0.1% 5-[3H]HT), 1 h). B, time course of 5-HT released from segments of adult guinea pig ileum following loading (100 μm 5-HT (0.1% 5-[3H]HT), 1 h). Data represent 20 segments of ileum from 4 different animals. C, 5-HT3 receptor resensitization in adult guinea pig ileum. Acute exposure to 5-HT (100 μm) was applied until full receptor desensitization observed and 5-HT3 receptor contractions (50 μm 2-ME) were determined after a 10-min resensitization period in the absence (−5-HT) or presence (+5-HT) of 5 μm 5-HT. Data represent 12 segments of ileum for each experiment on 3 separate occasions. *** signifies p = 0.0014 (paired t test, 2 tailed, n = 4). D, representative traces of guinea pig contractions indicating initial responses to ACh, 2-ME, and 5-HT (left traces) and subsequent 2-ME and ACh (right traces) responses after receptor desensitization to 5-HT (100 μm, <1 min) and subsequent recovery in the absence (upper panel) or presence (lower panel) of 5-HT (5 μm). Each panel (upper or lower) represents traces from a single continuous experiment.
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Figure 5: 5-HT release and its impact on receptor resensitization.A, time course of 5-HT release from COS-7 cells after loading (300 μm 5-HT (0.1% 5-[3H]HT), 1 h). B, time course of 5-HT released from segments of adult guinea pig ileum following loading (100 μm 5-HT (0.1% 5-[3H]HT), 1 h). Data represent 20 segments of ileum from 4 different animals. C, 5-HT3 receptor resensitization in adult guinea pig ileum. Acute exposure to 5-HT (100 μm) was applied until full receptor desensitization observed and 5-HT3 receptor contractions (50 μm 2-ME) were determined after a 10-min resensitization period in the absence (−5-HT) or presence (+5-HT) of 5 μm 5-HT. Data represent 12 segments of ileum for each experiment on 3 separate occasions. *** signifies p = 0.0014 (paired t test, 2 tailed, n = 4). D, representative traces of guinea pig contractions indicating initial responses to ACh, 2-ME, and 5-HT (left traces) and subsequent 2-ME and ACh (right traces) responses after receptor desensitization to 5-HT (100 μm, <1 min) and subsequent recovery in the absence (upper panel) or presence (lower panel) of 5-HT (5 μm). Each panel (upper or lower) represents traces from a single continuous experiment.

Mentions: To determine if cells are capable of releasing the sequestered 5-HT, we investigated the release of 5-[3H]HT from COS-7 cells and the guinea pig ileum. COS-7 cells (no SERT) were loaded with 5-HT (300 μm, 0.1% 5-[3H]HT, 60 min), excess 5-HT was washed off, and cells were chased for 120 min. Under the experimental conditions in which we observed down-regulation of function (Figs. 1 and 3), the release of 5-HT reached low micromolar levels (Fig. 5A). To determine if the same pool of 5-HT exists within cells in native tissue where high 5-HT level can occur, we investigated 5-HT uptake (300 μm, 0.1% 3H-5-HT, 60 min) in guinea pig ileum segments. A 7-cm segment was turned inside out and incubated with 5-HT (100 μm, 0.16% 3H-5-HT, 60 min). Five equal sections were cut, and 5-HT release was monitored (in 1.5 ml) from each. Under these conditions, 5-HT is sequestered and released from the ileum in the low micromolar range (Fig. 5B).


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)

5-HT release and its impact on receptor resensitization.A, time course of 5-HT release from COS-7 cells after loading (300 μm 5-HT (0.1% 5-[3H]HT), 1 h). B, time course of 5-HT released from segments of adult guinea pig ileum following loading (100 μm 5-HT (0.1% 5-[3H]HT), 1 h). Data represent 20 segments of ileum from 4 different animals. C, 5-HT3 receptor resensitization in adult guinea pig ileum. Acute exposure to 5-HT (100 μm) was applied until full receptor desensitization observed and 5-HT3 receptor contractions (50 μm 2-ME) were determined after a 10-min resensitization period in the absence (−5-HT) or presence (+5-HT) of 5 μm 5-HT. Data represent 12 segments of ileum for each experiment on 3 separate occasions. *** signifies p = 0.0014 (paired t test, 2 tailed, n = 4). D, representative traces of guinea pig contractions indicating initial responses to ACh, 2-ME, and 5-HT (left traces) and subsequent 2-ME and ACh (right traces) responses after receptor desensitization to 5-HT (100 μm, <1 min) and subsequent recovery in the absence (upper panel) or presence (lower panel) of 5-HT (5 μm). Each panel (upper or lower) represents traces from a single continuous experiment.
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Figure 5: 5-HT release and its impact on receptor resensitization.A, time course of 5-HT release from COS-7 cells after loading (300 μm 5-HT (0.1% 5-[3H]HT), 1 h). B, time course of 5-HT released from segments of adult guinea pig ileum following loading (100 μm 5-HT (0.1% 5-[3H]HT), 1 h). Data represent 20 segments of ileum from 4 different animals. C, 5-HT3 receptor resensitization in adult guinea pig ileum. Acute exposure to 5-HT (100 μm) was applied until full receptor desensitization observed and 5-HT3 receptor contractions (50 μm 2-ME) were determined after a 10-min resensitization period in the absence (−5-HT) or presence (+5-HT) of 5 μm 5-HT. Data represent 12 segments of ileum for each experiment on 3 separate occasions. *** signifies p = 0.0014 (paired t test, 2 tailed, n = 4). D, representative traces of guinea pig contractions indicating initial responses to ACh, 2-ME, and 5-HT (left traces) and subsequent 2-ME and ACh (right traces) responses after receptor desensitization to 5-HT (100 μm, <1 min) and subsequent recovery in the absence (upper panel) or presence (lower panel) of 5-HT (5 μm). Each panel (upper or lower) represents traces from a single continuous experiment.
Mentions: To determine if cells are capable of releasing the sequestered 5-HT, we investigated the release of 5-[3H]HT from COS-7 cells and the guinea pig ileum. COS-7 cells (no SERT) were loaded with 5-HT (300 μm, 0.1% 5-[3H]HT, 60 min), excess 5-HT was washed off, and cells were chased for 120 min. Under the experimental conditions in which we observed down-regulation of function (Figs. 1 and 3), the release of 5-HT reached low micromolar levels (Fig. 5A). To determine if the same pool of 5-HT exists within cells in native tissue where high 5-HT level can occur, we investigated 5-HT uptake (300 μm, 0.1% 3H-5-HT, 60 min) in guinea pig ileum segments. A 7-cm segment was turned inside out and incubated with 5-HT (100 μm, 0.16% 3H-5-HT, 60 min). Five equal sections were cut, and 5-HT release was monitored (in 1.5 ml) from each. Under these conditions, 5-HT is sequestered and released from the ileum in the low micromolar range (Fig. 5B).

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