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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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5-HT3 down-regulation of function in the guinea pig ileum occurs through endocytosis-independent mechanisms.A, sample trace of an organ bath experiment investigating the chronic effects of 5-HT. Dots represent brief exposure times whereby the indicated drug was washed out immediately after maximal contraction obtained. The arrow indicates prolonged (60 min) exposure with no washout. B, the contractile responses to 2-ME (50 μm) were measured in the tissue (Initial). After 60 min of exposure to 100 μm 5-HT (followed by 10 min washout), the resultant 2-ME responses were measured (post-5-HT). 2-ME responses were also tested 60 min post treatment to monitor long term recovery. Data represent an average of 28 independent tissue samples. *** signifies p < 0.001 (ANOVA). C, responses to 1 μm ACh before and after 5-HT treatment (60 min) and after a 60-min recovery in drug-free buffer. Data represent an average of 14 sections of tissue. D, tissue responses to 2-ME (50 μm) after chronic exposure to 5-HT (100 μm, 60 min) in the absence and presence of dynasore (DYN; 80 μm), nystatin (NYS; 21 μm), dynasore plus nystatin, or filipin (FLP; 7.6 μm). Data are an average of 8–33 sections of ileum.
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Figure 2: 5-HT3 down-regulation of function in the guinea pig ileum occurs through endocytosis-independent mechanisms.A, sample trace of an organ bath experiment investigating the chronic effects of 5-HT. Dots represent brief exposure times whereby the indicated drug was washed out immediately after maximal contraction obtained. The arrow indicates prolonged (60 min) exposure with no washout. B, the contractile responses to 2-ME (50 μm) were measured in the tissue (Initial). After 60 min of exposure to 100 μm 5-HT (followed by 10 min washout), the resultant 2-ME responses were measured (post-5-HT). 2-ME responses were also tested 60 min post treatment to monitor long term recovery. Data represent an average of 28 independent tissue samples. *** signifies p < 0.001 (ANOVA). C, responses to 1 μm ACh before and after 5-HT treatment (60 min) and after a 60-min recovery in drug-free buffer. Data represent an average of 14 sections of tissue. D, tissue responses to 2-ME (50 μm) after chronic exposure to 5-HT (100 μm, 60 min) in the absence and presence of dynasore (DYN; 80 μm), nystatin (NYS; 21 μm), dynasore plus nystatin, or filipin (FLP; 7.6 μm). Data are an average of 8–33 sections of ileum.

Mentions: As high levels of 5-HT are observed within the gut, we investigated whether chronic exposure to 5-HT led to a ligand-induced down-regulation of receptor function in the intact guinea pig ileum. Specific 5-HT3 receptor activation was monitored by the contractile responses induced by the selective agonist 2-ME. Acute exposure to 5-HT (100 μm) caused a robust contraction and rapid receptor desensitization (Fig. 2A, center trace) which recovered quickly after washing (<10 min, not shown). In contrast, chronic exposure to 5-HT (100 μm, 60 min), followed by washout in a drug-free buffer for 10 min (to allow receptor resensitization) caused a significant decrease (to 48.6 ± 25.0%, Fig. 2B) in the magnitude of subsequent 2-ME-evoked contractions (p < 0.001, ANOVA, n = 28) (Fig. 2A, right trace) compared with the pretreatment contraction (Fig. 2A, left trace). After removal of 5-HT (60 min) the responses to 2-ME were fully recoverable (Fig. 2B). To investigate the specificity of the 5-HT3 functional loss, contractions evoked by ACh were also quantified. In this case the magnitude of ACh responses were unaffected by chronic 5-HT treatment (p > 0.05; ANOVA, n = 14; Fig. 2C), verifying that the tissue was still capable of undergoing contractions and that the down-regulation was specific to 5-HT3 receptors. An individual example of 5-HT3 functional loss is shown (Fig. 2A) where a pre-pulse of 2-ME and ACh precedes 5-HT chronic (60 min) exposure (note early receptor desensitization). After the chronic exposure to 5-HT, 2-ME, but not ACh, responses were reduced.


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-HT3 down-regulation of function in the guinea pig ileum occurs through endocytosis-independent mechanisms.A, sample trace of an organ bath experiment investigating the chronic effects of 5-HT. Dots represent brief exposure times whereby the indicated drug was washed out immediately after maximal contraction obtained. The arrow indicates prolonged (60 min) exposure with no washout. B, the contractile responses to 2-ME (50 μm) were measured in the tissue (Initial). After 60 min of exposure to 100 μm 5-HT (followed by 10 min washout), the resultant 2-ME responses were measured (post-5-HT). 2-ME responses were also tested 60 min post treatment to monitor long term recovery. Data represent an average of 28 independent tissue samples. *** signifies p < 0.001 (ANOVA). C, responses to 1 μm ACh before and after 5-HT treatment (60 min) and after a 60-min recovery in drug-free buffer. Data represent an average of 14 sections of tissue. D, tissue responses to 2-ME (50 μm) after chronic exposure to 5-HT (100 μm, 60 min) in the absence and presence of dynasore (DYN; 80 μm), nystatin (NYS; 21 μm), dynasore plus nystatin, or filipin (FLP; 7.6 μm). Data are an average of 8–33 sections of ileum.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231679&req=5

Figure 2: 5-HT3 down-regulation of function in the guinea pig ileum occurs through endocytosis-independent mechanisms.A, sample trace of an organ bath experiment investigating the chronic effects of 5-HT. Dots represent brief exposure times whereby the indicated drug was washed out immediately after maximal contraction obtained. The arrow indicates prolonged (60 min) exposure with no washout. B, the contractile responses to 2-ME (50 μm) were measured in the tissue (Initial). After 60 min of exposure to 100 μm 5-HT (followed by 10 min washout), the resultant 2-ME responses were measured (post-5-HT). 2-ME responses were also tested 60 min post treatment to monitor long term recovery. Data represent an average of 28 independent tissue samples. *** signifies p < 0.001 (ANOVA). C, responses to 1 μm ACh before and after 5-HT treatment (60 min) and after a 60-min recovery in drug-free buffer. Data represent an average of 14 sections of tissue. D, tissue responses to 2-ME (50 μm) after chronic exposure to 5-HT (100 μm, 60 min) in the absence and presence of dynasore (DYN; 80 μm), nystatin (NYS; 21 μm), dynasore plus nystatin, or filipin (FLP; 7.6 μm). Data are an average of 8–33 sections of ileum.
Mentions: As high levels of 5-HT are observed within the gut, we investigated whether chronic exposure to 5-HT led to a ligand-induced down-regulation of receptor function in the intact guinea pig ileum. Specific 5-HT3 receptor activation was monitored by the contractile responses induced by the selective agonist 2-ME. Acute exposure to 5-HT (100 μm) caused a robust contraction and rapid receptor desensitization (Fig. 2A, center trace) which recovered quickly after washing (<10 min, not shown). In contrast, chronic exposure to 5-HT (100 μm, 60 min), followed by washout in a drug-free buffer for 10 min (to allow receptor resensitization) caused a significant decrease (to 48.6 ± 25.0%, Fig. 2B) in the magnitude of subsequent 2-ME-evoked contractions (p < 0.001, ANOVA, n = 28) (Fig. 2A, right trace) compared with the pretreatment contraction (Fig. 2A, left trace). After removal of 5-HT (60 min) the responses to 2-ME were fully recoverable (Fig. 2B). To investigate the specificity of the 5-HT3 functional loss, contractions evoked by ACh were also quantified. In this case the magnitude of ACh responses were unaffected by chronic 5-HT treatment (p > 0.05; ANOVA, n = 14; Fig. 2C), verifying that the tissue was still capable of undergoing contractions and that the down-regulation was specific to 5-HT3 receptors. An individual example of 5-HT3 functional loss is shown (Fig. 2A) where a pre-pulse of 2-ME and ACh precedes 5-HT chronic (60 min) exposure (note early receptor desensitization). After the chronic exposure to 5-HT, 2-ME, but not ACh, responses were reduced.

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