5-Hydroxytryptamine type 2A and 2C receptors linked to Na+/K+/Cl- cotransport
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1Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Abstract:
The goal of the present paper was to investigate 5-hydroxytryptamine (5HT)2A and 5HT2C receptor regulation of ion transport in fibroblast cell lines transfected with these receptors. Na+/K(+)-ATPase and Na+/K+/2Cl- cotransport were measured with 86Rb+ as a surrogate for K+ uptake. Serotonin agonists had no effect on Na+/K(+)-ATPase activity in either cell line. Bumetanide, an antagonist of Na+/K+/2Cl- cotransport, almost completely blocked ouabain-insensitive K+ uptake in both cell lines, with an IC50 of about 1 microM. 36Cl- uptake was 2-fold greater than 86Rb+ uptake, consistent with the expected 2:1 stoichiometry. In addition, the Cl-/HCO3- uptake blocker 4,4'-diisothiostilbene-2,2'-disulfonic acid had no effect on Cl- uptake. The 5HT2A/2C receptor agonist (-)-2,5-dimethoxy-4-bromoamphetamine increased ouabain-insensitive K+ uptake, and this effect was blocked by bumetanide. The receptor antagonists mianserin and mesulergine, but not spiperone, blocked (-)-2,5-dimethoxy-4-bromoamphetamine responses in fibroblasts transfected with 5HT2C receptors, and all three antagonists blocked the effects in cells expressing 5HT2A receptors. Ouabain-insensitive 22Na+ uptake was similarly affected. 5HT receptor-related actions were not observed in untransfected parent NIH/3T3 fibroblasts. Thus, we have demonstrated that 5HT2C and 5HT2A receptors are linked to activation of Na+/K+/2Cl- cotransport in transfected fibroblasts. This activity may be a factor in the pharmacological actions of 5HT agonists and antagonists.
Insights
This study shows that serotonin 5HT2A and 5HT2C receptors activate ion transport, specifically Na+/K+/2Cl- cotransport, in transfected cells. This finding may explain how serotonin drugs affect cells.
Area of Science:
- Pharmacology
- Cell Biology
- Molecular Biology
Background:
- Serotonin receptors (5HT) play crucial roles in cellular signaling.
- Ion transport mechanisms are vital for cell function and homeostasis.
- Understanding receptor-ligand interactions is key to drug development.
Purpose of the Study:
- To investigate the role of 5-hydroxytryptamine (5HT)2A and 5HT2C receptors in regulating ion transport.
- To determine if these receptors influence Na+/K(+)-ATPase and Na+/K+/2Cl- cotransport activity.
- To explore the effects of serotonin agonists and antagonists on ion transport in transfected fibroblast cell lines.
Main Methods:
- Utilized 86Rb+ as a surrogate for K+ uptake to measure Na+/K(+)-ATPase and Na+/K+/2Cl- cotransport.
- Employed 36Cl- uptake assays to confirm cotransporter activity.
- Administered specific serotonin receptor agonists and antagonists (e.g., (-)-2,5-dimethoxy-4-bromoamphetamine, bumetanide, mianserin, mesulergine, spiperone).
- Compared ion transport in transfected NIH/3T3 fibroblasts versus untransfected parent cells.
Main Results:
- Serotonin agonists did not affect Na+/K(+)-ATPase activity.
- Bumetanide, a Na+/K+/2Cl- cotransport antagonist, significantly inhibited K+ uptake (IC50 ~1 microM).
- 36Cl- uptake was consistent with the 2:1 stoichiometry of the Na+/K+/2Cl- cotransporter.
- The 5HT2A/2C receptor agonist increased ouabain-insensitive K+ uptake, an effect blocked by bumetanide.
- Specific antagonists differentially blocked agonist effects on 5HT2A and 5HT2C receptors.
- Ouabain-insensitive 22Na+ uptake was similarly modulated.
- No 5HT receptor-related actions were observed in untransfected cells.
Conclusions:
- 5HT2A and 5HT2C receptors are linked to the activation of Na+/K+/2Cl- cotransport in transfected fibroblasts.
- This receptor-mediated activation of ion transport may contribute to the pharmacological effects of serotonin agonists and antagonists.
- The findings provide insights into the cellular mechanisms underlying serotonin signaling pathways.