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Characterization of functional responses in A9 cells transfected with cloned rat 5-HT1C receptors
H W Boddeke1, B J Hoffman, J M Palacios
1Sandoz Pharma Ltd, Preclinical Research, Basle, Switzerland.
Abstract:
Functional responses to stimulation of rat 5-HT1C receptors expressed in A9 cells were studied using whole cell voltage clamp and calcium recording techniques. Stimulation of 5-HT1C receptors evoked outward currents clamped at -50 mV. The outward currents were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. 8-Bromo cyclic AMP (5 mmol/l) neither produced an effect per se nor affected the 5-HT-induced outward current in A9 cells, thus excluding cAMP as a second messenger involved in 5-HT1C receptor activation. Phorbol myristic acetate (PMA; 10 mumol/l) did not affect the electrical activity of the transfected A9 cells but reduced the 5-HT-induced current amplitude to 71 +/- 9% of the control value (n = 12). This indicates that activation of protein kinase C does not play a direct role in the 5-HT-induced response in these cells. The 5-HT induced currents mainly involved potassium ions, although a small contribution of chloride ions was also observed. The 5-HT-induced current was inhibited by the K+ channel blocking agents tetraethylammonium (1 mmol/l), apamin (0,5 mumol/l) and 4-aminopyridine (5 mmol/l). The 5-HT-induced currents recorded at -50 mV were unaffected by removal of extracellular calcium, but inclusion of the calcium chelator BAPTA (5 mmol/l) in the intracellular solutions abolished the current. Measurement with the calcium indicator Fluo-3 revealed a 5-HT-induced increase in intracellular calcium which was not affected by removal of extracellular calcium but declined after repeated stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Stimulating rat 5-HT1C receptors in A9 cells activates outward currents primarily via potassium ions. This response involves G-protein signaling and intracellular calcium increase, but not cyclic AMP or protein kinase C.
Area of Science:
- Neuroscience
- Molecular Pharmacology
Background:
- The 5-HT1C receptor is a serotonin receptor implicated in various central nervous system functions.
- Understanding the signaling pathways activated by 5-HT1C receptors is crucial for developing targeted therapeutics.
Purpose of the Study:
- To elucidate the specific ionic mechanisms and second messenger pathways involved in the functional response of rat 5-HT1C receptors expressed in A9 cells.
Main Methods:
- Whole-cell voltage clamp electrophysiology to record membrane currents.
- Calcium imaging using Fluo-3 to monitor intracellular calcium levels.
- Pharmacological manipulation using GTP analogs, cAMP, phorbol esters, and ion channel blockers.
Main Results:
- 5-HT1C receptor stimulation evoked outward currents at -50 mV, dependent on GTP and inhibited by GDP-beta-S, indicating G-protein involvement.
- The currents were primarily carried by potassium ions, with a minor chloride contribution, and were blocked by specific potassium channel inhibitors.
- Intracellular calcium levels increased upon 5-HT stimulation, independent of extracellular calcium, but sensitive to intracellular chelation.
Conclusions:
- 5-HT1C receptor activation in A9 cells triggers a G-protein mediated outward current, predominantly through potassium channels.
- Cyclic AMP and direct protein kinase C activation are not involved in this specific 5-HT1C receptor signaling pathway.
- An increase in intracellular calcium, originating from internal stores, is a key component of the 5-HT1C receptor response.