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Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells
J M Elliott1, N R Newberry, A J Cholewinski
1SmithKline Beecham Centre for Applied Neuro-psychobiology, Oxford University. U.K.
Abstract:
We have investigated the identity and intracellular cascade of responses resulting from activation of the endogenous 5-hydroxytryptamine receptor in the C6 rat glioma cell line. Sequence analysis of reverse transcription-polymerase chain reaction products derived from C6 glioma cell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine2A receptor. The binding of [3H]ketanserin to cell membranes demonstrated a significant correlation with the 5-hydroxytryptamine2A receptor in rat frontal cortex. On intact cells, 5-hydroxytryptamine stimulated a concentration-dependent increase in phosphatidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydroxytryptamine2A receptors. In whole-cell patch-clamp recordings, 5-hydroxytryptamine induced an outward current mediated predominantly by K+ ions (reversal potential = -80 mV). Using caged molecules containing Ca2+ or inositol 1,4,5-trisphosphate in the patch electrode solution, we found that rapid photolytic release of Ca2+ and particularly inositol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydroxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosphoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+]i and activation of an outward current was observed more frequently in differentiated cells. Prolonged exposure of the cells to 5-hydroxytryptamine led to a decrease in all responses and to the down-regulation of receptor number. We conclude that the rat C6 glioma cell expresses a 5-hydroxytryptamine2A receptor identical to that found in rat brain and that stimulation of the receptor in C6 cells leads to the activation of Ca2+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca2+ ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptamine compared to Ca2+ release and conductance change indicate that a complex relationship exists between the component parts of the receptor-activated cascade.
Insights
Rat C6 glioma cells express the 5-hydroxytryptamine2A receptor, activating a signaling cascade involving phosphatidyl inositide hydrolysis and calcium release. This leads to K+ channel activation, though differentiation impacts receptor density and response.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- The 5-hydroxytryptamine receptor (5-HTR) plays crucial roles in the central nervous system.
- Understanding 5-HTR signaling in glial cells like C6 rat glioma is important for neurological research.
Purpose of the Study:
- To identify the specific 5-HTR subtype in C6 rat glioma cells.
- To elucidate the intracellular signaling cascade triggered by 5-HTR activation in these cells.
- To investigate the effects of cell differentiation on 5-HTR responses.
Main Methods:
- Sequence analysis of reverse transcription-polymerase chain reaction (RT-PCR) products.
- Radioligand binding assays using [3H]ketanserin.
- Measurement of phosphatidyl inositide turnover and intracellular calcium ([Ca2+]) levels.
- Whole-cell patch-clamp electrophysiology.
- Experiments with caged Ca2+ and inositol 1,4,5-trisphosphate.
Main Results:
- C6 glioma cells express a 5-hydroxytryptamine2A receptor (5-HT2AR) identical to that in rat brain.
- 5-HT stimulation increases phosphatidyl inositide turnover and intracellular [Ca2+].
- 5-HT activates an outward potassium (K+) current, mediated by Ca2+-activated K+ channels, via phosphoinositide hydrolysis and subsequent Ca2+ rise.
- Undifferentiated cells show higher 5-HT2AR density and phosphoinositide response, while differentiated cells exhibit more frequent Ca2+ release and outward current.
- Prolonged 5-HT exposure leads to receptor down-regulation.
Conclusions:
- Rat C6 glioma cells possess functional 5-HT2ARs that initiate a signaling cascade involving phosphoinositide hydrolysis and Ca2+ mobilization.
- The activated cascade leads to the opening of Ca2+-activated K+ channels.
- Cell differentiation influences the interplay between receptor density, phosphoinositide signaling, and ion channel activation, indicating a complex regulatory relationship.
