Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells.
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.
Elliott
J M
JM
SmithKline Beecham Centre for Applied Neuro-psychobiology, Oxford University. U.K.
Newberry
N R
NR
Cholewinski
A J
AJ
Bartrup
J T
JT
Briddon
S J
SJ
Carey
J E
JE
Flanigan
T P
TP
Newton
R A
RA
Phipps
S L
SL
Reavley
A C
AC
D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005910:Glioma / Q000378:metabolism*; D007650:Ketanserin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011985:Receptors, Serotonin / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology
D012333:RNA, Messenger; D011985:Receptors, Serotonin; D012701:Serotonin; D007650:Ketanserin; D002118:Calcium