title: Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells.
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.
authors.affiliation: SmithKline Beecham Centre for Applied Neuro-psychobiology, Oxford University. U.K.
authors.forename: J M
authors.initials: JM
authors.lastname: Elliott
authors.forename: N R
authors.initials: NR
authors.lastname: Newberry
authors.forename: A J
authors.initials: AJ
authors.lastname: Cholewinski
authors.forename: J T
authors.initials: JT
authors.lastname: Bartrup
authors.forename: S J
authors.initials: SJ
authors.lastname: Briddon
authors.forename: J E
authors.initials: JE
authors.lastname: Carey
authors.forename: T P
authors.initials: TP
authors.lastname: Flanigan
authors.forename: R A
authors.initials: RA
authors.lastname: Newton
authors.forename: S L
authors.initials: SL
authors.lastname: Phipps
authors.forename: A C
authors.initials: AC
authors.lastname: Reavley
mesh_terms: 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
chemical_list: D012333:RNA, Messenger; D011985:Receptors, Serotonin; D012701:Serotonin; D007650:Ketanserin; D002118:Calcium