Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways

Human serotonin 5A (5-HT_5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT_5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca²⁺ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT_5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K⁺ was replaced with Cs⁺ but was not significantly inhibited by typical K⁺ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca²⁺ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT_5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT_5A receptors regulate intracellular Ca²⁺ mobilization which is probably a result of the IP3-sensitive Ca²⁺ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.