Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

Abstract

5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of ³²P_i into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC₅₀ of 1.2 ± 10^-7M. The phosphoinositide response was blocked by the 5-HT₂ antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by ˜30%. The 5-HT_1A agonist 8-hydroxy-2 (di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 μM) for 1 h increased the incorporation of [2-³H]myo-inositol into all phosphoinositides but not into inositol phosphates (IPs). Li⁺ alone at 10 mM increased labeling in inositol bisphosphate (IP₂) and trisphosphate (IP₃), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn²⁺ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-³H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 μM). Radioactivity in IP₂ and IP₃ was very low, was stimulated ˜50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP₃ but was increased only from 3 min on with a peak at 20 min, when the elevation was ˜40 times that in IP₃. The EC₅₀ value of 1.8 ± 10^-7M was comparable with that obtained from ³²P_i studies measuring labeled PI. Ketanserin and spiperone inhibited 5-HT-stimulated [2-³H]IP release with IC₅₀ values of 3.1 ± 10^-9 and 1.8 ± 10, ^-8M, respectively. This study demonstrates that phosphoinositide hydrolysis is enhanced by 5-HT in C6 glioma cells and that this phenomenon is linked to 5-HT₂-like binding sites.