CHARACTERIZATION OF THE 5-HYDROXYTRYPTAMINE(2A) RECEPTOR-ACTIVATED CASCADE IN RAT C6 GLIOMA-CELLS

We have investigated the identity and intracellular cascade of respons es resulting from activation of the endogenous 5-hydroxytryptamine rec eptor in the C6 rat glioma cell line. Sequence analysis of reverse tra nscription-polymerase chain reaction products derived from C6 glioma c ell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine(2A) receptor. The binding of [H-3]ketanserin to c ell membranes demonstrated a significant correlation with the 5-hydrox ytryptamine(2A) receptor in rat frontal cortex. On intact cells, 5-hyd roxytryptamine stimulated a concentration-dependent increase in phosph atidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydro xytryptamine(2A) receptors. In whole-cell patch-clamp recordings, 5-hy droxytryptamine 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 inosi tol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydr oxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosp hoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+](i) and activation of an outward curr ent was observed more frequently in differentiated cells. Prolonged ex posure of the cells to 5-hydroxytryptamine led to a decrease in all re sponses and to the down-regulation of receptor number. We conclude tha t the rat C6 glioma cell expresses a 5-hydroxytryptamine(2A) receptor identical to that found in rat brain and that stimulation of the recep tor in C6 cells leads to the activation of Ca2+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca2ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptami ne compared to Ca2+ release and conductance change indicate that a com plex relationship exists between the component parts of the receptor-a ctivated cascade.