The present study was undertaken to examine the involvement of 5-HT2A receptor-mediated second messengers in the agonist-induced up-regulation of 5-HT2A receptors in cerebellar granule cells. Stimulation of these cells with a 5-HT2A/5-HT2C agonist, (+/-)-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane for 16 hr resulted in a marked increase in [3H]ketanserin binding to 5-HT2A receptors in intact cells. (+/-)-(2, 5-dimethoxy-4-iodophenyl)-2- aminopropane up-regulated, but not basal levels of 5-HT2A binding sites, were largely attenuated by actinomycin D and cycloheximide, suggesting an essential role for de novo RNA and protein synthesis in this up-regulation process. This receptor up-regulation was 5-HT2A receptor-mediated but was unaffected by short-term pretreatment with phorbol dibutyrate to attenuate (+/-)-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane-induced phosphoinositide hydrolysis or by treatment with staurosporine to inhibit protein kinase C. In contrast, blockade of Ca2+ channels by LaCl3 or SK&F 96365 and depletion of extracellular Ca2+ by EGTA preferentially decreased the up-regulated 5-HT2A receptor levels, suggesting a role of Ca2+ influx in the receptor up-regulation. The (+/-)-(2, 5-dimethoxy-4- iodophenyl)-2-aminopropane up-regulation was also prevented by inhibitors of calmodulin, calmidazolium and W-7. Moreover, the effect of (+/-)-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane was blocked by KN-62, a selective Ca2+/calmodulin kinase inhibitor and by H-7 and staurosporine at concentrations high enough to be nonselective for a specific type of protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)