Stimulation of Calcium Influx and Calcium Cascade by Cyclic AMP in Cultured Carrot Cells

Treatment of cultured carrot (Daucus carota L.) cells with activators of adenylate cyclase, forskolin, and cholera toxin induced the biosynthesis of an antifungal isocoumarin, 6-methoxymellein, in the cells. Addition of dihutyryl cyclic AMP to carrot cell culture also stimulated the accumulation of the compound. The cyclic AMP-evoked 6-methoxymellein production was significantly depressed in the presence of certain inhibitors of calcium cascade such as Ca2+ channel blockers and inhibitors of calmodulin-dependent processes. In dibutyryl cyclic AMP- and forskolin-treated carrot cells, increase in cytosolic Ca2+ concentration was observed as monitored by the fluorescent calcium indicator fluo-3. Cyclic AMP-dependent Ca2+ influx into carrot cells was also confirmed with Ca2+-loaded vesicles prepared from the plasma membrane-rich fraction of the cells. Transient increase in Ca2+- and Ca2+/calmodulin-dependent protein kinase activity but not cyclic AMP-dependent protein phosphorylation was detected in the cells of high cyclic AMP concentration. Results obtained in the present work suggest that the increase in cyclic AMP content in carrot cells induces Ca2+ influx across plasma membrane without activating cyclic AMP-dependent protein kinase which, then, stimulates calcium cascade in the cells.