Modulation of mitochondrial Ca2+ in ECV304 endothelial cells by agents which elevate cAMP

In the human umbilical vein endothelial cell-derived cell line, ECV304, we have previously shown that the elevation of [Ca2+]m in response to agonist stimulation is dependent on Ca2+ influx, i.e. an ATP-induced sustained increase in [Ca2+]c results in a slow-onset, sustained elevation in [Ca2+]m [Lawrie A.M., Rizzuto R., Pozzan T., Simpson A.W.M. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753-10759]. In this study, we have investigated the effect of raising cAMP on ATP-evoked elevations in both [Ca2+]m and [Ca2+]c by: (i) activating adenylate cyclase with the forskolin analogue--forskolin 6-[3'-(N,N-dimethylaminopropionyl)]-HCl (1 microM) (FA); (ii) addition of membrane permeable dibutyryl-cAMP (100 microM) (dbcAMP); and (iii) a combination of FA plus inhibition of cAMP phosphodiesterase using RO-20-1724 (17.5 microM) (RO);. We have found that protocols aimed at elevating cAMP significantly reduce the ATP-evoked (1-10 microM) rise in [Ca2+]m (n = 14); however, the [Ca2+]c response to ATP was not affected (n = 33). This new evidence shows that a second messenger system, other than Ca2+ itself, may influence [Ca2+]m changes in response to agonist stimulation.