R-Ras Alters Ca2+ Homeostasis by Increasing the Ca2+ Leak across the Endoplasmic Reticular Membrane*

Evidence in the literature implicating both Ras-like Ras (R-Ras) and intracellular Ca2+ in programmed cell death and integrin-mediated adhesion prompted us to investigate the possibility that R-Ras alters cellular Ca2+handling. Chinese hamster ovary cells expressing the cholecystokinin (CCK)-A receptor were loaded with indo-1 to study the effects of constitutively active V38R-Ras and dominant negative N43R-Ras on the kinetics of the thapsigargin (Tg)- and CCK8-induced Ca2+ rises using high speed confocal microscopy. In the absence of extracellular Ca2+, both 1 μm Tg, a potent and selective inhibitor of the Ca2+ pump of the intracellular Ca2+ store, and 100 nmCCK8 evoked a transient rise in Ca2+, the size of which was decreased significantly after expression of V38R-Ras. At 0.1 nm, CCK8 evoked periodic Ca2+rises. The frequency of these Ca2+ oscillations was reduced significantly in V38R-Ras-expressing cells. In contrast to V38R-Ras, N43R-Ras did not alter the kinetics of the Tg- and CCK8-induced Ca2+ rises. The present findings are compatible with the idea that V38R-Ras expression increases the passive leak of Ca2+ of the store leading to a decrease in Ca2+ content of this store, which, in turn, leads to a decrease in frequency of the CCK8-induced cytosolic Ca2+ oscillations. The effect of V38R-Ras on the Ca2+ content of the intracellular Ca2+ store closely resembles that of the antiapoptotic protein Bcl-2 observed earlier. Together with reports on the role of dynamic Ca2+changes in integrin-mediated adhesion, this leads us to propose that the reduction in endoplasmic reticulum Ca2+ content may underlie the antiapoptotic effect of R-Ras, whereas the decrease in frequency of stimulus-induced Ca2+ oscillations may play a role in the inhibitory effect of R-Ras on stimulus-induced cell detachment and migration.