title: R-Ras alters Ca2+ homeostasis by increasing the Ca2+ leak across the endoplasmic reticular membrane.
abstract: Evidence in the literature implicating both Ras-like Ras (R-Ras) and intracellular Ca(2+) in programmed cell death and integrin-mediated adhesion prompted us to investigate the possibility that R-Ras alters cellular Ca(2+) 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 CCK(8)-induced Ca(2+) rises using high speed confocal microscopy. In the absence of extracellular Ca(2+), both 1 microm Tg, a potent and selective inhibitor of the Ca(2+) pump of the intracellular Ca(2+) store, and 100 nm CCK(8) evoked a transient rise in Ca(2+), the size of which was decreased significantly after expression of V38R-Ras. At 0.1 nm, CCK(8) evoked periodic Ca(2+) rises. The frequency of these Ca(2+) 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 CCK(8)-induced Ca(2+) rises. The present findings are compatible with the idea that V38R-Ras expression increases the passive leak of Ca(2+) of the store leading to a decrease in Ca(2+) content of this store, which, in turn, leads to a decrease in frequency of the CCK(8)-induced cytosolic Ca(2+) oscillations. The effect of V38R-Ras on the Ca(2+) content of the intracellular Ca(2+) store closely resembles that of the antiapoptotic protein Bcl-2 observed earlier. Together with reports on the role of dynamic Ca(2+) changes in integrin-mediated adhesion, this leads us to propose that the reduction in endoplasmic reticulum Ca(2+) content may underlie the antiapoptotic effect of R-Ras, whereas the decrease in frequency of stimulus-induced Ca(2+) oscillations may play a role in the inhibitory effect of R-Ras on stimulus-induced cell detachment and migration.
authors.affiliation: Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, The Netherlands.
authors.forename: Werner J H
authors.initials: WJ
authors.lastname: Koopman
authors.forename: Remko R
authors.initials: RR
authors.lastname: Bosch
authors.forename: Sjenet E
authors.initials: SE
authors.lastname: van Emst-de Vries
authors.forename: Marcel
authors.initials: M
authors.lastname: Spaargaren
authors.forename: Jan Joep H H M
authors.initials: JJ
authors.lastname: De Pont
authors.forename: Peter H G M
authors.initials: PH
authors.lastname: Willems
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement; D002469:Cell Separation; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D020558:GTP Phosphohydrolases / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D009991:Oscillometry; D051381:Rats; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013997:Time Factors; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D008164:Luminescent Proteins; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015544:Inositol 1,4,5-Trisphosphate; D020558:GTP Phosphohydrolases; D018631:ras Proteins; D002118:Calcium