Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells.
The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of approximately 30% in suspended (i.e., round) cells and approximately 60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indicating the role of calcium-regulated K+ and Cl- efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (approximately 0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na(+)-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl(-)-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.
Lo
C
C
MRC Group in Periodontal Physiology, University of Toronto, Canada.
Ferrier
J
J
Tenenbaum
H C
HC
McCulloch
C A
CA
D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane; D048429:Cell Size; D019204:GTP-Binding Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009995:Osmosis; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology; D012516:Osteosarcoma; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D000199:Actins; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins