REGULATION OF CELL-VOLUME AND INTRACELLULAR PH IN HYPOSMOTICALLY SWOLLEN RAT OSTEOSARCOMA CELLS

The maintenance of cell volume involves transduction of a volume-sensi ng signal into effecters of volume-regulatory transporters. After expo sure to anisotonic conditions, cells undergo compensatory volume chang es that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pH(i)) homeostasis may be compromised d uring these processes. We have studied pH(i) and some of the signal tr ansduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarc oma cells, ROS 17/2.8. Cells were loaded with BCECF; pH(i) and cell vo lume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) bu ffered hypotonic medium induced a rapid cell swelling followed by an i ncomplete RVD of similar to 30% in suspended (i.e., round) cells and s imilar to 60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and hig h external [K+], all of which should reduce the cell membrane electroc hemical gradient for K+. Inhibition of RVD was induced also by decreas ing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indica ting the role of calcium-regulated K+ and Cl- efflux during RVD. Depol ymerization of actin filaments by cytochalasin D prolonged the RVD thr ee-fold and nonspecific activation of GTP-binding proteins up-regulate d RVD. In attached cells the hypoosmolar-induced swelling caused a lar ge reduction in pH(i) (similar to 0.7 units), which was sustained as l ong as cells were in hypoosmotic medium. The reduction of pH(i) induce d by cell swelling was inhibited by Na+-free extracellular medium, oua bain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl--free medium. However, amiloride failed to inhibit the hypoosmol ar-induced reduction of pH(i). Collectively these data indicate that R VD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conduc tive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pH(i) homeostasis in duced by cell swelling may reflect the existence of cell volume sensin g mechanisms that operate through tyrosine kinases to regulate pH(i).