Growth factor activation of an amiloride-sensitive Na+/H+ exchange system in quiescent fibroblasts: coupling to ribosomal protein S6 phosphorylation.
Chinese hamster lung fibroblast cells (CCl39) enter the G0/G1 nonproliferative state after serum deprivation. In this report, we show that reinitiation of DNA synthesis by serum or the combination of purified human thrombin and insulin (1-10 microgram/ml) is preceded by very early stimulation of ionic fluxes (Na+/Rb+) and protein phosphorylation (27,000 daltons, 62,000 daltons, and the ribosomal S6 proteins). The potentiating action of insulin on thrombin-stimulated DNA synthesis is also observed on thrombin-stimulated Na+ influx, Rb+ influx, and protein S6 phosphorylation. Moreover, we demonstrate that CCl39 cells possess a Na+/H+ exchange system sensitive to amiloride. Half-maximal inhibition of growth factor-activated Na+ influx and Na+-dependent H+ efflux is obtained with 3-10 microM amiloride. Two lines of evidence indicate that the extrusion of H+ via the activation of the Na+/H+ exchanger is coupled to protein S6 phosphorylation: serum-stimulated phosphorylation is blocked by (i) amiloride at a concentration that abolishes serum-stimulated Na+ influx and (ii) protonophores that acidify the cell interior. The present data support the idea that the regulation of intracellular pH is a key event in the mechanism of growth factor action.
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication; D005347:Fibroblasts / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008168:Lung; D010766:Phosphorylation; D011719:Pyrazines / Q000494:pharmacology*; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins / Q000378:metabolism*; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D007328:Insulin; D011719:Pyrazines; D038601:Ribosomal Protein S6; D012269:Ribosomal Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium; D012413:Rubidium