Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series.
We have previously characterized in Chinese hamster lung fibroblasts a growth factor activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of growth factor-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity 20- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon growth factor action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for growth factor-induced DNA synthesis.
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication / Q000187:drug effects*; D006133:Growth Substances / Q000494:pharmacology*; D007399:Interphase / Q000187:drug effects; D007700:Kinetics; D008168:Lung; D011719:Pyrazines / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D006133:Growth Substances; D011719:Pyrazines; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D012964:Sodium