EFFECT OF THE NA+ H+ ANTIPORT INHIBITOR HOE-694 ON THE ANGIOTENSIN-II-INDUCED VASCULAR SMOOTH-MUSCLE CELL-GROWTH/

1 Hoe 694 (3-methylsulphonyl-4-piperidinobenzoyl)guanidine methanesulp honate) was characterized as a new, potent, non-amiloride inhibitor of the Na+/H+ exchanger. In order to elucidate the role of the Na+/H+ ex changer isoform 1 (NHE-1) in the regulation of vascular smooth muscle cell growth, we investigated the effects of different amiloride analog ues and of Hoe 694 on angiotensin II-induced cell growth. Since intrac ellular pH, the intracellular free Ca2+ concentration and the expressi on of the transcription factor c-fos seem to be involved in the regula tion of cell growth, the effects of the amiloride analogues and Hoe 69 4 on the angiotensin II-induced changes in these three parameters were examined. 2 Measurement of cytosolic Ca2+ and pH in cell monolayers w as performed using fura-2/AM and BCECF/AM, respectively. The effect of angiotensin II on cell growth was examined using (1) [H-3]-thymidine incorporation, (2) the bromo-2-deoxyuridine (BrdU) immunfluorescence a ssay, (3) the colorimetric determination of cell mitochondrial dehydro genase activity and (4) determination of cell number. Total RNA was ex tracted from cells by the guanidinium isothiocyanate/CsCl procedure. T he expression of c-fos was quantitated by Northern blotting. 3 Various amiloride analogues inhibited the angiotensin II-induced stimulation of the Na+/H+ exchanger, the increase in cytosolic Ca2+ and cell growt h but not the induction of c-fos mRNA. Hoe 694 (1-25 mu M) dose-depend ently inhibited the angiotensin II-induced stimulation of the Na+/H+ e xchanger but had no significant effects on cytosolic Ca2+, c-fos mRNA levels or cell growth. 4 Our findings support the concept that activat ion of the Na+/H+ exchanger is not essential for angiotensin II-induce d vascular smooth muscle cell growth.