CELLULAR MECHANISMS OF ACTION OF A OUABAIN-LIKE FACTOR IN VASCULAR SMOOTH-MUSCLE CELLS

Ouabain-like factor (OLF) has been implicated to play an important rol e in certain forms of hypertension. We isolated OLF from the urine of salt-loaded healthy subjects by stepwise chromatographic procedures. T he post-salt fraction (F IV) eluted from Sephadex G-25 was rechromatog raphed on Sephadex G-10. A late small-molecular-weight fraction F8 inh ibited Na-K-ATPase in vitro (OLF activity). The effects of OLF on intr acellular Ca2+ concentrations ([Ca2+]i) and pH (pH(i)) were examined i n cultures of vascular smooth-muscle cells using the fluorescent probe s fura-2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) , respectively. Preincubation with OLF increased basal [Ca2+]i, from 8 7 +/- 6 to 160 +/- 8 nM (p < 0.001) and enhanced arginine vasopressin- stimulated maximal [Ca2+]i (418 +/- 11 vs. 523 +/- 14 nM, p < 0.01). T his effect was similar to that of ouabain. OLF also induced a rapid tr ansient increase of [Ca2+]i (82 +/- 9 vs. 253 +/- 23 nM, p < 0.01); [C a2+]i returned to levels slightly above baseline within -4 min. OLF-st imulated [Ca2+]i was attenuated by verapamil (126 +/- 5 nM, p < 0.01) and was also reduced in Ca2+-free medium (104 +/- 9 nM, p < 0.01). As opposed to OLF, ouabain did not exhibit this fast transient effect on [Ca2+]i. Amiloride (10(-3)M) blocked the sustained effect of OLF on [C a2+]i, (77 +/- 11 vs. 86 +/- 12 nM, NS). OLF induced an increase of pH (i) from 7.09 +/- 0.03 to 7.28 +/- 0.04 (p < 0.002). These results dem onstrate that OLF mobilizes Call in VSMC, in part by mobilizing Ca2+ f rom intracellular stores, in part by stimulating transmembrane Ca2+ in flux. This may render VSMC more susceptible to vasopressor stimuli. Th e effects of OLF differ from those of ouabain, suggesting that OLF eit her is not ouabain or represents more than one compound affecting cell ular Ca2+ mobilization. In factor verse-phase high-performance liquid chromatography of F8 revealed at least two Na-K-ATPase inhibitors of d ifferent polarities, whose identification and differential effects on Ca2+ kinetics in vascular smooth muscle are presently under investigat ion.