EFFECTS OF URINARY OUABAIN-LIKE FACTORS AND VANADIUM DIASCORBATE ON CALCIUM MOBILIZATION IN PORCINE INNER MEDULLARY COLLECTING DUCT CELLS -COMPARISON WITH THE EFFECTS OF OUABAIN AND VASOPRESSIN

It is speculated that ouabain-like factors (OLF) play a role in the pa thogenesis of volume-dependent hypertension. In previous studies we is olated a more polar OLF-1 and a more apolar OLF-2 from the urine of he althy subjects after 5 days on a high sodium intake (>400 mmol/day) by gel chromatography (Sephadex G-25 and G-10) and reverse-phase HPLC. W e subsequently identified the chemical structure of OLF-2 as vanadium (V-IV) diascorbate. OLF-1, OLF-2, and vanadium diascorbate inhibited d ose-dependently porcine Na-K-ATPase in vitro. Because the inner medull ary collecting duct (IMCD) plays a crucial role in the long-term regul ation of body fluid volume, in the present study we investigated the e ffects of urinary OLF-1 and OLF-2, and of vanadium diascorbate in comp arison to ouabain and vasopressin (AVP) on calcium mobilization, ie, o n free calcium concentration [Ca2+](i), in cultured porcine IMCD cells . [Ca2+](i) was determined by the fura-2 method in IMCD cells isolated by hypotonic treatment and density gradient centrifugation from fresh porcine kidneys. Assuming an approximate molecular weight (MW) of 400 for OLF-1 and OLF-2, OLF-1 (10(-4) mol/L) produced a slow increase in [Ca2+](i) from 39 +/- 10 to 169 +/- 21 nmol/L (n = 7) after 4 min. Si milarly, OLF-2 (10(-4) mol/L) resulted in an increase in [Ca2+](i) fro m 74 +/- 20 to 216 +/- 52 nmol/L (n = 7) after 4 min. Vanadium diascor bate (MW 403) dose-dependently increased [Ca2+](i). At a concentration of 10(-6) mol/L it increased [Ca2+](i) from 46 +/- 5 to 149 +/- 9 nmo l/L (n = 5) after 4 min. A similar slow increase in [Ca2+](i) was foun d with ouabain (10-6 mol/L), which increased [Ca2+](i) from 61 +/- 22 to 180 +/- 29 nmol/L (n = 5) after 4 min in contrast to AVP (10(-7) mo l/L), which rapidly increased [Ca2+](i) from 48 +/- 10 to 299 +/- 32 n mol/L (n = 4) within 30 sec. Thus, OLF-1, OLF-2, and Vanadium diascorb ate, the active component of OLF-2, reveal similar effects as ouabain on IMCD cells, ie, they produce a slow increase in [Ca2+](i) as expect ed from inhibition of Na-K-ATPase. The physiologic or pathologic roles of these and additional OLF in body fluid and blood pressure regulati on and in hypertension have yet to be evaluated. Am J Hypertens 1998;1 1:1208-1213 (C) 1998 American Journal of Hypertension, Ltd.