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 pathogenesis of volume-dependent hypertension. In previous studies we isolated a more polar OLF-1 and a more apolar OLF-2 from the urine of healthy 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. We subsequently identified the chemical structure of OLF-2 as vanadium (VIV ) diascorbate. OLF-1, OLF-2, and vanadium diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. Because the inner medullary collecting duct (IMCD) plays a crucial role in the long-term regulation of body fluid volume, in the present study we investigated the effects of urinary OLF-1 and OLF-2, and of vanadium diascorbate in comparison to ouabain and vasopressin (AVP) on calcium mobilization, ie, on 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. Similarly, OLF-2 (10−4 mol/L) resulted in an increase in [Ca2+]i from 74 ± 20 to 216 ± 52 nmol/L (n = 7) after 4 min. Vanadium diascorbate (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 nmol/L (n = 5) after 4 min. A similar slow increase in [Ca2+]i was found 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 mol/L), which rapidly increased [Ca2+]i from 48 ± 10 to 299 ± 32 nmol/L (n = 4) within 30 sec. Thus, OLF-1, OLF-2, and Vanadium diascorbate, the active component of OLF-2, reveal similar effects as ouabain on IMCD cells, ie, they produce a slow increase in [Ca2+]i as expected from inhibition of Na-K-ATPase. The physiologic or pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.