Ca²⁺ uptake by endoplasmic reticulum of renal cortex. I. Ionic requirements and regulation in vitro

A subcellular fraction enriched in cytochrome c reductase (7.9-fold) and relatively de-enriched (0.64-fold) in Na⁺/K⁺-ATPase was prepared from canine kidney cortex by sucrose density gradient ultracentrifugation. It was shown by electron microscopy to consist primarily of a light fraction of endoplasmic reticulum (LER). LER vesicles displayed ATP-dependent ⁴⁵Ca²⁺ uptake that was insensitive to 10 mM KCN or NaN₃, and was promptly released by 20 μM A23187 or ionomycin. Inositol-1,4,5-trisphosphate (IP₃) appeared to produce a time-dependent release of ⁴⁵Ca²⁺. Vanadate inhibited ⁴⁵Ca²⁺ uptake with a K_i≈0.3 mM, further suggesting that the activity resided in the ER rather than the plasma membrane. ⁴⁵Ca²⁺ uptake by LER, at 5 μM total [Ca²⁺], displayed a strong dependence on divalent cations (Mg²⁺>Co²⁺>Mn²⁺≫Ba²⁺≥Cd²⁺≥Sr²⁺, present at 2 mM) as well as on monovalent cations (Na⁺≥K⁺+Na⁺ >K⁺>Li⁺>choline⁺), and anions (Cl^->acetate^-≥NO₃^-≥F^->H₂PO₄^-≫gluconate^-≥oxalate⁼≫SO₄⁼). It had a fairly narrow pH optimum (7.25-7.50). Preincubation (10 min) of LER vesicles with 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated LER Ca²⁺ uptake; this effect was enhanced in the presence of renal cytosol [5% (vol/vol)]. However, Ca²⁺ uptake was not affected by preincubation with dibutyryl cyclic AMP, calmodulin, or 1,25-(OH)₂-vitamin D₃, either in the absence or presence of renal cytosol. Thus, the Mg²⁺-ATP dependent ⁴⁵Ca²⁺ uptake activity of this canine renal cortical LER fraction displays modulation by IP₃, TPA, and pH that appears to be physiologically relevant.