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 Ki≈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.
- Endoplasmic reticulum