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 microM 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 approximately 0.3 mM, further suggesting that the activity resided in the ER rather than the plasma membrane. 45Ca2+ uptake by LER, at 5 microM total [Ca2+], displayed a strong dependence on divalent cations (Mg2+ greater than Co2+ greater than Mn2+ much greater than Ba2+ greater than or equal to Cd2+ greater than or equal to Sr2+, present at 2 mM) as well as on monovalent cations (Na+ greater than or equal to K+ + Na+ greater than K+ greater than Li+ greater than choline +), and anions (Cl- greater than acetate- greater than or equal to NO3- greater than or equal to F- greater than H2PO4- much greater than gluconate- greater than or equal to oxalate= much greater than 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)].