P-i inhibits the SR Ca2+ pump and stimulates pump-mediated Ca2+ leak in rabbit cardiac myocytes

Measurements of sarcoplasmic reticulum (SR) Ca2+ uptake were made from aliq uots of dissociated permeabilized ventricular myocytes using fura 2. Equili bration with 10 mM oxalate ensured a reproducible exponential decline of [C a2+] from 600 nM to a steady state of 100-200 nM after addition of Ca2+. In the presence of 5 mM ruthenium red, which blocks the ryanodine receptor, t he time course of the decline of [Ca2+] can be modeled by a Ca2+-dependent uptake process and a fixed Ca2+ leak. Partial inhibition of the Ca2+ pump w ith 1 mu M cyclopiazonic acid or 50 nM thapsigargin reduced the time consta nt for Ca2+ uptake but did not affect the SR Ca2+ leak. Addition of 10 mM i norganic phosphate (P-i) decreased the rate of Ca2+ accumulation by the SR and increased the Ca2+ leak rate. This effect was reversed on addition of 1 0 mM phosphocreatine. 10 mM P-i had no effect on Ca2+ leak from the SR afte r complete inhibition of the Ca2+ pump. In conclusion, P-i decreases the Ca 2+ uptake capacity of cardiac SR via a decrease in pump rate and an increas e in Ca2+ pump-dependent Ca2+ leak.