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

Abstract

Measurements of sarcoplasmic reticulum (SR) Ca2+ uptake were made from aliquots of dissociated permeabilized ventricular myocytes using fura 2. Equilibration with 10 mM oxalate ensured a reproducible exponential decline of [Ca2+] from 600 nM to a steady state of 100–200 nM after addition of Ca2+. In the presence of 5 μM ruthenium red, which blocks the ryanodine receptor, the 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 with 1 μM cyclopiazonic acid or 50 nM thapsigargin reduced the time constant for Ca2+ uptake but did not affect the SR Ca2+leak. Addition of 10 mM inorganic phosphate (Pi) decreased the rate of Ca2+ accumulation by the SR and increased the Ca2+ leak rate. This effect was reversed on addition of 10 mM phosphocreatine. 10 mM Pi had no effect on Ca2+ leak from the SR after complete inhibition of the Ca2+ pump. In conclusion, Pi decreases the Ca2+ uptake capacity of cardiac SR via a decrease in pump rate and an increase in Ca2+ pump-dependent Ca2+ leak.