LUMINAL CA2+ REGULATES PASSIVE CA2+ EFFLUX FROM THE INTRACELLULAR STORES OF HEPATOCYTES

Ca2+ uptake into the intracellular stores of permeabilized hepatocytes was entirely dependent on ATP and substantially inhibited by either i onomycin or thapsigargin, although both were required for complete inh ibition. Unidirectional efflux of Ca-45(2+) after removal of ATP from cells loaded to steady state (1.60 +/- 0.12 nmol/10(6) cells) was mono exponential and occurred with a half-time of 103 +/- 10 s. However, th e Ca-45(2+) content of the stores did not return to their pre-ATP leve l, but reached a plateau at 0.12 +/- 0.04 nmol/10(6) cells. A similar amount of Ca2+ was trapped within the stores when Ca2+ uptake was prev ented by thapsigargin and chelation of Ca2+; at all temperatures betwe en 2 degrees C and 37 degrees C; and after stores had first been loade d with unlabelled Ca2+. Simultaneous addition of inositol 1,4,5-trisph osphate (InsP(3)) and inhibition of Ca2+ uptake reduced the amount of trapped Ca2+ to a level consistent with InsP(3) rapidly and more compl etely emptying a fraction of the stores that could be only partially e mptied by the passive leak. After dilution of the specific activity of the Ca-45(2+) under conditions that maintained the steady-state activ ities of the pumps and leaks, the stores rapidly lost their entire Ca- 45(2+) content. We conclude that the channel responsible for mediating the leak of Ca2+ abruptly closes when the luminal [Ca2+] of the intra cellular stores falls below a critical threshold corresponding to abou t 7% of their steady-state loading. Whereas InsP(3) is capable of comp letely emptying a fraction of the stores, regulation of the passive le ak by luminal [Ca2+] is likely to prevent it from completely emptying them;such regulation may ensure that the many other functions of Ca2within the endoplasmic reticulum are not compromised.