Physiological concentrations of inorganic phosphate affect MgATP-dependent Ca²⁺ storage and inositol trisphosphate-induced Ca²⁺ efflux in microsomal vesicles from non-hepatic cells

1. MgATP-dependent ⁴⁵Ca²⁺ uptake by microsomes obtained from various non-hepatic tissues, namely rat brain, rat solid Morris hepatoma 3924A and human platelets, was measured in the presence of P(i) at low, cytosol-like, concentrations. 2. Increasing P(i) concentrations (0.5-3 mM) caused a progressive enlargement of the ⁴⁵Ca²⁺-storage capacity of all the microsomal fractions. 3. As a result of P(i) stimulation of Ca²⁺ uptake, ⁴⁵Ca²⁺ and [³²P]P(i) were co-accumulated by the three microsomal fractions. 4. The time course for ⁴⁵Ca²⁺ and [³²P]P(i) accumulation in brain microsomes revealed a biphasic ⁴⁵Ca²⁺ uptake: a rapid phase was followed by a second, slower, phase, which depended on the presence of P(i). During the P(i)-dependent phase, the uptake of ⁴⁵Ca²⁺ was paralleled by the uptake of [³²P]P(i). 5. The passive efflux of Ca²⁺ was paralleled by the efflux of P(i) and vice versa. In fact, the inhibition of active Ca²⁺ uptake by excess EGTA, or lowering the P(i) concentration of the incubation system by dilution, caused the release of ⁴⁵Ca²⁺ and [³²P]P(i) from ⁴⁵Ca²⁺ or [³²P]P(i) pre-loaded brain microsomes. The Ca²⁺ ionophore A23187 also released ⁴⁵Ca²⁺ and [³²P]P(i). 6. Ca²⁺ efflux by A23187 was rapid (t( 1/2 ) approx. 2 s) and independent of the extent of intravesicular Ca²⁺ loading, which indicates that Ca²⁺ and P(i) do not form intravesicular insoluble complexes. 7. The progressive increase in Ca²⁺ accumulation, depending on P(i) stimulation, resulted in a proportional increase in the amount of Ca²⁺ releasable by InsP₃ in the three non-hepatic microsomal fractions and in digitonin-permeabilized platelets. 8. Concomitantly to Ca²⁺ microsomal P(i) was also released by InsP₃.