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Abstract

In permeabilized parotid cells and in isolated membrane vesicles from parotid endoplasmic reticulum (ER), Mg-ATP-dependent Ca²⁺ uptake was measured using a Ca²⁺-specific macroelectrode and ⁴⁵Ca²⁺, respectively. Mg-ATP-dependent Ca²⁺ uptake was inhibited by vanadate (2 x 10^-3 mol/l) by approximately 45% in permeabilized cells and by approximately 70% in membrane vesicles from ER during the initial 10 min. After this lag phase, Ca²⁺ uptake increased and low steady-state free [Ca²⁺] of approximately 3 x 10^-7 mol/l was still reached in presence of vanadate within 30-40 min. Subsequent addition of inositol 1,4,5-trisphosphate (IP₃) caused a similar Ca²⁺ release compared with control. This indicates that in presence of vanadate an IP3-sensitive Ca²⁺ pool was filled. However, when protonophores, such as nigericin or carbonyl cyanide-m-chlorophenylhydrazone, were added in addition to vanadate, this low steady-state free [Ca²⁺] was not reached. ⁴⁵Ca²⁺ uptake was reduced by approximately 70% within 60 min, and IP₃ did not cause ⁴⁵Ca²⁺ release when given subsequently, indicating that filling of an IP₃-sensitive Ca²⁺ pool was prevented. Mg-ATP-driven H⁺ uptake into ER vesicles, as estimated with acridine orange, was abolished by protonophores and by the H⁺-ATPase blockers N-ethylmaleimide and Dio 9 but was unaltered by vanadate. Preincubation of ER vesicles in a medium without Ca²⁺, but with vanadate and with Mg-ATP to generate an H⁺ gradient, allowed demonstration of ⁴⁵Ca²⁺ uptake from a medium that did not contain ATP. The cation sequence in absence of vanadate for support of Mg-ATP-dependent ⁴⁵Ca²⁺uptake was K+⁺ greater than Na⁺ greater than Li⁺ = choline⁺ and, in presence of vanadate, was choline⁺ greater than Li⁺ = Na⁺ greater than K⁺. A preformed H⁺ gradient dissipated more rapidly in presence of K⁺ compared with choline⁺, probably due to an intrinsic K⁺ permeability of ER membrane. Our data indicate that both a Ca²⁺ and a H⁺ pump are located in a compartment of ER that is also sensitive to IP₃. Ca²⁺ uptake is coupled to an H⁺ gradient that is generated by the H⁺ pump and most likely occurs via Mg-ATP-driven Ca²⁺-H⁺ countertransport but to some extent can also operate in absence of ATP at the expense of the H⁺ gradient.