Bidirectional Activity of the Endoplasmic Reticulum Ca2+-ATPase of Bovine Adrenal Cortex

Abstract

It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor play major roles in the complex mechanisms by which agonists increase intracellular Ca2+ concentration. In these mechanisms, the endoplasmic reticulum Ca(2+)-ATPase has been attributed an accessory role of refilling the intracellular Ca2+ store. In the present study, the activity of the microsomal Ca(2+)-ATPase of bovine adrenal cortex was investigated. We show that the Ca(2+)-pumping activity of the Ca(2+)-ATPase is related to the ADP/ATP ratio. Our results also show that a brisk increase of the ADP/ATP ratio upon addition of exogenous ADP triggered a rapid release of Ca2+ from preloaded microsomes. ADP released Ca2+ in a dose-dependent manner with an EC50 of 2.98 +/- 0.78 mM. ADP-induced Ca2+ release was not prevented by heparin, ruling out the participation of the inositol 1,4,5-trisphosphate receptor. ADP-induced Ca2+ release could not be attributed to the mere inhibition of the Ca(2+)-ATPase, since the rate of ADP-induced Ca2+ release was 20 times faster than the rate of Ca2+ release induced by a maximal concentration of thapsigargin (2 microM). ADP-induced Ca2+ release experiments performed in the presence of [32P]PO4 revealed a concomitant production of [32P]ATP. ADP-induced [32P]ATP production was dose-dependent, with an EC50 of 5.50 +/- 0.70 mM. ADP-induced [32P]ATP production was prevented by ionomycin (10 microM) and by high concentrations of extramicrosomal Ca2+. These results demonstrate that the microsomal Ca(2+)-ATPase of adrenal cortex possesses a bidirectional activity that depends on ADP concentrations, the Ca2+ gradient across the microsomal membrane, and probably also ATP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

FK506 Blocks Intracellular Ca2+ Oscillations in Bovine Adrenal Glomerulosa Cells

“…InsP 3 Binding Assay of the Immunoprecipitated InsP 3 Receptor. Bovine adrenal cortex membranes were prepared as previously described (26). Membranes (1 mg of protein) were solubilized, and the InsP 3 receptor was immunoprecipitated as described in the previous section.…”

Synthesis, Acid−Base Behavior, and Binding Properties of 6-Modified myo-Inositol 1,4,5-Tris(phosphate)s

“…Ins(1,4,5)P 3 Derivatives Binding Assay. Bovine adrenal cortex microsomes were prepared as previously described …”

“…Bovine adrenal cortex microsomes were prepared as previously described. 46 Bovine adrenal microsomes were incubated for 30 min at 0 °C in a medium containing 25 mM Tris/HCl buffered at pH 8.5, 100 mM KCl, 20 mM NaCl, 5 mM KH2PO4, and 1 mM EDTA in a final volume of 500 µL with the appropriate concentration of [ 3 H]Ins(1,4,5)P3 (0.9 nM), Ins(1,4,5)P3, and Ins(1,4,5)P3 derivatives. Nonspecific binding was determined in the presence of 1 µM Ins(1,4,5)P3.…”

F‐actin‐based Ca signaling—a critical comparison with the current concept of Ca signaling

“…All hitherto identified TRP type channels, the best characterized and the most likely channel type involved in receptormediated Ca signaling, are not IP 3 -, ryanodine-, cADPribose-, or NAADP-sensitive. Instead, constitutionally open ion channels have been found in Ca-storing microsomal vesicles using the 45-Ca uptake technique showing rapid equilibration with external Ca, ATP, and IP 3 and other large anions via open cation and anion channels (Clairmont et al, 1992;Lange and Brandt, 1993b;Poitras et al, 1995;. Several studies have shown that Ca-storing vesicles of different origin exhibit a constitutionally open influx pathway for Ca, which clearly contradicts the hypothesis of an active ATP-dependent Ca pumping mechanism located in the vesicle membrane (Schmid et al, 1990;Lange and Brandt, 1993b).…”