Thapsigargin-sensitive Ca2+-ATPases account for Ca2+ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca2+ stores in adrenal chromaffin cells

Abstract

In chromaffin cells of adrenal medulla, heterogeneity of Ca2+ stores has been suggested with respect to the mechanisms of Ca2+ uptake and release. We have examined Ca(2+)-ATPases responsible for loading of Ca2+ stores in these cells for their sensitivity to thapsigargin, a highly selective inhibitor of the SERCA [sarco(endo)plasmic reticulum calcium ATPase] family of intracellular Ca2+ pumps. Using immunostaining, we studied the distribution of Ca(2+)-ATPases, and of receptors for inositol 1,4,5-trisphosphate (InsP3) and ryanodine, in the density-gradient fractions of microsomes from bovine adrenal medulla. In parallel, we examined distribution profiles of ATP-dependent Ca2+ uptake in the same fractions, along with subcellular markers for plasma membranes and endoplasmic reticulum (ER). Two Ca(2+)-ATPase-like proteins (116 and 100 kDa) were detected, consistent with the presence of SERCA 2b and SERCA 3 isoenzymes of Ca2+ pumps. The distribution of these putative Ca(2+)-ATPase iso-enzymes paralleled that of InsP3 and ryanodine receptors. This distribution of ER Ca(2+)-ATPases, as determined immunologically, was consistent with that of thapsigargin-sensitive, but not of thapsigargin-insensitive, ATP-dependent Ca2+ uptake. In contrast, the distribution profile of the thapsigargin-insensitive Ca2+ uptake was strongly correlated to that of plasma membranes, and co-distributed with plasma membrane Ca(2+)-ATPase detected immunologically. In isolated, permeabilized chromaffin cells, InsP3 and caffeine induced Ca2+ release following an ATP-dependent Ca2+ accumulation to the stores. This accumulation was abolished by thapsigargin. Together, these data strongly indicate that the thapsigargin-sensitive, presumably SERCA-type Ca(2+)-ATPases account for Ca2+ uptake to InsP3-sensitive, as well as to caffeine-sensitive, Ca2+ stores in bovine adrenal chromaffin cells.

Thapsigargin discriminates strongly between Ca2+‐ATPase phosphorylated intermediates with different subcellular distributions in bovine adrenal chromaffin cells

“…Since the absence of a phosphorylated protein at 116 kDa allowed a more accurate measurement of the radioactivity selectively associated with the 100 kDa E~P, we routinely omitted Mg 2÷ in the further analysis of the 100 kDa phosphorylated intermediate. The distribution of the 100 kDa E~P in the submicrosomal fractions was analyzed using the isopycnic, discontinuous sucrose gradient characterized recently [12]. The distribution was broad, indicating the presence of Ca2+-ATPase in ER subcompartments spanning different buoyant densities ( Fig.…”

“…We studied the formation of Ca2*-ATPase phosphorylated intermediates (E ~ P) [15,16] in a preparation ofmicrosomes from bovine adrenal medulla described earlier [12,13]. The microsomes were incubated with [~'-32p]ATP, followed by PAGE in an acid gel system and autoradiography.…”

“…SDS polyacrylamide gel electrophoresis (SDS PAGE) under acid conditions (to preserve Ca2+-ATPase E~P) and immunoblotting were carried out as described earlier [12,13]. After staining and drying, the gels were autoradiographed on the ECL Hyperfilm (Amersham).…”

“…Preparation of bovine adrenal medulla microsomes and submicrosomal fractions, and isolation of chromaffin cells were performed as described earlier [12].…”

Organelles Containing Inositol Trisphosphate Receptor Type 2 in Adrenal Medullary Cells

“…Furthermore, BODIPY-FL-thapsigargin -binding sites coincided with those labeled with the anti-calnexin Ab. We [23] and others [34] reported that thapsigargin depleted Ca 2+ ions from store sites, from which Ca 2+ mobilization occurred in response to InsP 3 -generating receptor agonists. Thus the coincidence of BODIPY-FL-thapsigargin-binding sites with calnexin-like immunoreactive sites is consistent with the notion that the ER alone is responsible for InsP 3 -sensitive store sites in chromaffin cells.…”