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

Abstract

We studied the effects of thapsigargin on the formation of the phosphorylated intermediates (E~Ps) of endoplasmic reticulum Ca2÷-ATPases in microsomes from bovine adrenal medulla. When submicrosomal fractions were separated on a sucrose gradient, two components of 100 kDa Ca2÷-ATPase E~P displaying distinct subeellular distributions were resolved. The first component was defined by Ca2÷-induced protection against thapsigargin inhibition. The second component did not display such protection, with a 3 orders of magnitude difference in thapsigargin inhibitory potency towards the 2 components. In the absence of Ca 2+, both E~P components were highly sensitive to thapsigargin inhibition, revealing the presence of high-affinity thapsigargio-binding sites characteristic of SERCA ATPases. These data demonstrate a new level of molecular heterogeneity among Ca2+-ATPases of endoplasmic reticulum, and provide the first evidence of differential subcellular localization of individual Ca 2+ pump subtypes in cells of neural origin.

cAMP-dependent protein kinase phosphorylates and activates nuclear Ca 2+ -ATPase

“…This finding suggests that it corresponds to the formation of a high-energy phosphoenzyme intermediate (36). Furthermore, it was abolished after pretreatment with thapsigargin, confirming that it corresponds to a SERCA-type enzyme (37).…”

“…SERCA2b is the ubiquitous ''housekeeping'' isoform found in the ER of all tissues (38). A heterogeneity of SERCA2b-type Ca 2ϩ -ATPases has been observed with respect to thapsigargin sensitivity (37,39). The NCA phosphoenzyme intermediate is found to be thapsigargin sensitive, which is analogous to the thapsigargin-sensitive 100-kDa SERCA phosphorylated intermediate described in bovine adrenal chromaffin cells.…”

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

“…Distinction among individual ER cisternae had therefore not been possible. Immunocytochemistry and subcellular fractionation studies, carried out in many nonmuscle cell types Volpe et al, 1991;Sharp et al, 1992;Villa et al, 1993;Lievremont et al, 1994;Caspersen and Treiman, 1995) including PC12 (Rooney and Meldolesi, 1996), documented heterogeneities in the distribution of various ER proteins participating in Ca2+ homeostasis, including pumps and channels (see Pozzan et al, 1994). Whether such molecular heterogeneities give rise to heterogeneous distributions of calcium within the ER had however not been established.…”

Pharmacological Modulation of Sarcoplasmic Reticulum Function in Smooth Muscle

“…However, its potency is quite variable across experimental systems (IC 50 values ranging from subnanomolar to micromolar) . Since the interaction between SERCA1 and thapsigargin is stoichiometric (Lytton et al, 1991;, it would follow that, if this relationship is also applied to the other SERCA isoforms, the apparent inhibitory potency of thapsigargin would be influenced by the number of SERCA molecules per cell or per microgram of microsomal preparation, as shown in various preparations (Papp et al, 1991;Caspersen and Treiman, 1995;Hussain et al, 1995). Likewise, as for other highly lipophilic drugs, thapsigargin interaction with SERCA should be highly dependent on the ratio (lipid ϩ SERCA protein)/thapsigargin (Heirwegh et al, 1988).…”