abstract
- The study of the relative roles of the plasma membrane (PM) and the sarcoplasmic reticulum (SR) Ca2+ pumps in the regulation of internal Ca2+ has been limited in the past due in part to the lack of selective inhibitors for either Ca2+ pump. Recently, three compounds have been discovered which appear to be selective SR Ca2+ pump inhibitors: thapsigargin (TSG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl)-1,4-benzohydroquinone. Contractility studies in various smooth muscle tissues have demonstrated that all three compounds inhibit repletion of the intracellular Ca2+ store, presumably due to inhibition of the SR Ca2+ pump. These functional studies however provided only indirect evidence for Ca2+ pump inhibition. Using the microsomal membrane fraction isolated from the smooth muscle of rat vas deferens, we investigated the effects of CPA and TSG of ATP-dependent Ca2+ uptake in order to obtain direct evidence about the mechanism of action of CPA and TSG on smooth muscle Ca2+ pumps. CPA and TSG potently inhibited oxalate-stimulated Ca2+ uptake in a concentration-dependent manner. Since oxalate-stimulated Ca2+ uptake is generally considered to be a property of the SR and not the PM Ca2+ pump, this directly demonstrates that CPA and TSG act by inhibiting the SR Ca2+ pump. We also demonstrated that inhibition of Ca2+ uptake in the presence of oxalate by CPA could be completely reversed while the effects of TSG could only be partially reversed.(ABSTRACT TRUNCATED AT 250 WORDS)