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. This corresponds well with contractility studies which report similar results. These pharmacological tools have also demonstrated the presence of multiple Ca2+ stores. Studies in vascular and ileal smooth muscle have shown that CPA blocked repletion of phenylephrine-, inositol 1,4,5-trisphosphate (IP3)- and caffeine-sensitive Ca2+ stores. On the other hand, TSG completely blocked repletion of IP3-sensitive Ca2+ stores but not caffeine-sensitive Ca2+ stores. These selective SR Ca2+ pump inhibitors will prove to be useful in the study of Ca2+ entry and Ca2+ stores in smooth muscle.

Keywords:
Thapsigargin, Cyclopiazonic acid, 2,5-Di-(tert-butyl)-1,4-benzohydroquinone, Smooth muscle, Ca2+ pumps, Sarcoplasmic reticulum
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© 1993 S. Karger AG, Basel
1994
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