Thapsigargin, a tumour-promoting sesquiterpene lactone, selectively inhibits the Ca(2+)-ATPase responsible for Ca2+ accumulation by the endoplasmic reticulum (ER). Mobilization of ER-sequestered Ca2+ to the cytosol and to the extracellular fluid subsequently ensues, with concomitant alteration of cellular functions. Thapsigargin was found to serve as a rapid, potent and efficacious inhibitor of amino acid incorporation in cultured mammalian cells. At concentrations mobilizing cell-associated Ca2+ to the extracellular fluid, thapsigargin provoked extensive inhibition of protein synthesis within 10 min. The inhibition in GH3 pituitary cells involved the synthesis of almost all polypeptides, was not associated with increased cytosolic free Ca2+ concentration ([Ca2+]i), and was not reversed at high extracellular Ca2+. The transient rise in [Ca2+]i triggered by ionomycin was diminished by thapsigargin. Polysomes failed to accumulate in the presence of the drug, indicative of impaired translational initiation. With longer (1-3 h) exposures to thapsigargin, recovery of translational activity was observed accompanied by increased synthesis of the ER protein glucose-regulated stress protein 78 or immunoglobulin heavy-chain binding protein ('GRP78/BiP') and its mRNA. Such inductions were comparable with those observed previously with Ca2+ ionophores which mobilize the cation from all intracellular sequestered sites. Actin mRNA concentrations declined significantly during such treatments. In HepG2 cells processing and secretion of the glycoprotein alpha 1-antitrypsin were rapidly suppressed by thapsigargin. Ca2+ sequestered specifically by the ER is concluded to be essential for optimal protein synthesis and processing. These rapid effects of thapsigargin on mRNA translation, protein processing and gene expression should be considered when evaluating potential mechanisms by which this tumour promoter influences cellular events.

Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells

http://www.jbc.org/content/267/25/17624.full.pdf

Thapsigargin activates a calcium influx pathway in the unfertilized mouse egg and suppresses repetitive calcium transients in the fertilized egg.

1. The interactions between IP3 receptor-mediated and Ca(2+)-induced Ca2+ release were investigated in cerebellar granule cell bodies, using the techniques of microfluorimetry and image analysis. 2. The IP3-sensitive Ca2+ release mechanism was activated using acetylcholine (ACh) and the selective metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD). Caffeine was used to activate, and ryanodine to inhibit, the Ca(2+)-induced Ca2+ release process. Thapsigargin was used to deplete intracellular Ca2+ stores. 3. Transient applications of caffeine (5-50 mM), ACPD (50-500 microM) and ACh (0.05-1 microM) mobilized intracellular Ca2+ ([Ca2+]i). Ca2+ mobilizing responses to 50 mM caffeine and 1 microM ACh increased with time in culture until day 4. However, beyond this period the responsiveness of cells to caffeine, but not to ACh, declined markedly. 4. Responses induced by ACPD and ACh were inhibited in the presence of caffeine at concentrations below those which mobilized Ca2+ (1-5 mM). This effect was not due to Ca2+ pool depletion, elevation of cAMP or inhibition of phosphodiesterases. 5. Prior challenge with ACh or ACPD inhibited Ca2+ mobilization induced by caffeine (50 mM). Transient exposure to caffeine inhibited subsequent responses to ACh through a mechanism which involved store depletion. 6. Thapsigargin (0.1-1 microM) inhibited, to a similar extent, Ca2+ mobilization induced by caffeine, ACPD and ACh. 7. Ryanodine (10 microM) antagonized Ca2+ mobilization induced by caffeine, ACh and ACPD. However, the ability of ryanodine to block inositol 1,4,5-trisphosphate-linked agonist responses varied considerably between cells. The sensitivity of ACh-induced responses to ryanodine correlated with the sensitivity of the cells to caffeine. 8. The possible explanations for the pronounced interactions between IP3 receptor-mediated and Ca(2+)-induced Ca2+ release processes in cerebellar granule cells are discussed.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1992.sp019360

Interactions between Ca2+ mobilizing mechanisms in cultured rat cerebellar granule cells

Intracellular Ca2+ pools in PC12 cells. A unique, rapidly exchanging pool is sensitive to both inositol 1,4,5-trisphosphate and caffeine-ryanodine.

Ca2+ extrusion across plasma membrane and Ca2+ uptake by intracellular stores.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.

/pdf/thapsigargin-but-not-caffeine-blocks-the-ability-of-2v6wla00oc.pdf

Thapsigargin, but not caffeine, blocks the ability of thyrotropin-releasing hormone to release Ca2+ from an intracellular store in GH4C1 pituitary cells.

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Thapsigargin, but not caffeine, blocks the ability of thyrotropin-releasing hormone to release Ca2+ from an intracellular store in GH4C1 pituitary cells.