A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases

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Abstract

Thapsigargin is the most widely used inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca2+-ATPases in mammalian cells. Over the past ten years, this guaianolide compound of plant origin has become a popular tool in a host of studies directed at elucidating the mechanisms of intracellular Ca2+ signalling. Its remarkable potency and selectivity have been instrumental in widening our view of the function of intracellular Ca2+ stores to include such key aspects as store-operated Ca2+ entry or the involvement of the stores in protein synthesis or cell growth. In this article Marek Treiman, Casper Caspersen and Søren Brøgger Christensen review the key pharmacological features of thapsigargin action; they also discuss some of the ways in which its unique properties have shown to be important for obtaining new insights into the biology of Ca2+ stores, and how these properties might encompass a therapeutic potential. In parallel, attention is drawn to some of the limitations and pitfalls encountered when working with thapsigargin.

Section snippets

Potency of thapsigargin towards SERCAs

The basic questions confronting a researcher intending to use thapsigargin include: What potency would the inhibitor be expected to display in the particular system? Can all Ca2+ pumping to the stores be inhibited? If more than one type of intracellular Ca2+-ATPase is involved, is there a possibility of differential inhibition?

The SERCA family comprises products of at least three genes[2]. The occurrence of SERCA1 is restricted to the fast-twitch skeletal muscle fibres. SERCA2a is expressed

Specificity of thapsigargin towards SERCAs

The apparent specificity of thapsigargin towards SERCAs is remarkable. Nevertheless, there is evidence suggesting that caution should be exercised, especially in the micromolar concentration range. For example, thapsigargin in this range appeared to inhibit the store-operated Ca2+ entry[35]. While 5 nm thapsigargin or 500 nm tBHQ depleted the Ins(1,4,5)P3-sensitive stores in GH3 pituitary cells, each of these agents at 5 μm was shown to block the L-type Ca2+ channels[36]. Such observations

Thapsigargin and Ca2+ release from intracellular stores

Release of Ca2+ from intracellular stores by thapsigargin accounts for most of its popularity in the literature. However, it achieves this effect indirectly, as opposed to ionophores such as calcimycin or ionomycin. Inhibition of SERCAs by thapsigargin prevents the pumps from counterbalancing the passive Ca2+ leak from the stores to the cytosol. Thus, the Ca2+-releasing action of thapsigargin depends on two factors simultaneously: the presence of thapsigargin-sensitive pumps and an appreciable

Thapsigargin and a role of SERCAs in generation of Ca2+ signals

One important question concerning SERCAs is whether their role in Ca2+-store-dependent signalling extends beyond refilling the stores after an agonist-mediated Ca2+ discharge. Is a cell's total ER pump capacity regulated in a moment-to-moment manner? Would such regulation affect the generation of Ca2+ signals and the cellular response to an agonist? In the heart, the activity of SERCA2a is regulated by an interaction with phospholamban, a membrane protein whose phosphorylation by cAMP-dependent

Thapsigargin and a role for Ca2+ stores in cell growth

The very tight binding of thapsigargin to the currently known SERCAs results in inhibition being practically irreversible, persisting after dilution[44] or removal of the excess inhibitor. This property of thapsigargin has been used to study the process of pump synthesis and Ca2+-pool reconstitution in smooth-muscle cells following an 18-hour application and subsequent removal of thapsigargin[45]. A close correlation had previously been observed between the activity of the pumps in keeping the

Thapsigargin: a drug lead?

The long-standing interest in the potential medical benefits of Thapsia garganica is attested by writings of Theophrastos (c. 372–287 bc) and by its use in traditional Arabian and European medicine for rheumatic pains, up to its listing in the French Pharmacopoeia of 1937. Modern work using purified thapsigargin uncovered its tumour-promoting action on mouse skin[46]. Following an initial rise, the fraction of mice developing tumours unexpectedly dropped before a much slower rise ensued after

Concluding remarks

Close to 20 years following its isolation[6], thapsigargin has come of age as a potent and remarkably specific tool for studying Ca2+ stores and their SERCAs. Provided that attention is paid to issues such as stoichiometry and irreversibility of binding, the indirect mode of Ca2+-store depletion, and a possibility – never to be discounted – of nonspecific effects, thapsigargin may be considered a highly useful addition to a pharmacologist's toolbox, and one possibly harbouring a therapeutic

Acknowledgements

Research in the authors' laboratory has been supported by The Danish Government Biotechnology Program and The Danish Medical Research Council.

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