The PI-PLC inhibitor U-73122 is a potent inhibitor of the SERCA pump in smooth muscle

Abstract

In this issue MacMillan and McCarron in 2010 demonstrated that the phospholipase C (PLC) inhibitor U-73122 can potently inhibit Ca²⁺ release from isolated smooth muscle cells independent of its effect on PLC. Their data suggest that the PLC inhibitor can block the sarcoplasmic/endoplasmic reticulum calcium ATPase pump in smooth muscle and cast doubt on the reliability of U-73122 as the main pharmacological tool to assess the role of the phosphotidyl inositol-PLC pathway in cellular signalling.

The initial discovery of the N-aminosteroid homologue of N-ethylmaleimide, U-73122, as an inhibitor of phospholipase C (PLC) by Bleasdale et al. (1990) and Smith et al. (1990), provided a tool to help assess the contribution of PLC to cellular signalling pathways in a variety of cell types. The data presented in their early papers demonstrated that U-73122 could reduce thrombin-induced inositol trisphosphate (IP₃) production in platelets and polymorphonuclear neutrophils. This inhibitory effect of U-73122 appeared to be dependent on the presence of a pyrroledione group, as replacement of this with pyrrolidinedione (to form U-73343) abolished the inhibitory effects of the molecule on IP₃ synthesis and Ca²⁺ release.

In the 20 years since the discovery of this molecule, approximately 2000 papers have been published, where the effects of U-73122 have been attributed to its inhibitory effects on PLC in a variety of cell types including smooth muscle (Ellershaw et al., 2002), interstitial cells of Cajal (Kim et al., 2003; Johnston et al., 2005) and pancreatic acinar cells (Yule and Williams, 1992). In the current issue, MacMillan and McCarron (2010) suggest that U-73122 interferes with Ca²⁺ handling in smooth muscle independent of an effect on PLC. Their data call into question the reliability of this molecule when used alone as a tool to investigate the contribution of PLC to cellular signalling.

In their study MacMillan and McCarron (2010) attempted to establish the contribution of IP₃ receptors to the propagation of agonist-evoked Ca²⁺ waves in guinea-pig, freshly dispersed colonic smooth muscle cells. They found that U-73122 abolished the excitatory effects of exogenous carbachol, consistent with the idea that IP₃ synthesis was inhibited via blockade of PLC. To test that these effects were attributable to an effect on PLC, the authors examined the effects of U-71322 on Ca²⁺ transients, which do not involve PLC activation, by either photo releasing caged IP₃ or by evoking Ca²⁺ release from ryanodine receptors using caffeine. Surprisingly, U-73122 abolished Ca²⁺ oscillations induced by both protocols, strongly suggesting that U-71322 can inhibit Ca²⁺ oscillations by a mechanism that does not involve PLC. The authors noted that the effects of U-71322 (such as reduced amplitude and rate of decay of the caffeine or IP₃-evoked Ca²⁺ transients, as well as elevated basal Ca²⁺ levels) were remarkably similar to the effect of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump inhibitor CPA. Therefore, this study suggests that U-73122 (but not its ‘inactive’ analogue, U-73343) is a potent inhibitor of the SERCA pump in smooth muscle.

These observations may help to explain one of the discrepancies in the original study (Bleasdale et al., 1990), which showed that U-71322 was able to inhibit agonist responses at concentrations lower than that needed to inhibit IP₃ production. For example, Ca²⁺ transients evoked by the thromboxane receptor agonist U-46619 were abolished by 2 µM U-73122 whereas IP₃ production was only inhibited by 50% in the presence of 10 µM U-73122.

Several other studies have suggested that U-73122 has effects unrelated to the inhibition of PLC including the depletion of intracellular stores in PC12 cells (Clementi et al., 1992), potentiation of IP₃-mediated Ca²⁺ release and direct stimulation of cation channels in excised patches from murine pancreatic acinar cells (Mogami et al., 1997). Taken together, the results of MacMillan and McCarron (2010) suggest that great care should be taken in the interpretation of experiments that use U-73122 as the primary pharmacological tool to assess the contribution of the phosphotidyl inositol-PLC pathway in cells.

Glossary

Abbreviations:

IP₃

inositol trisphosphate

PLC

phospholipase C

PI-PLC

phosphotidyl inositol-phospholipase C

SERCA

sarcoplasmic/endoplasmic reticulum calcium ATPase