The phospholipase C inhibitor U-73122 inhibits Ca²⁺ release from the intracellular sarcoplasmic reticulum Ca²⁺ store by inhibiting Ca²⁺ pumps in smooth muscle

Introduction

The cytosolic Ca²⁺ concentration ([Ca²⁺]_cyto) is critically regulated by the intracellular store (the sarcoplasmic reticulum, SR), which controls Ca²⁺ release (Bootman et al., 2001; McCarron et al., 2006). Two major routes of Ca²⁺ release from the SR exist. The first is the inositol 1,4,5-trisphosphate receptor (IP₃R), the other, the ryanodine receptor (RyR; nomenclature follows Alexander et al., 2009). In several cell types such as smooth muscle and non-excitable cells (e.g. epithelial cells and fibroblasts) IP₃R is the predominant Ca²⁺ release mechanism. IP₃Rs are activated by IP₃ generated via G-protein- or tyrosine kinase-linked receptor-dependent activation of phosphoinositide-specific phospholipase C (PI-PLC) (Bootman et al., 2001). Activation of PI-PLC catalyses the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) to generate diacylglycerol and IP₃. IP₃ subsequently releases Ca²⁺ from the SR to generate a local Ca²⁺ rise, which may itself induce further synthesis of IP₃ via a Ca²⁺-dependent positive feedback on PI-PLC (Bartlett et al., 2005). Central therefore to an understanding of the role of IP₃Rs in Ca²⁺ regulation and wave propagation is an appreciation of the control of the PI-PLC/IP₃ signalling pathway.

Investigation of the PI-PLC pathway in agonist-evoked Ca²⁺ signals has been aided by the development of specific, membrane-permeant, activators and inhibitors. Among several, U-73122 has been established as a selective pharmacological inhibitor of PI-PLC (Bleasdale et al., 1990; Smith et al., 1990) and is used widely. U-73122 inhibited PI hydrolysis and IP₃ synthesis in broken cell systems and reduced agonist-evoked [Ca²⁺]_cyto rises in intact cells, such as neutrophils (Bleasdale et al., 1990; Smith et al., 1990), neuroblastoma cells (Thompson et al., 1991), acinar cells (Yule and Williams, 1992) and platelets (Bleasdale et al., 1990). U-73122 has thus gained general acceptance as a specific PI-PLC inhibitor and inhibition of [Ca²⁺]_cyto rises in intact cells by U-73122 has been interpreted as evidence for a contribution of PI-PLC in the response in a number of studies (Yule and Williams, 1992; Hansen et al., 1995; Heemskerk et al., 1997), including smooth muscle (Zizzo et al., 2008; Frei et al., 2009).

In the present study, which examined the role of PI-PLC in IP₃-mediated Ca²⁺ regulation in smooth muscle, U-73122 potently inhibited IP₃-mediated Ca²⁺ release independently of PI-PLC activity.

Methods

Results

Phosphoinositide-specific phospholipase C is a key enzyme in the regulation of IP₃-mediated Ca²⁺ release from the SR. To clarify the role of PI-PLC in IP₃R-mediated Ca²⁺ regulation, the effect of the PI-PLC inhibitor, U-73122, on the IP₃-generating agonist CCh, was examined in voltage-clamped, single colonic myocytes. In these experiments, CCh was applied in a Ca²⁺-free bath solution to ensure that [Ca²⁺]_cyto rises derived from Ca²⁺ release from the SR. The IP₃-generating muscarinic agonist CCh (100 µM) reproducibly increased [Ca²⁺]_cyto (Figure 1). U-73122 (10 µM; Figure 1) significantly (P < 0.05) inhibited CCh-evoked [Ca²⁺]_cyto increases (ΔF/F₀) by 90 ± 3% [from 2.24 ± 0.4 to 0.27 ± 0.1 (n = 5)], a result that appears consistent with a requirement for PI-PLC generation of IP₃.

Unexpectedly, in subsequent experiments, U-73122 appeared to inhibit IP₃-mediated Ca²⁺ release independently of PI-PLC. In these experiments the signal transduction pathway that mediates IP₃ synthesis, that is, PI-PLC, was bypassed by photolysing caged IP₃. U-73122 (1 µM, Figure 2A) significantly (P < 0.05) decreased the IP₃-evoked [Ca²⁺]_cyto transient (ΔF/F₀) by 76 ± 6% from 2.78 ± 0.5 to 0.59 ± 0.1 (n = 5). U-73122 (10 µM, Figure 2B) significantly (P < 0.05) decreased the IP₃-evoked [Ca²⁺]_cyto transient (ΔF/F₀) by 91 ± 2% from 2.44 ± 0.4 to 0.24 ± 0.1 (n = 5). Interestingly, the inactive analogue, U-73343 (10 µM) (Bleasdale et al., 1990; Smith et al., 1990) used often as a control compound for U-73122, did not significantly (P > 0.05) alter IP₃-mediated [Ca²⁺]_cyto increases evoked by photolysis of caged IP₃. The [Ca²⁺]_cyto increase remained at 105 ± 9% of the control response [ΔF/F₀ from 1.68 ± 0.2 to 1.71 ± 0.2 (Figure 3, n = 5)]. As the [Ca²⁺]_cyto increase evoked by photolysis of caged IP₃ does not require IP₃ synthesis, these results suggest that U-73122-evoked inhibition of Ca ²⁺ release was not mediated by inhibition of PI-PLC activity.

The inhibition of IP₃-mediated Ca²⁺ release by U-73122 may arise from either a direct effect of the drug on IP₃-mediated Ca²⁺ release or indirectly via a reduction in the store's Ca²⁺ content. To distinguish between these possibilities, the effect of U-73122 on RyR-mediated Ca²⁺ release was examined. If the reduction in IP₃R-mediated Ca²⁺ release arose by depletion of the store of Ca²⁺ then U-73122 should also inhibit RyR-mediated Ca²⁺ release because both IP₃R and RyR access a single common Ca²⁺ store in this smooth muscle preparation (McCarron and Olson, 2008). U-73122 (10 µM, Figure 4A) significantly (P < 0.05) decreased the caffeine-evoked [Ca²⁺]_cyto transient ΔF/F₀ by 73 ± 11% from 1.69 ± 0.3 to 0.37 ± 0.1 (n = 5). Thus, U-73122 appears to have a general inhibitory effect on Ca²⁺ release from the intracellular SR Ca²⁺ store. Again the inactive analogue, U-73343 (10 µM), was without effect on caffeine-evoked Ca²⁺ release [(ΔF/F₀) from 2.05 ± 0.2 to 1.96 ± 0.2 (Figure 4B, n = 6, P > 0.05)]. These results suggest that the decrease in IP₃R- and RyR-mediated Ca²⁺ release is likely to be explained by a reduction in the store's Ca²⁺ content perhaps by U-73122's inhibition of the SR Ca²⁺ pump and that the control compound, U-73343, is without effect on the pump.

In support of the proposal that U-73122 inhibited the SR Ca²⁺ pump, an increase in steady-state [Ca²⁺]_cyto (measured as fluorescence) was observed following either 1 µM U-73122 [(F/F₀) from 1.26 ± 0.1 to 1.57 ± 0.2 (Figure 2A, n = 5, P < 0.05)] or 10 µM U-73122 [(F/F₀) from 1.04 ± 0.1 to 1.37 ± 0.1 (Figure 2B, n = 10, P < 0.05)]. Inhibition of the SR Ca²⁺ pump is known to increase steady-state [Ca²⁺]_cyto (Bradley et al., 2002). The SR Ca²⁺ pump inhibitor CPA was without effect on steady-state [Ca²⁺]_cyto following U-73122. In these experiments U-73122 elevated steady-state [Ca²⁺]_cyto and inhibited caffeine-evoked Ca²⁺ release (Figure 5A) after which CPA (10 µM) failed to further increase steady-state [Ca²⁺]_cyto[(F/F₀) from 1.19 ± 0.03 (U-73122) to 1.2 ± 0.04 (CPA, n = 4, P > 0.05)]. Similarly, CPA (10 µM) decreased the caffeine-evoked [Ca²⁺]_cyto transients and increased steady-state [Ca²⁺]_cyto (Figure 5B) after which U-73122 evoked no further increase in steady-state [Ca²⁺]_cyto[(F/F₀) from 1.21 ± 0.1 (CPA) to 1.19 ± 0.1 (U-73122, n = 3, P > 0.05)]. These results suggest that U-73122 and CPA each alter Ca²⁺ signals via a common mechanism.

Further support for a U-73122-evoked inhibition of the SR Ca²⁺ pump was found in the observation that the drug significantly slowed the rate of Ca²⁺ removal from the cytoplasm following each of IP₃- or caffeine-evoked Ca²⁺ release (Figures 2C and 4C). The 80–20% decay interval following IP₃- and caffeine-evoked Ca²⁺ release was 3.5 ± 0.7 s and 2.9 ± 0.3 s in controls and 5.9 ± 0.9 s and 4.7 ± 0.4 s in U-73122 (n = 10 and 5, P < 0.05) respectively. Taken together these data imply that U-73122 reduced the SR Ca²⁺ store content by inhibiting SR Ca²⁺ pump activity, in addition to inhibition of PI-PLC.

Discussion and conclusions

In the present study, our initial experiments found that U-73122 inhibited CCh-evoked [Ca²⁺]_cyto increases, a result that appeared consistent with the proposed mechanism of action of U-73122, that is, inhibiting PI-PLC activity (Bleasdale et al., 1990; Thompson et al., 1991). However, in subsequent experiments, U-73122 also inhibited Ca²⁺ release evoked by photolysed caged IP₃ and hydrostatically applied caffeine. Inhibition of PI-PLC activity is unlikely to account for the inhibition of SR Ca²⁺ release evoked by either photolysed caged IP₃ or caffeine as the former mechanism does not use the signal transduction pathway that mediates IP₃ synthesis and the latter's mechanism of action is independent of PI hydrolysis and IP₃ synthesis. A direct selective inhibitory effect of U-73122 on IP₃R is also unlikely as RyR-mediated Ca²⁺ release was also reduced by U-73122. The decrease in IP₃R- and RyR-mediated Ca²⁺ release can be explained by a reduction in the store's Ca²⁺ content by U-73122's inhibition of the SR Ca²⁺ pump; IP₃R and RyR access a common Ca²⁺ store in this smooth muscle type (McCarron and Olson, 2008). Support for this proposal is found in the observation that the SR Ca²⁺ pump inhibitor, CPA, failed to increase steady-state [Ca²⁺]_cyto after U-73122. U-73122 also failed to increase steady-state [Ca²⁺]_cyto after CPA. These experiments suggest that CPA and U-73122 have a common mechanism of action, that is, SR Ca²⁺ pump inhibition. U-73122 also slowed the rate of Ca²⁺ removal from the cytoplasm, an effect predictable from inhibition of the SR Ca²⁺ pump. Together the results are consistent with U-73122 inhibiting the SR Ca²⁺ pump to reduce IP₃R- and RyR-mediated Ca²⁺ release.

U-73122 is reported to specifically inhibit PI-PLC activity (Bleasdale et al., 1990; Smith et al., 1990). However, a discrepancy between the concentration of U-73122 required to inhibit PI-PLC and those inhibiting Ca²⁺ release was also observed in these initial reports suggesting that U-73122 specifically inhibited PI-PLC activity (Bleasdale et al., 1990; Smith et al., 1990). Importantly, higher concentrations of U-73122 are required to suppress enzyme activity than to inhibit agonist-induced Ca²⁺ release. For example, U-73122 inhibited IP₃-mediated Ca²⁺ release at concentrations of 1–2 µM, while the production of IP₃, used as an indicator of PI-PLC activity, may only be significantly reduced at higher concentrations, for example 10 µM (Alter et al., 1994; Hellberg et al., 1996; Pulcinelli et al., 1998). In platelets, the activity of PI-PLC was resistant to U-73122 up to 50 µM despite the drug inhibiting agonist-induced Ca²⁺ increases at much lower concentrations, for example 1 µM (Muto et al., 1997; Pulcinelli et al., 1998). The reported IC₅₀ value of U-73122 for PI-PLC inhibition was 40 µM whereas that for inhibiting the Ca²⁺ transient was 1 µM (Bleasdale et al., 1990). The results in the present study in smooth muscle show that U-73122 inhibited SR Ca²⁺ release at a concentration (1 µM) up to 50-fold lower than concentrations reported necessary to inhibit PI-PLC activity in other cell types (Smith et al., 1990; Muto et al., 1997; Pulcinelli et al., 1998; Hou et al., 2004).

The present findings in colonic smooth muscle are in agreement with other observations that suggest U-73122 may inhibit SR Ca²⁺ release independently of PI-PLC inhibition (Berven and Barritt, 1995; Grierson and Meldolesi, 1995; Hellberg et al., 1996; Pulcinelli et al., 1998). Inhibition of SR Ca²⁺ pump activity in liver microsomes and hepatocytes (De Moel et al., 1995) and emptying of the Ca²⁺ stores in PC12 cells by U-73122 have been reported (Clementi et al., 1992; Grierson and Meldolesi, 1995). U-73122 has also been shown to increase steady-state [Ca²⁺]_cyto (Smallridge et al., 1992) and to slow the rate of recovery of Ca²⁺ to resting levels following release (Grierson and Meldolesi, 1995) in other preparations, although the latter effect was attributed to PI-PLC inhibition (Grierson and Meldolesi, 1995). U-73122 has also been shown to inhibit Ca²⁺ release from RyR in the form of Ca²⁺ sparks and via IP₃R as Ca²⁺ puffs (Bayguinov et al., 2000; Liu et al., 2007).

The present results are consistent with inhibition of the SR Ca²⁺ pump providing a mechanism by which U-73122 inhibits IP₃-mediated Ca²⁺ release. In some experiments inhibition of IP₃-mediated Ca²⁺ release occurred before an increase in steady-state [Ca²⁺]_cyto was decreased. The latter is a common feature of SR Ca²⁺ pump inhibition. A slow Ca²⁺ leak from the SR by Ca²⁺ pump inhibition may have been initially compensated by Ca²⁺ extrusion from the cytoplasm via the plasma membrane Ca²⁺ pumps so that little change in resting [Ca²⁺]_cyto occurs although the SR Ca²⁺ content has begun to decline (see Figure 2). The reduced SR Ca²⁺ content was apparent when the cell was activated by, for example, IP₃ and so an inhibition of IP₃-mediated Ca²⁺ release occurred prior to an increase in steady-state [Ca²⁺]_cyto. As Ca²⁺ is repeatedly released from the store but not subsequently returned via the SR Ca²⁺ pumps (because of inhibition by U-73122), increased steady-state [Ca²⁺]_cyto levels result. Although a direct inhibitory effect of U-73122 on either IP₃R or RyR cannot be fully excluded, reduced IP₃R- and RyR-mediated Ca²⁺ release, an increase in steady-state [Ca²⁺]_cyto and a slowed rate of Ca²⁺ removal are each consistent with SR Ca²⁺ pump inhibition.

In conclusion, U-73122 inhibits SR Ca²⁺ release independently of PI-PLC and via inhibition of the SR Ca²⁺ pump. This additional effect of U-73122 on Ca²⁺ signalling may complicate interpretation of results when the compound is used to define PI-PLC function.

Statement of conflicts of interest

None.