We have shown that the sarco/endoplasmic reticulum calciumATPase inhibitor thapsigargin (Tg) is able to activate both store-operated calcium entry (SOCE) and RhoKinase-mediated calcium sensitisation in the mouse anococcygeus, and we suggested that intracellular calcium store depletion might act as a trigger for activation of the calcium sensitisation pathway (Ayman et al, 2003). Calcium-independent phospholipase A₂ (iPLA₂) has been reported to be activated by calcium store depletion (Wolf et al, 1997) and the consequential rise in arachidonic acid to produce calcium sensitisation (Guo et al, 2003), offering a link between these two key processes. We have therefore studied the effects of the iPLA₂ inhibitor, bromoenol lactone (BEL; Hazen et al, 1991)) on contractile responses to Tg and carbachol (CCh) in the mouse anococcygeus. Muscles from male mice (LACA; 25-35g) were set up for the recording of isometric tension as described previously (Ayman et al, 2003). Results are presented as mean ± SEM for at least 4 experiments. CCh concentration response curves were normalized with respect to the maximum response to CCh under control conditions. Statistical analysis was by Student’s t-test or ANOVA with Student-Newman-Keuls post hoc test. P<0.05 was considered significant.

BEL (10μM) produced a slow diminution of tone in tissues pre-contracted with CCh (50μM). After 1 hour, the response to CCh was 27 ± 7% (n=4) of that seen on immediate application of the agonist whilst in time-matched, solvent (DMSO) controls tension was reduced to 67% (n=2). Concentration-response curves were constructed to CCh (0.1–100 μM) before and after a 1-hour incubation with either DMSO or BEL (3, 10 or 30 μM). DMSO had no significant effect on the response to CCh (Max response in DMSO 93±5% of control; pEC₅₀ control 5.4±0.12, DMSO 5.3±0.13). Treatment of tissues with 3 or 10μM BEL produced a reduction in the maximum response to CCh (50±9% and 12±1% of control respectively) with no change in EC₅₀ (pEC₅₀ for CCh of 5.5 ± 0.06 and 4.8 ± 2.5 in presence of 3 and 10μM BEL respectively). Treatment of tissues with 30 μM BEL for 1 hour abolished the response to CCh. In contrast, BEL (10 μM) produced a rapid abolition of the contraction produced by Tg (100nM; n = 4) whilst DMSO had no effect. Cumulative concentration-response curves for BEL against Tg-induced tone yielded an IC₅₀ of approximately 25 nM. These data generally support a role for iPLA₂ in mediating contractions to Tg and CCh in the mouse anococcygeus, although the relative lack of sensitivity of the CCh contractions to BEL would suggest that the enzyme plays a minor role during agonist mediated contractions. Activation of iPLA₂ may explain the receptor-independent calcium sensitisation produced by Tg in this tissue.

Ayman, S. et al (2003) Br. J. Pharmacol., 139, 1532-1538
Guo, Z. et al (2003) J. Biol. Chem., 278, 1856-1863
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Wolf, M.J. et al (1997) J. Biol. Chem., 272, 1522-1526