Abstract

Circulating hormones stimulate the phospholipase Cβ (PLC)/Ca²⁺ influx pathway to regulate numerous cell functions, including vascular tone. It was proposed previously that Ca²⁺-independent phospholipase A₂ (iPLA₂)-dependent store-operated Ca²⁺ influx channels mediate hormone-induced contractions in isolated arteries, because bromoenol lactone (BEL), a potent irreversible inhibitor of iPLA₂, inhibited such contractions. However, the effects of BEL on other channels implicated in mediating hormone-induced vessel contractions, specifically voltage-gated Ca²⁺ (Ca_V1.2) and transient receptor potential canonical (TRPC) channels, have not been defined clearly. Using isometric tension measurements, we found that thapsigargin-induced contractions were ∼34% of those evoked by phenylephrine or KCl. BEL completely inhibited not only thapsigargin- but also phenylephrine- and KCl-induced ring contractions, suggesting that Ca_V1.2 and receptor-operated TRPC channels also may be sensitive to BEL. Therefore, we investigated the effects of BEL on heterologously expressed Ca_V1.2 and TRPC channels in human embryonic kidney cells, a model system that allows probing of individual protein function without interference from other signaling elements of native cells. We found that low micromolar concentrations of BEL inhibited Ca_V1.2, TRPC5, TRPC6, and heteromeric TRPC1–TRPC5 channels in an iPLA₂-independent manner. BEL also attenuated PLC activity, suggesting that the compound may inhibit TRPC channel activity in part by interfering with an initial PLC-dependent step required for TRPC channel activation. Conversely, BEL did not affect endogenous voltage-gated K⁺ channels in human embryonic kidney cells. Our findings support the hypothesis that iPLA₂-dependent store-operated Ca²⁺ influx channels and iPLA₂-independent hormone-operated TRPC channels can serve as smooth muscle depolarization triggers to activate Ca_V1.2 channels and to regulate vascular tone.