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+ (CaV1.2) 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 CaV1.2 and receptor-operated TRPC channels also may be sensitive to BEL. Therefore, we investigated the effects of BEL on heterologously expressed CaV1.2 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 CaV1.2, 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 CaV1.2 channels and to regulate vascular tone.
Footnotes
This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL083381].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.183673.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- PLC
- phospholipase Cβ
- BEL
- bromoenol lactone
- CaV1.2
- dihydropyridine-sensitive L-type voltage-gated Ca2+ channels
- cPLA2
- Ca2+-dependent cytosolic phospholipase A2
- DAG
- diacylglycerol
- DMSO
- dimethyl sulfoxide
- GFP
- green fluorescent protein
- GFP-PH
- the phospholipase Cδ PH domain-green fluorescent protein fusion
- HEK
- human embryonic kidney
- IP3
- inositol trisphosphate
- iPLA2
- Ca2+-independent phospholipase A2
- PACOCF3
- palmitoyl trifluoromethyl ketone
- PIP2
- phosphatidylinositol 4,5-bisphosphate
- SOC
- store-operated Ca2+ influx
- TRP
- transient receptor potential
- TRPC
- transient receptor potential canonical
- NMDG
- N-methyl-d-glucamine
- U73122
- 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione.
- Received May 6, 2011.
- Accepted July 26, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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