PLA2 and TRPV4 channels regulate endothelial calcium in cerebral arteries.
We previously demonstrated that endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in cerebral arteries are significantly reduced by inhibitors of PLA(2). In this study we examined possible mechanisms by which PLA(2) regulates endothelium-dependent dilation, specifically whether PLA(2) is involved in endothelial Ca(2+) regulation through stimulation of TRPV4 channels. Studies were carried out with middle cerebral arteries (MCA) or freshly isolated MCA endothelial cells (EC) of male Long-Evans rats. Nitro-l-arginine methyl ester (l-NAME) and indomethacin were present throughout. In pressurized MCA, luminally delivered UTP produced increased EC intracellular Ca(2+) concentration ([Ca(2+)](i)) and MCA dilation. Incubation with PACOCF(3), a PLA(2) inhibitor, significantly reduced both EC [Ca(2+)](i) and dilation responses to UTP. EC [Ca(2+)](i) was also partially reduced by a transient receptor potential vanilloid (TRPV) channel blocker, ruthenium red. Manganese quenching experiments demonstrated Ca(2+) influx across the luminal and abluminal face of the endothelium in response to UTP. Interestingly, PLA(2)-sensitive Ca(2+) influx occurred primarily across the abluminal face. Luminal application of arachidonic acid, the primary product of PLA(2) and a demonstrated activator of certain TRPV channels, increased both EC [Ca(2+)](i) and MCA diameter. TRPV4 mRNA and protein was demonstrated in the endothelium by RT-PCR and immunofluorescence, respectively. Finally, application of 4alpha-phorbol 12,13-didecanoate (4alphaPDD), a TRPV4 channel activator, produced an increase in EC [Ca(2+)](i) that was significantly reduced in the presence of ruthenium red. We conclude that PLA(2) is involved in EC Ca(2+) regulation through its regulation of TRPV4 channels. Furthermore, the PLA(2)-sensitive component of Ca(2+) influx may be polarized to the abluminal face of the endothelium.
Marrelli
Sean P
SP
Department of Anesthesiology, Baylor College of Medicine, Houston, TX 77030, USA. marrelli@bcm.edu
O'neil
Roger G
RG
Brown
Rachel C
RC
Bryan
Robert M
RM
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D066298:In Vitro Techniques; D008345:Manganese / Q000378:metabolism; D020768:Middle Cerebral Artery / Q000187:drug effects / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000502:physiology*
D017931:DNA Primers; D050916:TRPV Cation Channels; C486577:Trpv4 protein, rat; D012430:Ruthenium Red; D016718:Arachidonic Acid; D008345:Manganese; D052250:Nitric Oxide Synthase Type III; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium