Localized TRPA1 channel Ca2+ signals stimulated by reactive oxygen species promote cerebral artery dilation.

Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca(2+)-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca(2+) signals representing Ca(2+) influx through single TRPA1 channels, which we called "TRPA1 sparklets." TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca(2+) entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca(2+) channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca(2+)-sensitive K(+) channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca(2+) influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.

Robust internal elastic lamina fenestration in skeletal muscle arteries.

Holes within the internal elastic lamina (IEL) of blood vessels are sites of fenestration allowing for passage of diffusible vasoactive substances and interface of endothelial cell membrane projections with underlying vascular smooth muscle. Endothelial projections are sites of dynamic Ca(2+) events leading to endothelium dependent hyperpolarization (EDH)-mediated relaxations and the activity of these events increase as vessel diameter decreases. We tested the hypothesis that IEL fenestration is greater in distal vs. proximal arteries in skeletal muscle, and is unlike other vascular beds (mesentery). We also determined ion channel protein composition within the endothelium of intramuscular and non-intramuscular skeletal muscle arteries. Popliteal arteries, subsequent gastrocnemius feed arteries, and first and second order intramuscular arterioles from rat hindlimb were isolated, cut longitudinally, fixed, and imaged using confocal microscopy. Quantitative analysis revealed a significantly larger total fenestration area in second and first order arterioles vs. feed and popliteal arteries (58% and 16% vs. 5% and 3%; N = 10 images/artery), due to a noticeably greater average size of holes (9.5 and 3.9 µm(2) vs 1.5 and 1.9 µm(2)). Next, we investigated via immunolabeling procedures whether proteins involved in EDH often embedded in endothelial cell projections were disparate between arterial segments. Specific proteins involved in EDH, such as inositol trisphosphate receptors, small and intermediate conductance Ca(2+)-activated K(+) channels, and the canonical (C) transient receptor potential (TRP) channel TRPC3 were present in both popliteal and first order intramuscular arterioles. However due to larger IEL fenestration in first order arterioles, a larger spanning area of EDH proteins is observed proximal to the smooth muscle cell plasma membrane. These observations highlight the robust area of fenestration within intramuscular arterioles and indicate that the anatomical architecture and endothelial cell hyperpolarizing apparatus for distinct vasodilatory signaling is potentially present.

Basal protein kinase Cδ activity is required for membrane localization and activity of TRPM4 channels in cerebral artery smooth muscle cells.

The melastatin (M) transient receptor potential channel (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. We recently reported that PKCδ activity influences smooth muscle cell excitability by promoting translocation of TRPM4 channel protein to the plasma membrane. Here we further investigate the relationship between membrane localization of TRPM4 protein and channel activity in native cerebral arterial myocytes. We find that TRPM4 immunolabeling is primarily located at or near the plasma membrane of freshly isolated cerebral artery smooth muscle cells. However, siRNA mediated downregulation of PKCδ or brief (15 min) inhibition of PKCδ activity with rottlerin causes TRPM4 protein to move away from the plasma membrane and into the cytosol. In addition, we find that PKCδ inhibition diminishes TRPM4-dependent currents in smooth muscle cells patch clamped in the amphotericin B perforated patch configuration. We conclude that TRPM4 channels are mobile in native cerebral myocytes and that basal PKCδ activity supports excitability of these cells by maintaining localization TRPM4 protein at the plasma membrane.