Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles

Abstract

Cerebral parenchymal arterioles (PA) regulate blood flow between pial arteries on the surface of the brain and the deeper microcirculation. Regulation of PA contractility differs from that of pial arteries and is not completely understood. Here, we investigated the hypothesis that the Ca(2+) permeable vanilloid transient receptor potential (TRPV) channel TRPV3 can mediate endothelium-dependent dilation of cerebral PA. Using total internal reflection fluorescence microscopy (TIRFM), we found that carvacrol, a monoterpenoid compound derived from oregano, increased the frequency of unitary Ca(2+) influx events through TRPV3 channels (TRPV3 sparklets) in endothelial cells from pial arteries and PAs. Carvacrol-induced TRPV3 sparklets were inhibited by the selective TRPV3 blocker isopentenyl pyrophosphate (IPP). TRPV3 sparklets have a greater unitary amplitude (ΔF/F0 = 0.20) than previously characterized TRPV4 (ΔF/F0 = 0.06) or TRPA1 (ΔF/F0 = 0.13) sparklets, suggesting that TRPV3-mediated Ca(2+) influx could have a robust influence on cerebrovascular tone. In pressure myography experiments, carvacrol caused dilation of cerebral PA that was blocked by IPP. Carvacrol-induced dilation was nearly abolished by removal of the endothelium and block of intermediate (IK) and small-conductance Ca(2+)-activated K(+) (SK) channels. Together, these data suggest that TRPV3 sparklets cause dilation of cerebral parenchymal arterioles by activating IK and SK channels in the endothelium.

Keywords: endothelium-dependent hyperpolarization; parenchymal arterioles; transient receptor potential channel; transient receptor potential sparklet; vanilloid transient receptor potential 3.

Fig. 1.

Vanilloid transient receptor potential 3 (TRPV3) channels are present in parenchymal arterioles (PAs) and primary pial artery endothelial cells. A, left: representative image showing TRPV3 immunolabeling in the endothelium of a cerebral PA (red). A, right: no signal was detected when the primary antibody was omitted. Scale bar, 16 μm. Images are representative of 3 independent experiments. B: platelet endothelial cell adhesion molecule-1 (PECAM-1) (green; left), a marker of endothelial cells, and TRPV3 (red; middle) coimmunofluorescence in freshly isolated endothelial cell tubes from PAs. Note the colocalization in the merged image (right). Scale bar, 16 μm. Images are representative of 3 independent experiments. C: freshly dissociated smooth muscle cells from PAs positive for smooth muscle myosin heavy chain (SM-MHC) (green; left) and TRPV3 (red; middle). Scale bar, 16 μm. D: RT-PCR for TRPV3 (160 bp) and endothelial nitric oxide synthase (eNOS; 117 bp) in cerebral arteries (CA) and primary CA endothelial cells (EC). Images are representative of 3 independent experiments. E: immunocytochemistry for TRPV3 (left) and eNOS (right) in primary pial artery EC. No signal was detected when the primary antibody was omitted. Scale bar, 4 μm. Images are representative of 3 independent experiments. NTC, no cDNA template control.

Fig. 2.

TRPV3 sparklets in primary pial artery EC. A: pseudocolor time lapse of a TRPV3 sparklet (green) in a primary pial artery EC recorded using total internal reflection fluorescence microscopy. Scale bar, 4 μm. B: TRPV3 agonist carvacrol induces a concentration-dependent increase in the frequency of sparklets (EC₅₀ = 8.5 μM); n = 5–12 cells/concentration. C: summary data indicating that carvacrol-induced (30 μM) TRPV3 sparklets are dependent on extracellular Ca²⁺; n = 9–16 cells/group. *P ≤ 0.05 vs. control (not exposed to carvacrol). D: summary data indicating that the carvacrol-induced increase (30 μM) in TRPV3 sparklet frequency is attenuated by IPP (10 μM); n = 12–19 cells/group, *P ≤ 0.05 vs. baseline, vehicle. E: the TRPA1 blocker HC-030031 (10 μM) has no effect on the carvacrol-induced increase (30 μM) in TRPV3 sparklet frequency; n = 9–15 cells/group, *P ≤ 0.05 vs. vehicle.

Fig. 3.

Carvacrol recruits previously inactive TRPV3 channels. A: outlines of a total internal reflection fluorescence image of a primary pial artery EC showing active sparklet sites before (green; left) and after (red; middle) the addition of carvacrol (30 μM). The merged image (right) indicates recruitment of new sparklet sites upon carvacrol addition. B: summary data of the frequency of sparklets per cell before and after the addition of carvacrol (30 μM); n = 10 cells. C: summary data for the frequency of sparklets per active site before and after the addition of carvacrol (30 μM); n = 10 cells. D: total number of active sparklet sites per cell before and after treatment with carvacrol (30 μM); n = 10 cells. *P ≤ 0.05 vs. before carvacrol.

Fig. 4.

Characteristics of TRPV3 sparklets in pial artery EC. A: representative traces of fluorescence intensity (F/F₀) over time for 2 active sites on a CA EC (insets i and ii, left). The majority of carvacrol-induced TRPV3 sparklets (30 μM) had an amplitude of 1.20 F/F₀. Scale bar, 4 μm; inset i and ii scale bars (right), 2 μm. B: histograms of TRPV3 sparklet amplitudes (left), durations (middle), and spatial spreads (right). One prominent peak was observed at an amplitude of F/F₀ = 1.20, as seen in the traces of fluorescence in A; n = 1,641 total events.

Fig. 5.

Characteristics of TRPV3 sparklets in primary PA EC. A, left: pseudocolored image (F/F₀) of 2 distinct sparklet sites (insets i and ii) in the same EC after addition of carvacrol (30 μM). Scale bar, 5 μm. A, right: representative traces of fluorescence intensity (F/F₀) over time for the sparklets shown in images i and ii above trace. The majority of carvacrol-induced TRPV3 sparklets (30 μM) had an amplitude of 1.20 F/F₀. Scale bar, 3 μm. B: histograms of TRPV3 sparklet amplitudes (left), durations (middle), and spatial spreads (right). One prominent peak was observed at an amplitude of F/F₀ = 1.20, as seen in the traces of fluorescence in A; n = 256 total events.

Fig. 6.

TRPV3 channel activation dilates cerebral PA. A: representative recording of a pressurized PA after exposure to carvacrol (10 μM) or IPP (3 μM) + carvacrol. B: summary data of the change in PA diameter (Δdiameter) after exposure to carvacrol in the absence and presence of isopentenyl pyrophosphate (IPP; n = 5). C: IPP had no effect on basal myogenic tone of cerebral PA (n = 5). D: removal of the endothelium almost completely abolished carvacrol-induced dilation of PAs (n = 6). *P ≤ 0.05 vs. carvacrol.

Fig. 7.

Intermediate (IK) and small-conductance Ca²⁺-activated K⁺ (SK) channel inhibition abolishes carvacrol-induced dilation of cerebral PAs. A: representative recording and summary data of carvacrol-induced (10 μM) PA dilation after NO synthase and cyclooxygenase inhibition by preincubation with N^ω-nitro-l-arginine methyl ester hydrochloride (l-NAME; 100 μM) and indomethacin (10 μM), respectively (n = 6). B and C: representative recordings and summary data demonstrating that blockade of either IK inhibition with TRAM-34 (1 μM) or SK inhibition with apamin (1 μM) attenuates carvacrol-induced PA dilation (n = 6). D: representative trace and summary indicating that dual inhibition of both SK and IK channels abolishes carvacrol-induced dilation (n = 6). *P ≤ 0.05 vs. carvacrol.