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. 2012 Sep 1;95(4):439-47.
doi: 10.1093/cvr/cvs208. Epub 2012 Jun 21.

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity

Sevvandi Senadheera  1 , Youngsoo Kim, T Hilton Grayson, Sianne Toemoe, Mikhail Y Kochukov, Joel Abramowitz, Gary D Housley, Rebecca L Bertrand, Preet S Chadha, Paul P Bertrand, Timothy V Murphy, Marianne Tare, Lutz Birnbaumer, Sean P Marrelli, Shaun L Sandow
Affiliations

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity

Sevvandi Senadheera et al. Cardiovasc Res. .

Abstract

Aims: Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery.

Methods and results: TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation.

Conclusion: The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

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Figures

Figure 1
Figure 1
TRPC3 localization and expression in rat mesenteric artery. Localized holes (dark spots; examples with arrows; A), are present in the internal elastic lamina (IEL; A) between the vascular endothelium and smooth muscle. TRPC3 presence in endothelial cells (EC; B and C) and apparent absence in smooth muscle cells (C, inset) was demonstrated using TRPC3 antibody batches AN-02, 03, and 07 (Supplementary material online, Table S1; AN-07, as example used here). Low-level diffuse TRPC3 is localized to the endothelial membrane (B), while overlay of IEL and TRPC3 label (C) shows strong TRPC3 expression at a proportion of IEL holes at the IEL-SM focal plane border (examples indicated with arrows with asterisks; see also Tables 1 and 2), as potential myoendothelial microdomain sites, noting that not all such sites have localized TRPC3 densities (example, arrow with no asterisk). Peptide block abolished staining (A, inset), with no labelling being present when incubated in the secondary antibody alone (data not shown). Perivascular nerve labelling (compare morphology with Milner et al.) acts as a positive control (B, inset, examples indicated with arrows). The TRPC3 antibody (AN-07) conjugated to 10 nm colloidal gold (D–F) confirms TRPC3 localization to myoendothelial contact regions (F, examples arrowed), as potential myoendothelial microdomain signalling sites, as well as at other sites within the endothelium (B, examples indicated with arrows). Vessel region in (A–C) correspond. Longitudinal vessel axis (A–C), left to right, n= 6 and 3, each from a different animal (A–C and D–F, respectively). The characteristics of rat mesenteric artery TRPC3 and its primary endothelial expression were examined using western blotting and the TRPC3 antibody (ACC-016; AN-07 used here; Supplementary material online, Table S1). Monoglycosylated TRPC3 is present as a band at ∼120 kDa (G2, box; +EC; see also Goel et al.), while the band at approximately >220 kDa (G1, box; see also Dietrich et al.) probably represents an undissociated aggregate of the TRPC3 tetrameric channel complex. TRPC3 expression was normalized to actin (at ∼43 kDa; lower bands, G), with endothelial removal (−EC; G, upper right column) reducing TRPC3 expression at approximately 120 and >220 kDa by approximately two-fold each (H, upper and lower, respectively; *P< 0.05, significant; values being mean ± SEM). Two lanes per artery were run with tissue from three different rats, 12 in total, with 20 μg of protein per lane being loaded. von Willebrand factor (vWF) labelling verified endothelial removal in whole-mount preparations (G, lower panels). Scale bars, 25 μm (A–C and G), 50 μm (B, inset), 5 μm (D), 2 μm (E), 1 μm (F).
Figure 2
Figure 2
TRPC3 in endothelium-dependent relaxation and endothelial cell hyperpolarization in rat mesenteric artery. In pressurized rat mesenteric arteries, relaxation to ACh (1 nM–100 μM) was examined in the presence and absence of Pyr3 (0.3 μM, A; 1 μM, A–C) with apamin (50 nM; B), or TRAM-34 (1 μM; C), or combined apamin and TRAM-34, to determine the relative contribution of TRPC3, SKCa, and IKCa. L-NAME (100 μM), ODQ (10 μM), and indomethacin (10 μM) were present in all experiments. n= 5–7 experiments, each from different animals; P< 0.05 indicates difference in *pEC50, or #Emax, relative to the ACh control (see Table 3 for drug characteristics). The endothelial cell hyperpolarization evoked by ACh [1 μM; 100%; n= 6; E(i)] in the presence of L-NAME (100 μM) and indomethacin (10 μM) was recorded in the presence of Pyr3 alone [1 μM; n= 6; E(ii)], Pyr3 and TRAM-34 [5 μM; n= 4; E(iii)], and Pyr3, TRAM-34, and apamin [100 nM, n= 4; D and E(iv)]. Example endothelial cell membrane potential recordings show hyperpolarization before and after the addition of Pyr3, TRAM-34, and apamin [E(ii–iv)]. Pyr3 (1 μM) reduced the hyperpolarization to ACh [D and E(ii–iv)], with the remaining hyperpolarization having a complex nature [D and E(ii–iv)], consisting of an initial rapid component [C1, with arrow in E(ii,iii)] followed by a second slower component [C2, with arrow in E(ii,iii)]. In the presence of all blockers, ACh evoked endothelial cell depolarization (E, far right) trace. Inset shows the example of dye-filled endothelial cells (Ea, inset, arrow) indicates cells from which recording was made. Scale bar, 50 μm, with the longitudinal vessel axis, left to right.
Figure 3
Figure 3
The vasodilator signalling mechanism at myoendothelial contact sites in rat mesenteric artery. At sites of close contact between the endothelium and smooth muscle, localized gap junction connexins (Cx), endoplasmic reticulum (ER) 1,4,5-triphosphate receptors (IP3R), and intermediate-conductance calcium-activated potassium channels (IKCa) occur in close proximity to TRPC3. The localization and differential distribution of these channels and receptors suggests that these myoendothelial microdomains enable transfer of a connexin (Cx)-dependent endothelial hyperpolarizing current (1) and/or localized K+ activity (2 and 3), with the net effect being smooth muscle hyperpolarization and endothelium-dependent relaxation (modified from Sandow et al.;, see also Figure 1 and Supplementary material online, FigureS7). TRPC3-dependent calcium influx may activate myoendothelial KCa directly (i), and/or refill the IP3R-mediated ER store (ii). Inward rectifying potassium channels (Kir) are exclusive to the endothelium,, and are activated by potassium in a feedback with KCa and Na+/K+-ATPase activity. DAG, diacylglycerol; Em, membrane potential; MEGJ, myoendothelial gap junction; PLCβ, phospholipase C-beta; VDCC, voltage-dependent calcium channel.
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