Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov;9(11):1491-1503.
doi: 10.15252/emmm.201707725.

Treatment of hypertension by increasing impaired endothelial TRPV4-KCa2.3 interaction

Dongxu He  1   2 , Qiongxi Pan  1   2 , Zhen Chen  1   2 , Chunyuan Sun  1   2 , Peng Zhang  1   2 , Aiqin Mao  1   2 , Yaodan Zhu  1   2 , Hongjuan Li  1   2 , Chunxiao Lu  1   2 , Mingxu Xie  1   2 , Yin Zhou  1   2 , Daoming Shen  1   2 , Chunlei Tang  1   2 , Zhenyu Yang  3 , Jian Jin  1   2 , Xiaoqiang Yao  4 , Bernd Nilius  5 , Xin Ma  6   2
Affiliations

Treatment of hypertension by increasing impaired endothelial TRPV4-KCa2.3 interaction

Dongxu He et al. EMBO Mol Med. 2017 Nov.

Abstract

The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta-blockers, constipation by Ca2+ channel blockers, and dry cough by ACEI, lead to non-compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca2+-activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high-salt diet, N(G)-nitro-l-arginine intake, or angiotensin II delivery, showed decreased TRPV4-KCa2.3 interaction in ECs. Perturbation of the TRPV4-KCa2.3 interaction in mouse ECs by overexpressing full-length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small-molecule drug, JNc-440, which showed affinity for both TRPV4 and KCa2.3. JNc-440 significantly strengthened the TRPV4-KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc-440 specifically targeted the impaired TRPV4-KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4-KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development.

Keywords: KCa2.3; TRPV4; artery; endothelium; hypertension.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Impairment of the physical and functional interaction of TRPV4‐KCa2.3 channels in hypertension
  1. A

    The hypothesis investigated in this study was that endothelial TRPV4‐KCa2.3 interaction modulates blood pressure, and impairment of the interaction is related to hypertension. So, strengthening the interaction with a small‐molecule drug, JNc‐440, would show antihypertensive effects.

  2. B

    Immuno‐FRET experiments in arterial sections from normotensive and hypertensive humans. Left: representative figures for FRET. White dotted line, autofluorescence of elastin underneath the endothelium; right: analysis of 21 normotensive and 15 hypertensive individuals (# P < 0.0001, unpaired t‐test vs. control). Scale bar, 20 μm.

  3. C–E

    Immuno‐FRET experiments in arterial sections from (C) mice on a high‐salt diet (8% NaCl), (D) mice with L‐NNA intake (0.5 g/l in the drinking water), or (E) mice with AngII delivery (infused at 500 ng/kg/min) (# P < 0.0001, unpaired t‐test vs. control).

  4. F

    Fluorescent K+ efflux results showing the TRPV4 agonist GSK1016790A (GSK)‐induced K+ efflux in primary cultured ECs from normal mice and mice on a high‐salt diet (left). Six mice were tested for each treatment and analyzed (right). ECs from TRPV4‐knockout (KO) mice were used as negative controls [# P < 0.0001, two‐way ANOVA (GSK 30 nM, high salt vs. control; GSK 100 nM, high salt vs. control)].

  5. G

    Modulation of vasodilation by GSK1016790A (GSK), acetylcholine (ACh), and sodium nitroprusside (SNP). Left: vasodilation assays by wire myograph showing GSK1016790A (GSK)‐induced dilation in freshly isolated arterial segments from normal mice and mice on a high‐salt diet. Second from left: five mice were tested for each treatment and statistically analyzed (# P < 0.0001 vs. control, two‐way ANOVA). Rightmost two panels: statistical data of vasodilation for ACh (n = 3/treatment) and SNP (n = 3/treatment) treatment in freshly isolated arterial segments from normal mice and mice on a high‐salt diet (*P < 0.0001, # P = 0.0004 vs. control, two‐way ANOVA).

  6. H

    FRET in HEK cells co‐expressing CFP‐tagged TRPV4 and YFP‐tagged KCa2.3 showing the critical role of the AR2 and C‐terminal 17‐amino acid (17C) regions in the interaction. TRPV4 was deleted at the different AR and CaMBD regions, whereas KCa2.3 was deleted at the 17C, α1, α2, Loop, and CAMBD regions. NC, negative control, assessed in cells co‐expressing CFP and YFP as separate molecules; PC, positive control, assessed in cells expressing YFP‐CFP dimer (# P < 0.0001 vs. wild‐type control (TRPV4‐KCa2.3), one‐way ANOVA).

  7. I

    Stochastic optical reconstruction images of the indicated constructs in overexpressing HEK cells (scale bars, 10 μm).

Data information: Data are given as the mean ± SD.
Figure 2
Figure 2. Increasing the TRPV4‐KCa2.3 interaction lowers blood pressure, but dissociation of TRPV4‐KCa2.3 interaction increases blood pressure
  1. A–H

    Mice were intravenously injected with AAV‐Flt1‐KCa2.3 and the AAV‐Flt1 vector served as a control. (A) Representative en face (left) and cross‐sectional (right) fluorescence images of AAV‐Flt1‐KCa2.3 or vector (flag‐tag) expression in AAV‐Flt1‐infected mice. Green, autofluorescence of elastin underneath the endothelium (excitation, 488 nm); red, anti‐flag. Scale bars, 10 μm. (B) Seven mice were tested for the effects of AAV‐Flt1‐KCa2.3 or vector in immuno‐FRET experiments (# P < 0.0001 vs. control, unpaired t‐test). (C, F) K+ efflux from primary cultured ECs (# P < 0.0001 vs. control, unpaired t‐test). (D, G) Vasodilation assays by wire myography (GSK1016790A (GSK) and ACh in freshly isolated arterial segments) (*P = 0.047, # P < 0.0001 vs. control, two‐way ANOVA). (E, H) Time course of the changes in mean arterial pressure (∆MAP) in wild‐type (C–E) and TRPV4‐knockout (KO) (F–H) mice after tail injection of AAV‐Flt1‐KCa2.3, AAV‐Flt1 vector, or vehicle (injection solution).

  2. I–O

    Mice were intravenously injected with AAV‐Flt1‐KCa2.3‐17C; the AAV‐Flt1 vector served as the control. (I) Mice were tested for effects of AAV‐Flt1‐KCa2.3‐17C or vector with immuno‐FRET experiments (# P < 0.0001 vs. control, unpaired t‐test). (J, M) K+ efflux from primary cultured ECs (# P < 0.0001 vs. control, unpaired t‐test). (K, N) Vasodilation assays by wire myography (GSK1016790A (GSK) and ACh) in freshly isolated arterial segments) (*P = 0.004, # P < 0.0001 vs. control, two‐way ANOVA). (L, O) Time course of the changes in mean arterial pressure (∆MAP) in wild‐type (J–L) and TRPV4‐knockout (KO) (M–O) mice after tail injection of AAV‐Flt1‐KCa2.3‐17C, AAV‐Flt1 vector, or vehicle (injection solution).

Data information: Data are mean ± SD.
Figure 3
Figure 3. Increasing the TRPV4‐KCa2.3 interaction with JNc‐440 is antihypertensive
  1. A

    Left: structural formula of JNc‐440. Right: affinity of JNc‐440 for both TRPV4 and KCa2.3. Biotinylated JNc‐440 (JNc‐440‐biotin) pulled down TRPV4 or KCa2.3 in primary cultured ECs.

  2. B–G

    (B) Immuno‐FRET experiments in arterial sections, (C) K+ efflux from primary cultured ECs, (D) vasodilation assays by wire myography (GSK and ACh) in freshly isolated arterial segments, and (E) mean arterial pressure. (F) Effect of JNc‐440 on blood pressure in normotensive mice. (G) Effect of JNc‐440 (1 mg/kg; daily for 1 week) on blood pressure in TRPV4‐knockout (KO) mice treated with a high‐salt diet, L‐NNA, or AngII. WT mice were treated with a high‐salt diet, L‐NNA, or AngII and then intravenously injected with JNc‐440 (1 mg/kg) or vehicle for 2 h (B–F) or daily for 1 week (G). Data are mean ± SD. (B, C) # P < 0.0001 vs. control, unpaired t‐test. (D) *P = 0.025 (high‐salt diet), *P = 0.008 (L‐NNA treatment), or *P = 0.021 (AngII treatment), # P < 0.0001 vs. control, two‐way ANOVA. (E) *P = 0.001, # P < 0.0001 vs. control, two‐way ANOVA.

Source data are available online for this figure.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adebiyi A, Zhao G, Narayanan D, Thomas‐Gatewood CM, Bannister JP, Jaggar JH (2010) Isoform‐selective physical coupling of TRPC3 channels to IP3 receptors in smooth muscle cells regulates arterial contractility. Circ Res 106: 1603–1612 - PMC - PubMed
    1. Adebiyi A, Thomas‐Gatewood CM, Leo MD, Kidd MW, Neeb ZP, Jaggar JH (2012) An elevation in physical coupling of type 1 inositol 1,4,5‐trisphosphate (IP3) receptors to transient receptor potential 3 (TRPC3) channels constricts mesenteric arteries in genetic hypertension. Hypertension 60: 1213–1219 - PMC - PubMed
    1. Bagher P, Beleznai T, Kansui Y, Mitchell R, Garland CJ, Dora KA (2012) Low intravascular pressure activates endothelial cell TRPV4 channels, local Ca2+ events, and IKCa channels, reducing arteriolar tone. Proc Natl Acad Sci USA 109: 18174–18179 - PMC - PubMed
    1. Barron LA, Giardina JB, Granger JP, Khalil RA (2001) High‐salt diet enhances vascular reactivity in pregnant rats with normal and reduced uterine perfusion pressure. Hypertension 38: 730–735 - PubMed
    1. Brahler S, Kaistha A, Schmidt VJ, Wolfle SE, Busch C, Kaistha BP, Kacik M, Hasenau AL, Grgic I, Si H, et al. (2009) Genetic deficit of SK3 and IK1 channels disrupts the endothelium‐derived hyperpolarizing factor vasodilator pathway and causes hypertension. Circulation 119: 2323–2332 - PubMed

Publication types

MeSH terms

-