1. Biochemistry and Chemical Biology
  2. Cell Biology

Endothelial pannexin 1–TRPV4 channel signaling lowers pulmonary arterial pressure in mice

  1. Zdravka Daneva
  2. Matteo Ottolini
  3. Yen Lin Chen
  4. Eliska Klimentova
  5. Maniselvan Kuppusamy
  6. Soham A Shah
  7. Richard D Minshall
  8. Cheikh I Seye
  9. Victor E Laubach
  10. Brant E Isakson
  11. Swapnil K Sonkusare  Is a corresponding author
  1. Robert M. Berne Cardiovascular Research Center, University of Virginia, United States
  2. Department of Pharmacology, University of Virginia, United States
  3. Department of Biomedical Engineering, University of Virginia, United States
  4. Department of Anesthesiology, Department of Pharmacology, University of Illinois, United States
  5. Department of Biochemistry, University of Missouri-Columbia, United States
  6. Department of Surgery, University of Virginia, United States
  7. Department of Molecular Physiology and Biological Physics, University of Virginia, United States
https://doi.org/10.7554/eLife.67777
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Cite this article
  1. Zdravka Daneva
  2. Matteo Ottolini
  3. Yen Lin Chen
  4. Eliska Klimentova
  5. Maniselvan Kuppusamy
  6. Soham A Shah
  7. Richard D Minshall
  8. Cheikh I Seye
  9. Victor E Laubach
  10. Brant E Isakson
  11. Swapnil K Sonkusare
(2021)
Endothelial pannexin 1–TRPV4 channel signaling lowers pulmonary arterial pressure in mice
eLife 10:e67777.
https://doi.org/10.7554/eLife.67777
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6 figures, 2 tables and 1 additional file

Figures

Figure 1 with 2 supplements
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ATP efflux through Panx1EC ATP activates TRPV4EC channels in pulmonary arteries (PAs) and lowers pulmonary arterial pressure (PAP).

(A) Left: immunofluorescence images of en face fourth-order PAs from Panx1fl/fl and Panx1 cKO-EC mice. CD31 immunofluorescence indicates ECs. Center: representative traces showing TRPV4EC sparklet …

Figure 1—figure supplement 1
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Panx1SMC mRNA levels in mesenteric arteries from Panx1fl/fl and Panx1 cKO-SMC mice.

Data presented as a fold change from Panx1fl/fl (n = 5; ***p<0.001 vs. Panx1fl/fl; t-test).

Figure 1—figure supplement 2
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TRPV4EC sparklet activity (NPO) per site and TRPV4 sparklet sites per cell in en face preparations of pulmonary arteries (PAs) from Panx1fl/fl and Panx1 cKO-EC mice in response to 30 nmol/L GSK101.

Experiments were performed in Fluo-4-loaded PAs in the presence of cyclopiazonic acid (CPA; 20 μmol/L), included to eliminate Ca2+ release from intracellular stores. ‘N’ is the number of channels …

Figure 2 with 3 supplements
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Endothelial Panx1–TRPV4 signaling lowers myogenic and agonist-induced constriction of pulmonary arteries (PAs).

(A) Top: an image showing the left lung and the order system used to isolate fourth-order PAs in this study; bottom: an image of a fourth-order PA cannulated and pressurized at 15 mm Hg. (B) …

Figure 2—source data 1

Endothelial TRPV4 knockout increases U46619-induced constriction of PAs.

https://cdn.elifesciences.org/articles/67777/elife-67777-fig2-data1-v2.xlsx
Figure 2—source data 2

Endothelial Panx1 knockout increases U46619-induced constriction of PAs.

https://cdn.elifesciences.org/articles/67777/elife-67777-fig2-data2-v2.xlsx
Figure 2—source data 3

Shear stress increases ATP efflux through endothelial Panx1 in PAs.

https://cdn.elifesciences.org/articles/67777/elife-67777-fig2-data3-v2.xlsx
Figure 2—source data 4

Endothelial TRPV4 channel does not contribute to shear stress-induced increase in luminal ATP.

https://cdn.elifesciences.org/articles/67777/elife-67777-fig2-data4-v2.xlsx
Figure 2—figure supplement 1
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Percent myogenic constriction in small pulmonary arteries (PAs; 50–100 μm internal diameter) and large PAs (>200 μm internal diameter; n = 6–10; ***p<0.001).
Figure 2—figure supplement 2
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Percent constriction of pulmonary arteries (PAs) from Panx1fl/fl and Panx1fl/fl plus apyrase (10 U/mL) mice in response to U46619 (U466; 1–100 nmol/L; n = 5; **p<0.01 vs. Panx1fl/fl; two-way ANOVA).
Figure 2—figure supplement 2—source data 1

Apyrase increases U46619-induced constriction of PAs.

https://cdn.elifesciences.org/articles/67777/elife-67777-fig2-figsupp2-data1-v2.xlsx
Figure 2—figure supplement 3
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Left: representative diameter traces showing ATP (1 μmol/L)-induced dilation of pulmonary arteries (PAs) from Trpv4fl/fl and Trpv4 cKO-EC mice, pre-constricted with the thromboxane A2 receptor analog U46619 (50 nmol/L).

Fourth-order PAs were pressurized to 15 mm Hg. Center: percent dilation of PAs from Trpv4fl/fl and Trpv4 cKO-EC mice in response to ATP (1 μmol/L; n = 5–10; ***p<0.001 vs. Trpv4fl/fl [ATP 1 μmol/L]; …

Figure 3
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Endothelial P2Y2R-TRPV4 channel signaling lowers pulmonary artery (PA) contractility and pulmonary arterial pressure (PAP).

(A) Left: immunofluorescence images of en face fourth-order PAs from P2ry2fl/fl and P2ry2 cKO-EC mice. CD31 immunofluorescence indicates ECs. Right: effects of ATP (1 μmol/L) on TRPV4EC sparklet …

Figure 3—source data 1

Endothelial P2Y2R knockout increases U46619-induced constriction of PAs.

https://cdn.elifesciences.org/articles/67777/elife-67777-fig3-data1-v2.xlsx
Figure 4 with 2 supplements
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Cav-1EC provides a signaling scaffold for Panx1EC–P2Y2REC–TRPV4EC signaling in pulmonary arteries (PAs).

(A) Left: representative traces showing TRPV4EC sparklets in en face preparations of PAs from Cav1fl/fl and Cav1 cKO-EC mice in the absence or presence of ATP (1 μmol/L). Dotted lines are quantal …

Figure 4—figure supplement 1
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Representative proximity ligation assay (PLA) images showing EC nuclei, TRPV4EC:P2Y2REC and Panx1EC:P2Y2REC co-localization in fourth-order pulmonary arteries (PAs) from P2ry2 cKO-EC mice.
Figure 4—figure supplement 2
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Left: representative proximity ligation assay (PLA) images showing EC nuclei and Cav-1EC:P2Y1EC co-localization in fourth-order pulmonary arteries (PAs) from Cav1fl/fl mice.

Right: quantification of Cav-1EC:P2Y1EC co-localization in PAs from Cav1fl/fl mice (n = 5).

Figure 5 with 2 supplements
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ATP activates TRPV4EC channels via phospholipase C–diacylglycerol–protein kinase C (PLC–DAG–PKC) signaling in pulmonary arteries (PAs).

(A) Left: representative traces showing TRPV4EC sparklet activity in en face preparations of PAs from C57BL6/J mice before and after treatment with ATP (1 μmol/L). Right: effects of U73122 (PLC …

Figure 5—figure supplement 1
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A multi-Gaussian to all-points histogram obtained using sparklet traces from X-Rhod-1-loaded pulmonary arteries (PAs), showing quantal (evenly spaced) ΔF/F0 levels of 0.21.
Figure 5—figure supplement 2
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Left: scatterplot showing TRPV4 sparklet activity, expressed as NPO per site, before and after light activation, in the presence of TRPV4 inhibitor GSK2193874 (GSK219; 100 nmol/L, n = 4).

‘N’ is the number of channels per site and ‘PO’ is the open state probability of the channel. Right: scatterplot showing TRPV4 sparklet activity, expressed as sparklet sites per cell, before and …

Figure 6
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Localization of PKCα with Cav-1EC increases the activity of TRPV4EC channels in pulmonary arteries (PAs).

(A) Top: representative merged images of proximity ligation assays (PLAs) showing endothelial cell (EC) nuclei and Cav-1EC:PKC co-localization (white puncta) in fourth-order PAs from Cav1fl/fl and Ca…

Tables

Table 1
Fulton index and functional MRI analysis of cardiac function in Panx1fl/fl and Panx1 cKO-EC mice.
Panx1fl/flPanx1 cKO-EC
Fulton index0.23 ± 0.010.26 ± 0.03
EDV (µL)46.9 ± 2.750.9 ± 2.9
ESV (µL)14.8 ± 1.713.1 ± 1.4
EF (%)68.9 ± 2.074.3 ± 2.3
SV (µL)32.2 ± 1.337.8 ± 2.4
R-R (ms)127.1 ± 5.5130.8 ± 2.5
CO (mL/min)15.2 ± 0.617.3 ± 1.2
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Mus musculus)C57BL/6JThe Jackson LaboratoryStock no: 000664
Genetic reagent (M. musculus)Trpv4conditional knockout in ECDr. Swapnil SonkusarePMID:32008372 
Genetic reagent (M. musculus)Trpv4 conditional knockout in SMCDr. Swapnil SonkusarePMID:33879616 
Genetic reagent (M. musculus)Panx1 conditional knockout in ECDr. Brant IsaksonPMID:26242575 
Genetic reagent (M. musculus)Panx1 conditional knockout in SMCDr. Brant IsaksonPMID:25690012 
Genetic reagent (M. musculus)Cav1 conditional knockout in ECDr. Swapnil SonkusarePMID:33879616Dr. Richard MinshallPMID:22323292 
Genetic reagent (M. musculus)P2ry2fl/fl miceDr. Cheikh SeyePMID:27856454 
Genetic reagent (M. musculus)Cdh5-Optoα1AR-
IRES-lacZ		CHROMus
(Cornell University, USA)		 
AntibodyTRPV4 antibody (aa100-150), (mouse polyclonal)LifeSpan Bioscience IncCat. #: LS-C94498;RRID:AB_2893149(1:200)
AntibodyAnti-caveolin-1 antibody - caveolae marker (rabbit polyclonal)Abcam plcCat. #: Ab2910;RRID:AB_303405(1:500)
AntibodyCaveolin-1 antibody (7C8) (mouse monoclonal)Novus Biologicals, LLCCat. #: NB100-615;RRID:AB_10003431(1:200)
AntibodyPKC (mouse monoclonal)Santa Cruz
Biotechnology, Inc		Cat. #: SC-17769;RRID:AB_628139(1:250)
AntibodyPanx1 (rabbit polyclonal)Alomone LabsCat. #: ACC-234;RRID:AB_2340917(1:100)
AntibodyP2Y2R (rabbit polyclonal)Alomone LabsCat. #: APR-010;RRID:AB_2040078(1:250)
AntibodyP2Y1R (rabbit polyclonal)Alomone LabsCat. #: APR-009;RRID:AB_2040070(1:100)
Chemical compound, drugGSK2193874Tocris BioscienceCat. #: 5106/5
Chemical compound, drugCyclopiazonic acid (CPA)Tocris BioscienceCat. #: 1235/10
Chemical compound, drugGSK1016790ATocris BioscienceCat. #: 6433/10
Chemical compound, drugPhorbol 12-myristate 13-acetate (PMA)Tocris BioscienceCat. #: 1201/1
Chemical compound, drugAR-C 118925XXTocris BioscienceCat. #: 4890/5
Chemical compound, drug2-Thio UTP tetrasodium saltTocris BioscienceCat. #: 3280/1
Chemical compound, drugMRS2179Tocris BioscienceCat. #: 0900/10
Chemical compound, drugU-73122Tocris BioscienceCat. #: 1268/10
Chemical compound, drugNS309Tocris BioscienceCat. #: 3895/10
Chemical compound, drugARL-67156Tocris BioscienceCat. #: 1283/10
OtherFluo-4-AMInvitrogenCat. #: F14201
Chemical compound, drug1-O-9Z-octadecenoyl-2-O-acetyl-sn-glycerol (OAG)Cayman ChemicalsCat. #: 62600
Chemical compound, drugPPADSCayman ChemicalsCat. #: 14537
Chemical compound, drugGö-6976Cayman Chemicals Cat. #: 13310
Chemical compound, drugJNJ-47965567Cayman ChemicalsCat. #: 21895
Chemical compound, drugU46619Cayman ChemicalsCat. #: 16452
Chemical compound, drugTamoxifenSigma-AldrichCat. #: T5648
Peptide, recombinant proteinApyraseSigma-AldrichCat. #: A6535
Software, algorithmLabChart8ADInstruments
https://www.adinstruments.com/products/labchart		RRID:SCR_017551
Software, algorithmSegment version 2.0 R5292Twilio(http://segment.heiberg.se) 
Software, algorithmIonOptixIonOptix, LLC (


https://www.ionoptix.com/products/software/ionwizard-core-and-analysis/)		 
Software, algorithmSparkAnDr. Adrian Bonev,
University of Vermont,
Burlington, VT, USA
PMID:22556255		 
Software, algorithmClampFit10.3Molecular Devices
(https://www.moleculardevices.com/)		RRID:SCR_011323
Software, algorithmImageJNational Institutes of Health
(https://imagej.nih.gov/ij/)		RRID:SCR_003070
Software, algorithmPatchMaster v2x90 programHarvard Bioscience
https://www.harvardbioscience.com/		RRID:SCR_000034
Software, algorithmFitMaster v2x73.2Harvard Bioscience
https://www.harvardbioscience.com/		RRID:SCR_016233
Software, algorithmMATLAB R2018aMathWorks
https://www.mathworks.com/products/matlab.html		RRID:SCR_013499
Software, algorithmCorelDraw Graphics Suite X7CorelDraw
(https://www.coreldraw.com/en)		RRID:SCR_014235
Software, algorithmGraphPad Prism 8.3.0GraphPad Software, Inc
(https://www.graphpad.com/)		RRID:SCR_002798
Software, algorithmGLIMMPSE software
(https://glimmpse.samplesizeshop.org/)		RRID:SCR_016297
Software, algorithmBiorenderhttp://biorender.comRRID:SCR_018361

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/67777/elife-67777-transrepform1-v2.pdf

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