Cell Biology

Roles of G-protein coupled receptors and mechanosensitive ion channels in pressure-induced chronotropy of lymphatic vessels

Michael J Davis author has email address
Hae Jin Kim
Min Li
Jorge A Castorena-Gonzalez
Soumiya Pal
Timothy L Domeier
Joshua P Scallan
Scott Earley
Scott D Zawieja

Department of Medical Pharmacology & Physiology, University of Missouri, Columbia, United States
Department of Pharmacology, Tulane University School of Medicine, New Orleans, United States
Department of Molecular Pharmacology & Physiology, University of South Florida, Morisani School of Medicine, Tampa, United States
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, United States

https://doi.org/10.7554/eLife.109466.1

Open access
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Figures and data

Quantification of the F-P relationship.
Steps for quantitative assessment of pressure-induced chronotropy. A) Representative recording of pressure and diameter changes in a WT popliteal lymphatic vessel during inflow (Pin) and outflow (Pout) pressure steps between 0.5 and 5 cmH₂O. Frequencies stated at the bottom of the trace were determined off-line using a custom peak detection program written in LabVIEW. B) Plot of frequency vs pressure for the vessel in A, showing how frequency reaches a plateau above 5 cmH₂O. C) Fit of the frequency data with a first-order equation and calculation of the frequency difference (ΔF) between 5 and 0.5 cmH₂O. D) Summary of contraction data for this representative WT vessel over the stated pressure range; see Methods for description of parameter calculations.

Consequences of ANO1 deletion and/or inhibition.
A) UMAP plot of Ano1 expression in the various cell populations within the walls of murine IALVs that were thoroughly cleaned prior to dissociation and scRNAseq analysis, as described in (Zawieja et al., 2025). The LMC cluster (composed of at least two subclusters of 978 cells) was identified by the expression of canonical smooth muscle cell markers Acta2 (SM α-actin), Myh11 (myosin heavy chain 11), Itga8 (integrin alpha 8). Gray color in this and subsequent figures represents Acta2 expression, red/brown in this panel indicates Ano1 expression. Bubble plot shows Ano1 expression in the LMC and other cell clusters in terms of percent cells in an individual cluster expressing Ano1 (size of dot) and the Ano1 expression level relative to Acta2 (color of dot). B) Slope of frequency vs pressure (F-P) for WT (C57Bl/6) vessels with / without treatment by the ANO1 inhibitor Ani9 (1 μM), for Ano1^f/f control vessels, for Ano1 smKO vessels and for the latter treated with Ani9. C) Summary plots of contraction parameters as a function of pressure for the 5 respective groups of vessels. WT N=15, n=20; WT+Ani9 N=5, n=6; Ano1 smKO N=14, n=23; Ano1^f/f N=8, n=16.

Consequences of Piezo1 deletion.
A) UMAP and bubble plots of Piezo1 and Piezo2 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Piezo1^f/f, Piezo1 smKO, Piezo1 nestin KO, Piezo1 lecKO mice. C) Summary plots of contraction parameters as a function of pressure for the 4 respective groups of vessels. Piezo1^f/f N=9, n=17; Piezo1 smKO N=7, n=13; Piezo1 nestin KO N=5, n=10; Piezo1 lecKO N=5, n=6.

Consequences of Trpc6 and/or Trpc3 deletion.
A) UMAP and bubble plots of Trpc6 and Trpc3 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from SV129 controls, Trpc6^−/−, Trpc3^−/− and Trpc6^−/−;Trpc3^−/− mice. C) Summary plots of contraction parameters as a function of pressure for the 4 respective groups of vessels. SV129 control N=8, n=11; Trpc6^−/− N=5, n=10; Trpc3^−/− N=7, n=11; Trpc6^−/−;Trpc3^−/− N=5, n=10.

Consequences of Trpm4 deletion.
A) UMAP and bubble plots of Trpm4 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Trpm4^f/f and Trpm4 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Trpm4 smKO N=6, n=13; Trpm4^f/f N=4, n=7.

Consequences of Pkd2 (TRPP1) deletion.
UMAP and bubble plots of Pkd2 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Pkd2 ^f/f and Pkd2 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Pkd2 smKO N=6, n=13; Pkd2 ^f/f N=4, n=8.

Consequences of Trpv4 deletion.
UMAP and bubble plots of Trpv4 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Trpv4^f/f and Trpv4 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. TRPV4 smKO N=5, n=10; TRPV4^f/f N=3, n=7.

Consequences of Trpv2 inhibition.
UMAP and bubble plots of Trpv2 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the TRPV2 inhibitors tranilast (10 μM) and SET2 (1 μM). C) Summary plots of contraction parameters as a function of pressure for the three respective groups of vessels. WT+tranilast N=5, n=10; WT+SET2 N=3, n=6.

Consequences of ENaC inhibition.
UMAP and bubble plots of ENaC subsunit expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the ENaC inhibitors amiloride (5 μM) and benzamil (1 μM). WT N=15, n=20, WT+amiloride N=5, n=9; WT+benzamil N=6, n=11.

Consequences of Slc8a1 (NCX1) deletion.
UMAP and bubble plots of Slc8a1 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Slc8a1^f/f and Slc8a1 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Slc8a1^f/f N=7, n=12; Slc8a1 smKO N=7, n=13.

Consequences of HCN inhibition.
UMAP and bubble plots of Hcn gene family expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the HCN inhibitors ivabradine (3 μM) and zatebradine (3 μM). WT N=15, n=20, WT+zatebradine. N=5, n=10; WT+ivabradine N=5, n=10.

Consequences of Kcnq1 deletion.
UMAP and bubble plots of Kcnq4 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT and Kcnq4 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Kcnq4 smKO N=5, n=7.

Consequences of Kir2.x inhibition.
UMAP and bubble plots of Kcnj2 (Kir2.1), Kcnj12 (Kir2.2) and Kcnj8 (Kir6.1) expression in various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the Kir2 inhibitor BaCl₂ (100 μM). WT N=15, n=20, WT+BaCl₂ N=4, n=11.

Consequences of Cacna1c (Cav1.2) and Cacna1g/Cacna1h (Cav3.1/3.2) deletion.
UMAP and bubble plots of the expression of voltage-gated Ca²⁺ channels and their accessory subunits in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Cacan1c^f/f, Cacna1c smKO and Cacna1g^−/−;Cacna1h^−/− mice. C) Summary plots of contraction parameters as a function of pressure for the three respective groups of vessels. Cacna1c: alpha-1c, Cacna2d1: α2/δ1, Cacnb2: β2, Cacnb3: β3, Cacna1d: alpha-1d (Ca_v1.3), Cacna1a: Ca_v2.1 (P/Q), Cacna1g: Ca_v3.1, Cacna1h: Ca_v3.2. Cacna1c^f/f N=10, n=20; Cacna1c smKO N=14, n=24; Cacna1g^−/−;Cacna1h^−/− N=16, n=25.

Consequences of Itpr1 (IP₃R1) deletion.
UMAP and bubble plots of Ip3r1 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Itpr1^f/f and Itpr1 smKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Itpr1 smKO N=5, n=8; Itpr1^f/f N=5, n=10.

Consequences of Gnaq and/or Gna11 deletion.
UMAP and bubble plots of Gnaq and Gna11 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Gnaq^f/f;Gna11^+/+, Myh11-CreER^T2;Gnaq^f/f (Gnaq smKO), Gna11^−/− and Myh11-CreER^T2;Gnaq^f/f;Gna11^−/− DKO mice. C) Summary plots of contraction parameters as a function of pressure for the four respective groups of vessels. Gnaq^f/f;Gna11^+/+ control N=3, n=6; Gnaq smKO N=4, n=8; Gna11^−/− N=3, n=6; Myh11-CreER^T2;Gnaq^f/f;Gna11^−/−N=8, n=13.

Consequences of Gna12/Gna13 deletion.
UMAP and bubble plots of Gna12 and Gna13 expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from Gna12^f/f;Gna13^+/+ and Myh11-CreER^T2;Gna12^f/f;Gna13^−/− DKO mice. C) Summary plots of contraction parameters as a function of pressure for the two respective groups of vessels. Gna12^f/f;Gna13^+/+ controls N=5, n=10; Myh11-CreER^T2;Gna12^f/f;Gna13^−/−N=7, N=15.

Consequences of Agtr1 (AT₁R) deletion/inhibition.
UMAP and bubble plots of Agtr1a expression in the various IALV cell clusters. B) Slope of F-P relationship for popliteal lymphatics from WT mice with/without treatment with the AT₁R inhibitor losartan (10 μM) and Agtr1a^−/− vessels. WT+losartan N=5, n=10; Agtr1a^−/− N=3, n=6.

Proposed mechanotransduction mechanism underlying pressure-induced chronotropy.
Summary diagram depicting the primary signaling pathway through which changes in pressure regulate the frequency of the ionic pacemaker. GNAQ/GNA11-coupled GPCRs are the primary, but not exclusive mechanosensor. ANO1 is the primary, but not exclusive effector. Kv11 provides at least one source of repolarizing signal (Kim et al., 2023).

Genes for which deletion/inhibition produced significant attenuation or enhancement of the F-P relationship.

Consequences of deletion/inhibition of ion channels on contraction amplitude and/or tone.*

Primers used for qPCR.

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