Research ArticleVASCULAR BIOLOGY

Voltage-dependent Ca2+ entry into smooth muscle during contraction promotes endothelium-mediated feedback vasodilation in arterioles

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Science Signaling  04 Jul 2017:
Vol. 10, Issue 486, eaal3806
DOI: 10.1126/scisignal.aal3806
  • Fig. 1 Direct activation of L-type VDCCs in arterioles with BayK increases Ca2+ events in endothelial cells.

    (A and B) Confocal fluorescence images of endothelial cells (ECs) loaded with the Ca2+ indicator OGB-1 viewed at the bottom plane in pressurized arterioles with myogenic tone in the absence (A) and presence (B) of nifedipine (Nif), at baseline and with the indicated concentrations of the L-type VDCC agonist BayK (movie S1); scale bar, 30 μm. Representative fluorescence intensity data shown as line scans [corresponding to white lines 1 to 3 in (A) or (B)] and fluorescence traces [F/F0; corresponding to colored subcellular regions of interest (ROIs) in the images]. (C and D) Bar graphs summarize the effect of BayK on the frequency of EC Ca2+ events in active cells (C) and the overall percentage of active cells (D) in the absence and presence of Nif. Data are means ± SEM (≥10 ECs in each field of view from n = 3 to 6 arterioles from different animals); *P < 0.05 compared with baseline; #P < 0.05 compared to control; §P < 0.05 compared to 3 nM BayK.

  • Fig. 2 VECTors occur in myoendothelial microdomains and are amplified by IP3Rs in endothelial cells.

    (A) Confocal fluorescence images of endothelial cells (ECs) loaded with heparin-Cy5 (yellow) and the corresponding IP3R1 fluorescence in the same arteriole (magenta). Nuclear staining (cyan) is indicated by dashed lines. Representative of three experiments; scale bar, 15 μm. (B) Simultaneous confocal fluorescence images of the internal elastic lamina labeled with AF-633 and ECs loaded with the Ca2+ indicator OGB-1 in a pressurized arteriole with myogenic tone during treatment with heparin and BayK; scale bar, 15 μm. (C) Representative fluorescence intensity data shown as line scans [corresponding to white lines Cell1 and Cell2 in (B)] and fluorescence traces [F/F0; corresponding to colored subcellular ROIs in (B)]. Examples of VECTors are indicated by arrowheads. W, wave. (D) Bar graph summarizes the effects of BayK on the percentage of EC Ca2+ events in the myoendothelial (ME) microdomain visible as holes through the internal elastic lamina. (E to H) Bar graphs summarize the effect of heparin on the frequency of EC Ca2+ events in active cells during BayK (E), the overall percentage of active cells (F), the percentage of these events that propagated along cells and were considered to be waves (G), and subsequent responses to the endothelium-dependent TRPV4 agonist GSK1016790A (GSK) (H). Data are means ± SEM (n = 4 arterioles from different animals); *P < 0.05 compared with baseline; P < 0.05 compared to baseline in the presence of heparin; #P < 0.05 compared to control. (E to G) Control data used for statistical comparisons for BayK are shown in Fig. 1; see (1) for control GSK data. Experiments using 5-kDa heparin were only included if the EC Ca2+ response to acetylcholine was markedly reduced (fig. S6D).

  • Fig. 3 Lack of L-type VDCCs in arteriolar endothelium.

    (A) Bright-field images of a freshly isolated (left) and pinned (right) endothelial cell (EC) tube isolated from a skeletal muscle arteriole; scale bar, 100 μm. (B and C) Confocal fluorescence images of two tubes loaded with Fluo-4 to detect Ca2+; scale bar, 30 μm. Representative fluorescence intensity data showing both line scans [corresponding to white lines 1 and 2 in (B) and (C)] and fluorescence traces [F/F0; corresponding to subcellular ROIs on 1 and 2 (colored squares)] in response to either BayK (B; movie S2) or phenylephrine (PE) (C, movie S3) and the muscarinic agonist acetylcholine (ACh). (D and E) Bar graphs summarize the effects of BayK, KCl, phenylephrine, and ACh on the frequency of EC Ca2+ events (D) and the percentage of active cells (E). Data are means ± SEM (n = 3 to 9 EC tubes from different animals); *P < 0.05 compared with baseline. (F and G) Bar graphs summarize the gene expression for (F) EC (PECAM-1, Pecam1) and smooth muscle cell (α–smooth muscle actin, Acta2) markers; and (G) L- and T-type CaV channel isoforms (CaV1.2, Cacna1c; Cav3.1, Cacna1g; and CaV3.2, Cacna1h) in arterioles and EC tubes. Data are means ± SEM; n = 4 sets of pooled mRNA samples from four animals. (H and I) Immunofluorescence for CaV1.2 (yellow) in a pressurized arteriole; scale bar, 20 μm. Vascular smooth muscle cells (VSMCs) have vertically aligned nuclei (blue), and ECs have horizontally aligned nuclei (blue). Representative of three arterioles from different animals.

  • Fig. 4 Depolarization opens VDCCs in smooth muscle and increases Ca2+ activity in endothelial cells.

    (A and B) Confocal fluorescence images of endothelial cells (ECs) loaded with the Ca2+ indicator OGB-1 in pressurized skeletal muscle arterioles with myogenic tone in the absence (A) and presence (B) of nifedipine (Nif), at baseline and with KCl (see also movie S4); scale bar, 30 μm. Representative fluorescence intensity data shown as line scans [corresponding to white lines 1 and 2 in (A) or (B)] and fluorescence traces (F/F0; corresponding to colored subcellular ROIs in the images). (C to F) Bar graphs summarize the effect of the KV channel blocker 4-aminopyridine (4-AP; movie S5) and KCl on the frequency of EC Ca2+ events in active cells (C) and the overall percentage of active cells (D), as well as the effects of KCl on the frequency of vascular smooth muscle (VSM) Ca2+ events (E) and the percentage of these events that propagated along cells and were considered to be waves (F) in the absence and presence of Nif. Data are means ± SEM (≥10 ECs or VSMCs in each field of view from n = 3 to 6 arterioles from different animals); *P < 0.05 compared with control baseline; P < 0.05 compared to baseline in the presence of 1 μM Nif; #P < 0.05 compared to control.

  • Fig. 5 Activation of α1-adrenoceptors in smooth muscle increases Ca2+ activity in endothelial cells.

    (A and B) Confocal fluorescence images of endothelial cells (ECs) loaded with the Ca2+ indicator OGB-1 from pressurized arterioles with myogenic tone in the absence (A) and presence (B) of nifedipine (Nif) at baseline and with the α1-adrenoceptor agonist phenylephrine (PE) (movie S6); scale bar, 30 μm. (C and D) Representative fluorescence intensity data shown as line scans [corresponding to white lines 1 and 2 in (A) or (B)] and fluorescence traces (F/F0; corresponding to colored subcellular ROIs in the images). (C and D) Bar graphs summarize the effects of PE on the frequency of EC Ca2+ events in active cells (C) and the overall percentage active cells (D) in the absence and presence of Nif. Data are means ± SEM (≥10 ECs in each field of view from n = 3 to 4 arterioles from different animals); *P < 0.05 compared with control baseline; P < 0.05 compared to baseline in the presence of 1 μM Nif; #P < 0.05 compared to control.

  • Fig. 6 IKCa channels in endothelial cells suppress vasoconstriction and initiate vasomotion.

    (A to C) Representative diameter traces in pressurized arterioles for (A) single, cumulative exposures to the L-type VDCC agonist BayK before and after incubation (in a separate arteriole) with 1 μM TRAM-34 to block IKCa channels, as well as consecutive additions of (B) 25 mM isotonic KCl to the superfusion solution in the absence (orange) and presence (brown) of nifedipine (Nif) (1 μM); and (C) the α1-adrenoceptor agonist phenylephrine in the absence (green) and presence of 1 μM TRAM-34 (purple). (D) Bar graphs summarize the effect of the three vasoconstrictors under control conditions; and with 100 μM l-NAME (to block nitric oxide synthase); 100 nM apamin (to block SKCa channels); 1 μM TRAM-34; apamin and TRAM-34 (Ap + TR); apamin, TRAM-34, and l-NAME (LN + Ap + TR); 100 nM iberiotoxin (IbTx, an inhibitor of BKCa channels); or in endothelium-denuded arterioles (−EC). Data are means ± SEM (n = 3 to 28 arterioles from different animals); *P < 0.05 compared to the corresponding control response for each vasoconstrictor; 5-min average is indicated by gray bars in (A) to (C). Figure S12 provides more details regarding these experiments.

  • Fig. 7 Defining the trigger point for activation of endothelial cell (EC) Ca2+ events during vasoconstriction.

    (A) Confocal images of the top and bottom walls of an arteriole stained with the elastin dye AF-633, indicating the points on the internal elastic lamina (IEL, gray crosses) tracked in the y axis over time and the superimposed transmitted light image (Trans); scale bar, 50 μm. (B) Original traces showing the tracked IEL in three arterioles (colored lines 1 to 3). The difference between the two traces reflect internal diameter (black dashed line) and are plotted for each arteriole. Phenylephrine (PE) was added to the superfusion solution for the period indicated by the bar. (C) Simultaneously acquired confocal images of ECs loaded with the Ca2+ indicator OGB-1 and elastin stained with AF-633. Representative fluorescence intensity data are shown as EC Ca2+ events and elastin fluorescence corresponding to colored boxes spanning the full range of movement; a.u., arbitrary units. Increased EC Ca2+ was observed at the point indicated by the black arrow (inset, 3× zoom of gray box). The tracked movement of the IEL is overlaid for comparison (blue trace in each panel) and corresponds to crosses shown in the images before (magenta) and during (lighter blue) vasoconstriction to 3 μM phenylephrine. EEL, external elastic lamina; scale bar, 30 μm; blue arrows represent 0 μm in the y axis. (D) Representative time courses of wall movement in response to KCl and phenylephrine in the same arteriole. The starting positions (magenta crosses) reflect myogenic tone, and the threshold to trigger vasoconstrictor-mediated activation of EC Ca2+ events is indicated by light blue crosses. Bar graphs summarize the distance moved (extrapolated to an approximated change in diameter) to reach the trigger point for EC Ca2+ activation for each vasoconstrictor. Data are means ± SEM of n = 6 arterioles from different animals. Myogenic tone, 53.4 ± 2.3%.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/486/eaal3806/DC1

    Fig. S1. Myogenic tone relies on L-type VDCCs in arterioles.

    Fig. S2. Methods for analyzing endothelial cell Ca2+ events in cannulated arterioles.

    Fig. S3. Block of arteriole constriction using blebbistatin.

    Fig. S4. Characteristics of arteriolar endothelial cell Ca2+ events.

    Fig. S5. Profile of Ca2+ release pathways in arteriolar endothelial cells.

    Fig. S6. Loading heparin into arteriolar endothelial cells inhibits IP3Rs.

    Fig. S7. Dichotomous effects of 2-APB.

    Fig. S8. Observations on the use of cell-permeant inhibitors of phospholipase C and IP3Rs.

    Fig. S9. Effect of ryanodine on arteriolar Ca2+ events.

    Fig. S10. Effect of nifedipine on vasoconstriction to phenylephrine.

    Fig. S11. Simultaneous imaging of endothelial and smooth muscle cell Ca2+ events at the arteriolar midplane.

    Fig. S12. Characteristics of arteriole vasoconstriction to phenylephrine.

    Fig. S13. Intercellular Ca2+ circuit in skeletal muscle arterioles.

    Table S1. Effect of nifedipine on vasoconstriction to BayK and KCl.

    Table S2. Details of rat genes detected with TaqMan probes.

    Movie S1. Time course of endothelial cell Ca2+ events in response to BayK in a pressurized arteriole.

    Movie S2. Time course of endothelial cell Ca2+ events in response to BayK in an isolated tube.

    Movie S3. Time course of endothelial cell Ca2+ events in response to phenylephrine in an isolated tube.

    Movie S4. Time course of endothelial cell Ca2+ events in response to KCl in a pressurized arteriole.

    Movie S5. Time course of endothelial cell Ca2+ events in response to 4-aminopyridine in a pressurized arteriole.

    Movie S6. Time course of endothelial cell Ca2+ events in response to phenylephrine in a pressurized arteriole.

  • Supplementary Materials for:

    Voltage-dependent Ca2+ entry into smooth muscle during contraction promotes endothelium-mediated feedback vasodilation in arterioles

    Christopher J. Garland, Pooneh Bagher, Chloe Powell, Xi Ye, Hamish A.L. Lemmey, Lyudmyla Borysova, Kim A. Dora*

    *Corresponding author. Email: kim.dora{at}pharm.ox.ac.uk

    This PDF file includes:

    • Fig. S1. Myogenic tone relies on L-type VDCCs in arterioles.
    • Fig. S2. Methods for analyzing endothelial cell Ca2+ events in cannulated arterioles.
    • Fig. S3. Block of arteriole constriction using blebbistatin.
    • Fig. S4. Characteristics of arteriolar endothelial cell Ca2+ events.
    • Fig. S5. Profile of Ca2+ release pathways in arteriolar endothelial cells.
    • Fig. S6. Loading heparin into arteriolar endothelial cells inhibits IP3Rs.
    • Fig. S7. Dichotomous effects of 2-APB.
    • Fig. S8. Observations on the use of cell-permeant inhibitors of phospholipase C and IP3Rs.
    • Fig. S9. Effect of ryanodine on arteriolar Ca2+ events.
    • Fig. S10. Effect of nifedipine on vasoconstriction to phenylephrine.
    • Fig. S11. Simultaneous imaging of endothelial and smooth muscle cell Ca2+ events at the arteriolar midplane.
    • Fig. S12. Characteristics of arteriole vasoconstriction to phenylephrine.
    • Fig. S13. Intercellular Ca2+ circuit in skeletal muscle arterioles.
    • Table S1. Effect of nifedipine on vasoconstriction to BayK and KCl.
    • Table S2. Details of rat genes detected with TaqMan probes.
    • Legends for movies S1 to S6

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.56 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Time course of endothelial cell Ca2+ events in response to BayK in a pressurized arteriole.
    • Movie S2 (.mov format). Time course of endothelial cell Ca2+ events in response to BayK in an isolated tube.
    • Movie S3 (.mov format). Time course of endothelial cell Ca2+ events in response to phenylephrine in an isolated tube.
    • Movie S4 (.mov format). Time course of endothelial cell Ca2+ events in response to KCl in a pressurized arteriole.
    • Movie S5 (.mov format). Time course of endothelial cell Ca2+ events in response to 4-aminopyridine in a pressurized arteriole.
    • Movie S6 (.mov format). Time course of endothelial cell Ca2+ events in response to phenylephrine in a pressurized arteriole.

    Citation: C. J. Garland, P. Bagher, C. Powell, X. Ye, H. A.L. Lemmey, L. Borysova, K. A. Dora, Voltage-dependent Ca2+ entry into smooth muscle during contraction promotes endotheliummediated feedback vasodilation in arterioles. Sci. Signal. 10, eaal3806 (2017).

    © 2017 American Association for the Advancement of Science

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