A Gs-coupled purinergic receptor boosts Ca2+ influx and vascular contractility during diabetic hyperglycemia
- University of California, Davis, United States
- University of Louisville, United States
- Sasse Surgical Associates, United States
- University of Nevada, United States
- VA Northern California Healthcare System, United States
Figures
Figure 1 with 2 supplements
Extracellular nucleotides promote vasoconstriction, CaV1.2 activity and Ser1928 phosphorylation in response to 20 mM D-glucose in murine arterial myocytes.
(A) Representative diameter recordings and summary arterial tone data from pressurized (60 mmHg) wt mouse cerebral arteries before and after application of 20 mM D-glucose in the absence (n = 6 …
-
Figure 1—source data 1
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 1A and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.010
-
Figure 1—source data 2
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 1B.
- https://doi.org/10.7554/eLife.42214.011
-
Figure 1—source data 3
Excel spreadsheet containing the individual numeric values of pSer1928/CaV1.2 relative density analyzed in Figure 1C.
- https://doi.org/10.7554/eLife.42214.012
Figure 1—figure supplement 1
K+-induced arterial constriction in the absence and presence of apyrase, no changes in arterial tone, IBa and pSer1928 in response to 20 mM mannitol, and full-length blots corresponding to Figure 1C.
(A) Bar plot of % constriction in response to high K+ (60 mM) from wt mouse cerebral arteries exposed to 10 mM D-glucose in the absence and presence of apyrase (apy; 0.32 U/ml; n = 6 arteries from …
-
Figure 1—figure supplement 1—source data 1
Excel spreadsheet containing the individual numeric values of 60 mM K+-induced % constriction analyzed in Figure 1—figure supplement 1A.
- https://doi.org/10.7554/eLife.42214.004
-
Figure 1—figure supplement 1—source data 2
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 1—figure supplement 1B and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.005
-
Figure 1—figure supplement 1—source data 3
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 1—figure supplement 1C.
- https://doi.org/10.7554/eLife.42214.006
-
Figure 1—figure supplement 1—source data 4
Excel spreadsheet containing the individual numeric values of pSer1928/α-tubulin relative density analyzed in Figure 1—figure supplement 1E.
- https://doi.org/10.7554/eLife.42214.007
Figure 1—figure supplement 2
Enhanced IBa in response to elevated glucose is prevented by continuous bath perfusion.
(A) Representative IBa recordings from the same cell and (B) summary IBa data from wt mouse cerebral arterial myocytes induced by step depolarizations from −70 to +10 mV during exposure to 10 mM …
-
Figure 1—figure supplement 2—source data 1
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 1—figure supplement 2B.
- https://doi.org/10.7554/eLife.42214.009
Figure 2 with 1 supplement
P2Y11 protein and distribution in arterial myocytes.
(A) Representative blot of immunoreactive bands of expected molecular weight for endogenous P2Y11 (~40 kDa), overexpressed P2Y11-GFP (~70 kDa), and β-actin (~43 kDa) in untransfected, …
-
Figure 2—source data 1
Excel spreadsheet containing the individual numeric values of P2Y11/ β-actin relative density corresponding to values reported in legend of Figure 2B.
- https://doi.org/10.7554/eLife.42214.016
Figure 2—figure supplement 1
Full-length blot for Figure 2C, knock down of P2Y11 in arterial lysates, P2Y11 immunoreactivity in isolated mouse arterial lysates, negative controls for immunofluorescence experiments in Figure 2D and P2Y11 antibody control.
(A) Full-length blot corresponding to Figure 2C. Red box indicates the crop region displayed in main figure. (B) Representative blots of immunoreactive bands of expected molecular weight for P2Y11 …
-
Figure 2—figure supplement 1—source data 1
Excel spreadsheet containing the individual numeric values of P2Y11/ β-actin relative density analyzed in Figure 2—figure supplement 1B.
- https://doi.org/10.7554/eLife.42214.015
Figure 3 with 3 supplements
Nanometer organization of P2Y11 with CaV1.2 and PKAcat in human arterial myocytes.
Representative conventional TIRF images (top) and corresponding GSD reconstruction maps (bottom) from human arterial myocytes labeled for (A) P2Y11 and CaV1.2 and (B) P2Y11 and PKAcat. Lower panels …
-
Figure 3—source data 1
Excel spreadsheet containing the individual numeric values of frequency distribution histograms for cluster area in Figure 3C.
- https://doi.org/10.7554/eLife.42214.025
-
Figure 3—source data 2
Excel spreadsheet containing the individual numeric values for cluster density in Figure 3D.
- https://doi.org/10.7554/eLife.42214.026
-
Figure 3—source data 3
Excel spreadsheet containing the individual numeric values of frequency distribution histograms for intermolecular distance in Figure 3G.
- https://doi.org/10.7554/eLife.42214.027
-
Figure 3—source data 4
Excel spreadsheet containing the individual numeric values of frequency distribution histograms for intermolecular distance in Figure 3H.
- https://doi.org/10.7554/eLife.42214.028
-
Figure 3—source data 5
Excel spreadsheet containing the individual numeric values for % of P2Y11 overlap in Figure 3I.
- https://doi.org/10.7554/eLife.42214.029
Figure 3—figure supplement 1
Validation for PKAcat primary antibody.
Representative confocal images of PKAcat-associated fluorescence (top) and differential interference contrast (DIC, bottom) in wt mouse arterial myocytes stained with an anti-PKAcat antibody (- PKAca…
Figure 3—figure supplement 2
Nanometer organization of P2Y11, CaV1.2 and PKAcat in murine arterial myocytes.
Representative TIRF images (top) and corresponding GSD reconstruction maps (bottom) from murine arterial myocytes labeled for (A) P2Y11 and CaV1.2 and (B) P2Y11 and PKAcat. Lower panels show …
-
Figure 3—figure supplement 2—source data 1
Excel spreadsheet containing the individual numeric values of frequency distribution histograms for cluster area in Figure 3—figure supplement 2C.
- https://doi.org/10.7554/eLife.42214.020
-
Figure 3—figure supplement 2—source data 2
Excel spreadsheet containing the individual numeric values for cluster density in Figure 3—figure supplement 2D.
- https://doi.org/10.7554/eLife.42214.021
-
Figure 3—figure supplement 2—source data 3
Excel spreadsheet containing the individual numeric values of frequency distribution histograms for intermolecular distance in Figure 3—figure supplement 2G.
- https://doi.org/10.7554/eLife.42214.022
-
Figure 3—figure supplement 2—source data 4
Excel spreadsheet containing the individual numeric values of frequency distribution histograms for intermolecular distance in Figure 3—figure supplement 2H.
- https://doi.org/10.7554/eLife.42214.023
Figure 3—figure supplement 3
Negative controls for GSD images in human and murine arterial myocytes, and experimental and randomized reconstruction maps.
Representative TIRF images (top) and corresponding GSD reconstruction maps (bottom) from freshly dissociated (A) human (n = 6 cells from four humans) and (B) mouse (n = 6 cells from six mice) …
Figure 4 with 1 supplement
P2Y11 associates with CaV1.2 and PKAcat in human and murine arterial myocytes.
(A) Exemplary fluorescence PLA (red)/DAPI (blue) and differential interference contrast (right) images of human arterial myocytes labeled for P2Y11 + CaV1.2 and P2Y11 + PKAcat. (B) Quantification of …
-
Figure 4—source data 1
Excel spreadsheet containing the individual numeric values of puncta/µm2 graphs in Figure 4B.
- https://doi.org/10.7554/eLife.42214.032
-
Figure 4—source data 2
Excel spreadsheet containing the individual numeric values of puncta/µm2 graphs in Figure 4D.
- https://doi.org/10.7554/eLife.42214.033
Figure 4—figure supplement 1
Negative controls for PLA in human and murine arterial myocytes.
Fluorescence PLA (red)/DAPI (blue) (left) and differential interference contrast (right) images of negative controls for human (A) and murine (B) arterial myocytes labeled with one 1° antibody (CaV1.…
Figure 5 with 1 supplement
Glucose and the P2Y11 agonist NF546 increase sarcolemmal cAMP synthesis in arterial myocytes, and these effects are prevented by the P2Y11 antagonist NF340.
(A) Time course of YFP/CFP (donor/acceptor) FRET ratios (normalized to resting levels before treatment) in human arterial myocytes expressing the ICUE3-PM biosensor in response to 15 mM D-glucose …
-
Figure 5—source data 1
Excel spreadsheet containing the individual numeric values for maximum FRET responses in Figure 5B.
- https://doi.org/10.7554/eLife.42214.039
-
Figure 5—source data 2
Excel spreadsheet containing the individual numeric values for maximum FRET responses in Figure 5D.
- https://doi.org/10.7554/eLife.42214.040
Figure 5—figure supplement 1
The P2Y11 agonist NF546 increases cAMP and this is blocked in the presence of the P2Y11 inhibitor NF340 but not with the P2Y1 inhibitor MSR2179 or P2Y6 inhibitor MRS2578 in tsA-201 cells, and no increases in cAMP in arterial myocytes with mannitol.
(A) Time course of YFP/CFP (donor/acceptor) FRET ratios (normalized to resting levels before treatment) in tsA-201 cells expressing the ICUE3-PM biosensor in response to 500 nM NF546 (black; n = 19 …
-
Figure 5—figure supplement 1—source data 1
Excel spreadsheet containing the individual numeric values for maximum FRET responses in Figure 5—figure supplement 1A.
- https://doi.org/10.7554/eLife.42214.036
-
Figure 5—figure supplement 1—source data 2
Excel spreadsheet containing the individual numeric values for maximum FRET responses in Figure 5—figure supplement 1B.
- https://doi.org/10.7554/eLife.42214.037
-
Figure 5—figure supplement 1—source data 3
Excel spreadsheet containing the individual numeric values for maximum FRET responses in Figure 5—figure supplement 1C.
- https://doi.org/10.7554/eLife.42214.038
Figure 6 with 1 supplement
The P2Y11 inhibitor NF340 prevents glucose-induced elevations in Ser1928 phosphorylation, LTCC activity, and vasoconstriction in human and murine arterial myocytes.
(A) Representative IBa recordings from the same cell and (B) summary current density data obtained from freshly dissociated human arterial myocytes before and after increasing extracellular …
-
Figure 6—source data 1
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 6B.
- https://doi.org/10.7554/eLife.42214.046
-
Figure 6—source data 2
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 6D.
- https://doi.org/10.7554/eLife.42214.047
-
Figure 6—source data 3
Excel spreadsheet containing the individual numeric values of pSer1928/CaV1.2 relative density analyzed in Figure 6E.
- https://doi.org/10.7554/eLife.42214.048
-
Figure 6—source data 4
Excel spreadsheet containing the individual numeric values of pSer1928/CaV1.2 relative density analyzed in Figure 6F.
- https://doi.org/10.7554/eLife.42214.049
-
Figure 6—source data 5
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 6H and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.050
-
Figure 6—source data 6
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 6J and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.051
Figure 6—figure supplement 1
Full-length blots for Figure 6E and F, high K+induced constriction in arteries pretreated with NF340 and MRS2578 corresponding to data in Figure 6, and glucose-induced vasoconstriction is not prevented or inhibited by a selective P2Y1 antagonist.
Representative full-length blots corresponding to (A) Figure 6E and (B) Figure 6F. Red boxes indicate the crop region displayed in the main figure. (C) Bar plot of % constriction in response to 60 …
-
Figure 6—figure supplement 1—source data 1
Excel spreadsheet containing the individual numeric values of 60 mM K+-induced % constriction analyzed in Figure 6—figure supplement 1C.
- https://doi.org/10.7554/eLife.42214.043
-
Figure 6—figure supplement 1—source data 2
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 6—figure supplement 1D and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.044
-
Figure 6—figure supplement 1—source data 3
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 6—figure supplement 1E and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.045
Figure 7 with 1 supplement
The P2Y11 agonist NF546 increases Ser1928 phosphorylation, LTCC activity, and induces vasoconstriction.
(A) Representative IBa recordings from the same cell (top) and summary IBa data (bottom) from freshly dissociated human arterial myocytes in response to step depolarizations from −70 to +10 mV …
-
Figure 7—source data 1
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 7A.
- https://doi.org/10.7554/eLife.42214.057
-
Figure 7—source data 2
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 7B.
- https://doi.org/10.7554/eLife.42214.058
-
Figure 7—source data 3
Excel spreadsheet containing the individual numeric values of pSer1928/CaV1.2 relative density analyzed in Figure 7C.
- https://doi.org/10.7554/eLife.42214.059
-
Figure 7—source data 4
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 7D.
- https://doi.org/10.7554/eLife.42214.060
-
Figure 7—source data 5
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 7E and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.061
-
Figure 7—source data 6
Excel spreadsheet containing the individual numeric values of % arterial tone analyzed in Figure 7F and corresponding raw diameters.
- https://doi.org/10.7554/eLife.42214.062
Figure 7—figure supplement 1
D-glucose and NF546 elicit changes in IBa of similar magnitude in mouse arterial myocytes, ATPγS increases IBa in arterial myocytes, full-length blots corresponding to data in Figure 7C, and high K+-induced constriction in wt and S1928A arteries treated with NF546 corresponding to data in Figure 7.
(A) Summary data showing changes in IBa in response to elevating D-glucose from 10 mM to 20 mM (−1.9 ± 0.2 pA/pF; n = 11 cells from five mice) or after application of the P2Y11 agonist NF546 (500 nM;…
-
Figure 7—figure supplement 1—source data 1
Excel spreadsheet containing the individual numeric values of change in current density analyzed in Figure 7—figure supplement 1A.
- https://doi.org/10.7554/eLife.42214.054
-
Figure 7—figure supplement 1—source data 2
Excel spreadsheet containing the individual numeric values of current density analyzed in Figure 7—figure supplement 1B.
- https://doi.org/10.7554/eLife.42214.055
-
Figure 7—figure supplement 1—source data 3
Excel spreadsheet containing the individual numeric values of 60 mM K+-induced % constriction analyzed in Figure 7—figure supplement 1D.
- https://doi.org/10.7554/eLife.42214.056
Figure 8 with 1 supplement
Augmented LTCC activity and Ser1928 phosphorylation in response to chronic extracellular glucose elevations are prevented in the presence of the P2Y11 antagonist NF340.
(A) Representative single LTCC recordings obtained during a 2 s step depolarization from −80 to −30 mV and (B) bar plot of LTCC nPo in arterial myocytes isolated from mouse cerebral arteries …
-
Figure 8—source data 1
Excel spreadsheet containing the individual numeric values of LTCC nPo analyzed in Figure 8B.
- https://doi.org/10.7554/eLife.42214.066
-
Figure 8—source data 2
Excel spreadsheet containing the individual numeric values of pSer1928/CaV1.2 relative density analyzed in Figure 8C.
- https://doi.org/10.7554/eLife.42214.067
Figure 8—figure supplement 1
Full-length blots corresponding to data in Figure 8C and unchanged LTCC nPo in response to chronic elevations in extracellular glucose in S1928A arterial myocytes.
(A) Full-length blots corresponding to Figure 8C. Red boxes indicate the crop region displayed in the main figure. (B) Representative single LTCC recordings obtained during a 2 s step depolarization …
-
Figure 8—figure supplement 1—source data 1
Excel spreadsheet containing the individual numeric values of LTCC nPo analyzed in Figure 8—figure supplement 1B.
Titles for supplementary files.
- https://doi.org/10.7554/eLife.42214.065
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Mus musculus), C57BL/6J | wild-type | Jackson Laboratories | stock # 000664 | |
| Strain (Mus musculus) | S1928A | (Lemke et al., 2008) | ||
| Cell line (human embryonal kidney) | tsA-201 | Sigma-Aldrich | 96121229 | SV40 transformed |
| Oligodeoxynucleotide, sense | P2Y11 SNS ODN | Integrated DNA Technologies | human P2Y11 sequence (NC_000019.10): 5’-CAACGTCTCGGGTAAGGAGAA-3’ and 5’-ATGAGGAAGGAAACGTGGGT-3’ | last three bases on the 3’ end were phosphoro-thioated |
| Oligodeoxynucleotide, antisense | P2Y11 ANS ODN | Integrated DNA Technologies | human P2Y11 sequence (NC_000019.10): 5’-CAAGGCCACCCTAACCACTG-3’ and 5’-CTCTCCCTTCCCTGCGTTA-3’ | last three bases on the 3’ end were phosphoro-thioated |
| DNA construct | human P2Y11 | UMR cDNA Resource Center | www.cDNA.org; clone ID P2Y1100000 | tagged with GFP at C- terminus |
| Antibody | anti-FP1 (CaV1.2; custom rabbit) | (Davare et al., 2000) | dilutions: 1:100 for immunoblot and PLA; 10 µg/mL for GSD | |
| Antibody | anti-CH3P (pSer1928, custom rabbit) | (Davare et al., 2000) | 1:50-1:100 dilutions | |
| Antibody | anti-β-actin (mouse monoclonal) | Abcam | ab8226 RRID: AB_30637 | 1:1000 dilution |
| Antibody | anti-α-tubulin (mouse monoclonal) | Active Motif | 39527; clone 5-B-1–2 | 1:500 dilution |
| Antibody | anti-P2Y11 (rabbit polyclonal) | Abcam | ab180739 | 1:100-1:200 dilutions |
| Antibody | anti-P2Y11 (goat polyclonal) | Santa Cruz Biotechnology | sc-69588; clone C-18 RRID: AB_21559 | dilutions: 1:100 for stainings (PLA, classical) and 10 µg/mL for GSD |
| Antibody | anti-PKAcatα, β, γ (mouse monoclonal) | Santa Cruz Biotechnology | sc-28315; clone A-2 | 1:200 dilution |
| Antibody | PKAcatα, β, γ blocking peptide | Santa Cruz Biotechnology | sc-28315 P; clone A-2 | 1:20 dilution for 1 µg of primary antibody |
| Antibody | anti-PKAcat α, β, γ (rabbit polyclonal) | Santa Cruz Biotechnology | sc-28892; clone H-95 | dilutions: 1:200 for PLA and 10 µg/mL for GSD |
| Antibody | Alexa Fluor 488 conjugate of wheat germ agglutinin | Life Technologies | W7024 | |
| Antibody | Alexa Fluor 568-conjugated donkey anti-goat | Molecular Probes | A11057 RRID: AB_142581 | 5 mg/mL dilution |
| Antibody | Alexa Fluor 568-conjugated donkey anti-mouse | Molecular Probes | A10037 | 5 mg/mL dilution |
| Antibody | Alexa Fluor 568-conjugated donkey anti-rabbit | Molecular Probes | A11011 RRID: AB_143157 | 2 µg/mL dilution |
| Antibody | Alexa Fluor 647-conjugated donkey anti-goat | Molecular Probes | A21447 RRID: AB_141844 | 2 µg/mL dilution |
| Antibody | goat anti-rabbit IgG (H + L)-horseradish peroxidase conjugate | Bio-Rad | 170–6515 RRID: AB_11125142 | 1:10000 dilution |
| Antibody | goat anti-mouse IgG (H + L)-horseradish peroxidase conjugate | Bio-Rad | 170–6516 | 1:10000 dilution |
| Chemical compound, drug | sodium pentobarbital (Fatal-Plus) | Vortech Pharma-ceuticals | NDC 0298-9373-68 | |
| Chemical compound, drug | mannitol | Fisher Scientific | BP686 | |
| Chemical compound, drug | NF340 | Santa Cruz Biotechnology | sc-361274 | |
| Chemical compound, drug | NF546 | Tocris | 3892 | |
| Chemical compound, drug | apyrase | New England Biolabs | M0398L | |
| Chemical compound, drug | nifedipine | Sigma-Aldrich | N7634 | |
| Chemical compound, drug | Bay K-8644 | Sigma-Aldrich | 71145-03-4 | |
| Chemical compound, drug | forskolin | Sigma-Aldrich | F6886 | |
| Chemical compound, drug | amphotericin B | Sigma-Aldrich | A4888 | |
| Chemical compound, drug | MRS2578 | Santa Cruz Biotechnology | sc-204103A | |
| Chemical compound, drug | MRS2179 | Tocris | 1454889-37-2 | |
| Chemical compound, drug | protein kinase A inhbitor (PKI) | Sigma-Aldrich | P9115 | fragment 14–22, myristoylated trifluoroacetate salt |
| Chemical compound, drug | Rp-Adenosine 3’,5’-cyclic monophos-phorothioate triethylam-monium salt (rpcAMP) | Sigma-Aldrich | A165 | |
| Chemical compound, drug | Adenosine 5’-[γ-thio] triphosphate tetralithium salt (ATPγS) | Sigma-Aldrich | A1388 | |
| Software, algorithm | GraphPad Prism | GraphPad Prism, RRID: SCR_002798 | ||
| Software, algorithm | ImageJ | Fiji, RRID: SCR_002285 | ||
| Software, algorithm | pCLAMP10 | Molecular Devices | electrophysiology | |
| Software, algorithm | LASAF | Leica | GSD | |
| Software, algorithm | IonOptix | IonOptix | arterial diameter recordings | |
| Software, algorithm | Metaflor | Molecular Devices | FRET |
Additional files
-
Supplementary file 1
K+-induced constriction and baseline and passive diameters of arteries from wild type and S1928A mice.
- https://doi.org/10.7554/eLife.42214.068
-
Supplementary file 2
Arterial tone from wild type and S1928A mouse arteries.
- https://doi.org/10.7554/eLife.42214.069
-
Supplementary file 3
Human nondiabetic patients undergoing surgical sleeve gastrectomy.
- https://doi.org/10.7554/eLife.42214.070
-
Transparent reporting form
- https://doi.org/10.7554/eLife.42214.071
