1. Arterioscler Thromb Vasc Biol
Atrial Natriuretic Peptide–Mediated Inhibition of Microcirculatory Endothelial Ca2+ and Permeability Response to Histamine Involves cGMP-Dependent Protein Kinase I and TRPC6 ChannelsSignificance
by Wen Chen, Heike Oberwinkler, Franziska Werner, Birgit Gaßner, Hitoshi Nakagawa, Robert Feil, Franz Hofmann, Jens Schlossmann, Alexander Dietrich, Thomas Gudermann, Motohiro Nishida, Sabrina Del Galdo, Thomas Wieland, and Michaela Kuhn
Arterioscler Thromb Vasc Biol
Volume 33(9):
August 14, 2013
Copyright © American Heart Association, Inc. All rights reserved.

2. Atrial natriuretic peptide (ANP) prevents the activation of perivascular mast cells and mast cell–induced vascular leakage of fluorescein isothiocyanate (FITC)-dextran in the mouse cremaster microcirculation.
Atrial natriuretic peptide (ANP) prevents the activation of perivascular mast cells and mast cell–induced vascular leakage of fluorescein isothiocyanate (FITC)-dextran in the mouse cremaster microcirculation. A, Time course of changes in net integrated optical intensity (IOI; an index of permeability) during 20 minutes of continuous local ANP (100 nmol/L) or vehicle superfusion followed by a 10-minute superfusion of the mast cell secretagogue C48/80 (5 μg/mL). B, Number of extravasated degranulated mast cells (stained with ruthenium red) after superfusion with vehicle or C48/80 (with/without ANP pretreatment; n=6 mice per treatment; *P<0.05 vs vehicle). Top in A and B, Representative original photographs.
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

3. Atrial natriuretic peptide (ANP), via the endothelial guanylyl cyclase-A (GC-A) receptor, prevents histamine-induced acute vascular leakage of fluorescein isothiocyanate (FITC)-dextran.
Atrial natriuretic peptide (ANP), via the endothelial guanylyl cyclase-A (GC-A) receptor, prevents histamine-induced acute vascular leakage of fluorescein isothiocyanate (FITC)-dextran. Time course of changes in permeability during 20 minutes of continuous local ANP (100 nmol/L) or vehicle and additional 10 minutes of histamine (2 μmol/L) superfusion in control mice (GC-Afl/fl; A) and in mice with conditional, endothelial GC-A deletion (EC GC-A KO; B; n=7–8 mice per genotype and treatment; *P<0.05 vs vehicle). Top in A and B, Representative original photographs.
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

4. The antihistaminergic actions of atrial natriuretic peptide (ANP) are mediated by endothelial cGMP–dependent protein kinase I (cGKI).
The antihistaminergic actions of atrial natriuretic peptide (ANP) are mediated by endothelial cGMP–dependent protein kinase I (cGKI). A, cGKI was detected in microvascular lung endothelial cells (MLECs) from control mice (cGKIfl/fl) but was almost absent in cultured MLEC preparations from EC cGKI KO mice (cGKIfl/fl; Tie2-Cre; n=3). B, Representative Western blots showing that cGKI expression levels in whole brain and heart protein extracts were not different between genotypes. C and D, Intravital microscopy, m. cremaster. Time course of changes in permeability during 20 minutes of continuous local ANP (100 nmol/L) or vehicle and additional 10 minutes of histamine (2 μmol/L) superfusion in control (cGKIfl/fl; C) and in EC cGKI KO mice (D; n=6 mice per genotype and treatment; *P<0.05 vs vehicle). Right in C and D, Representative original photographs.
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

5. Atrial natriuretic peptide (ANP) stimulates cGMP/cGKI signaling in cultured human dermal microvascular endothelial cells (HDMECs) but does not prevent histamine-induced activation of RhoA/ROCK.
Atrial natriuretic peptide (ANP) stimulates cGMP/cGKI signaling in cultured human dermal microvascular endothelial cells (HDMECs) but does not prevent histamine-induced activation of RhoA/ROCK. A, Effect of ANP on cyclic GMP content (in the presence of IBMX). B, Expression and ANP (10 and 100 nmol/L; 30 minutes)-induced activation of cGKI (cGKI-dependent phosphorylation of Ser239 in vasodilator-stimulated phosphoprotein [VASP]) with/without pretreatment with the cGKI inhibitor Rp-8-PET-cGMPs (100 μmol/L; 15 minutes). As also shown, ANP does not stimulate cAMP/PKA-dependent phosphorylation of VASP at Ser157. Top, Representative Western blot. Bottom, Results from 3 independent experiments. *P<0.05 compared with vehicle. C, Rho-A activation by histamine (10 μmol/L; 30 seconds) with/without ANP pretreatment (1 μmol/L; 10 minutes) was analyzed by effector pull-down assay. A representative assay is shown. D, Histamine (2 μmol/L; 30 minutes)-induced RhoA/ROCK-dependent phosphorylation of MYPT1; HDMECs were pretreated with vehicle or ANP (10 or 100 nmol/L) during 30 minutes (all n=3; *P<0.05 compared with vehicle).
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

6. In human dermal microvascular endothelial cells (HDMECs), atrial natriuretic peptide (ANP)/cGMP/cGKI signaling diminishes the Ca2+i responses to histamine.
In human dermal microvascular endothelial cells (HDMECs), atrial natriuretic peptide (ANP)/cGMP/cGKI signaling diminishes the Ca2+i responses to histamine. A, HDMECs were loaded with a calcium-sensitive fluorophore, and basal fluorescence was detected. The cells were pretreated with vehicle or ANP (100 nmol/L) during 15 minutes. After subsequent stimulation with 2 μmol/L histamine, the fluorescence intensity was recorded every 5 seconds for 1000 seconds. Measurements were performed in 10 replicates, n=3. B, Reverse transcription polymerase chain reaction analyses of inositol 1,4,5-trisphosphate receptor I–associated protein (IRAG), transient receptor potential canonical (TRPC) 3, TRPC6 and regulator of G-protein signaling (RGS)2 mRNA expression in human umbilical vein endothelial cells (HUVECs) and HDMECs. mRNA from human lung was used as positive control.
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

7. Transient receptor potential canonical (TRPC) 6 channels are essential for the endothelial calcium and hyperpermeability responses to histamine.
Transient receptor potential canonical (TRPC) 6 channels are essential for the endothelial calcium and hyperpermeability responses to histamine. A, Human dermal microvascular endothelial cells (HDMECs) were loaded with a calcium-sensitive fluorophore, and basal fluorescence was detected. The cells were pretreated with vehicle or SKF96365 (10 μmol/L) during 15 minutes. After subsequent stimulation with 2 μmol/L histamine, the fluorescence intensity was recorded every 5 seconds for 1000 seconds. Measurements were performed in 10 replicates, n=2 independent experiments. B, Intravital microscopy, m. cremaster. Time course of changes in permeability in response to histamine (2 μmol/L; 10 minutes) superfusion in wild-type as compared with TRPC6−/− mice (n=5 mice per genotype and treatment; *P<0.05 vs wild-type mice). Right in A and B, Representative original tracings and pictures.
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

8. Atrial natriuretic peptide (ANP), via cGKI, induces an inhibitory phosphorylation of transient receptor potential canonical (TRPC) 6 proteins (in vitro) and attenuates the hyperpermeability responses to the TRPC6 activator, hyperforin (in vivo).
Atrial natriuretic peptide (ANP), via cGKI, induces an inhibitory phosphorylation of transient receptor potential canonical (TRPC) 6 proteins (in vitro) and attenuates the hyperpermeability responses to the TRPC6 activator, hyperforin (in vivo). A, cGKI-dependent phosphorylation of Thr69 in TRPC6 by ANP. Phosphorylation of TRPC6 at Thr69 induced by ANP (10 nmol/L; 30 minutes incubation) in HEK 293 cells coexpressing guanylyl cyclase-A (GC-A), cGKI, and TRPC6 as well as in wild-type MLECs. The immunoreactive band is almost absent in TRPC3-expressing HEK 293 cells and TRPC6-deficient (KO) MLECs. Top, Original western. Bottom, Ratio of P-TRPC6/GAPDH. B and C, Intravital microscopy, m. cremaster. Time course of changes in net integrated optical intensity (IOI; an index of permeability) in response to hyperforin (local superfusion with 10 μmol/L during 5 minutes) in TRPC6−/− and respective control mice (B); and in control mice with and without local ANP (100 nmol/L) pretreatment (C; n=6–9 mice per pretreatment; *P<0.05 vs vehicle). Top in B and C, Representative original photographs.
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.

9. PDE5 inhibition enhances endothelial cGMP and barrier responses to ANP
PDE5 inhibition enhances endothelial cGMP and barrier responses to ANP. A, Effect of ANP on cyclic GMP content in the absence or presence of sildenafil (1 μmol/L; 15 minutes pretreatment) or IBMX (0.5 mmol/L).
PDE5 inhibition enhances endothelial cGMP and barrier responses to ANP. A, Effect of ANP on cyclic GMP content in the absence or presence of sildenafil (1 μmol/L; 15 minutes pretreatment) or IBMX (0.5 mmol/L). B and C, Intravital microscopy, m. cremaster. Time course of changes in net integrated optical intensity (IOI; an index of permeability) during 30 minutes of continuous local sildenafil (1 μmol/L) or vehicle and additional 10 minutes of histamine (2 μmol/L) superfusion in control mice (GC-Afl/fl; B) and in mice with conditional, endothelial GC-A deletion (EC GC-A KO; C). Left, Representative original photographs. D, Effect of sildenafil and histamine on venular diameters in control and EC GC-A KO mice (diameters represent the average of 5 measurements taken at 5 locations on each vessel at the same time, before and during subsequent application of sildenafil and histamine). All n=6 mice per genotype and treatment; *P<0.05 vs vehicle or baseline.
Wen Chen et al. Arterioscler Thromb Vasc Biol. 2013;33:
Copyright © American Heart Association, Inc. All rights reserved.