Age Impairs Soluble Guanylyl Cyclase Function in Mouse Mesenteric Arteries

Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10−6 mol L−1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10−6 mol L−1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.

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Hyperpolarization of murine small caliber mesenteric arteries by activation of endothelial proteinase-activated receptor 2

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y04-121 ◽

2004 ◽

Vol 82 (12) ◽

pp. 1103-1112 ◽

Cited By ~ 26

Author(s):

John J McGuire ◽

Morley D Hollenberg ◽

Brian M Bennett ◽

Chris R Triggle

Keyword(s):

Smooth Muscle ◽

Membrane Potential ◽

Vascular Smooth Muscle ◽

Blood Vessels ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Isometric Tension ◽

Mesenteric Arteries ◽

Induced Hypotension ◽

Protease Activated Receptor 2

Activation of endothelial proteinase-activated receptor 2 (PAR-2) relaxes vascular smooth muscle (VSM) and causes hypotension by nitric oxide (NO)–prostanoid-dependent and -independent mechanisms. We investigated whether endothelium-dependent hyperpolarization of VSM was the mechanism whereby resistance caliber arteries vasodilated independently of NO. VSM membrane potentials and isometric tension were measured concurrently to correlate the electrophysiological and mechanical changes in murine small caliber mesenteric arteries. In uncontracted arteries, the PAR-2 agonist, SLIGRL-NH2 (0.1 to 10 µmol/L), hyperpolarized the VSM membrane potential only in endothelium-intact arterial preparations. This response was unaltered by treatment of arteries with inhibitors of NO synthases (L-NAME), soluble guanylyl cyclase (ODQ), and cyclooxygenases (indomethacin). L-NAME, ODQ, and indomethacin also failed to inhibit SLIGRL-NH2-induced hyperpolarization and of cirazoline-contracted mesenteric arteries. However, in blood vessels that were depolarized and contracted with 30 mmol/L KCl, the effects of the SLIGRL-NH2 on membrane potential and tension were not observed. SLIGRL-NH2-induced hyperpolarization and relaxation was inhibited completely by the combination of apamin plus charybdotoxin, but only partially inhibited after treatment with the combination of barium plus ouabain, suggesting an important role for SKCa and IKCa channels and a lesser role for Kir channels and Na+/K+ ATPases in the hyperpolarization response. We concluded that activation of endothelial PAR-2 hyperpolarized the vascular smooth muscle (VSM) cells of small caliber arteries, without requiring the activation of NO synthases, cyclooxygenases, or soluble guanylyl cyclase. Indeed, this hyperpolarization may be a primary mechanism for PAR-2–induced hypotension in vivo.Key words: proteinase-activated receptor 2, protease-activated receptor 2, endothelium, vascular smooth muscle, hyperpolarization, blood vessels, transgenic mice, vasoactive peptides.

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Regulatory interaction of cytoskeleton-associated proteins with soluble guanylyl cyclase in vascular smooth muscle

Intrinsic Activity ◽

10.25006/ia.6.s1-a1.3 ◽

2018 ◽

Vol 6 (Suppl. 1) ◽

pp. A1.3

Author(s):

Alexander Kollau

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Regulatory Interaction ◽

Associated Proteins

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Aldosterone increases oxidant stress to inhibit soluble Guanylyl Cyclase activity in vascular smooth muscle cells

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.758.26 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Bradley Allen Maron ◽

Ying‐yi Zhang ◽

Shiow‐Shih Tang ◽

Joseph Loscalzo ◽

Jane A. Leopold

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Guanylyl Cyclase ◽

Oxidant Stress ◽

Soluble Guanylyl Cyclase ◽

Muscle Cells ◽

Cyclase Activity ◽

Guanylyl Cyclase Activity

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TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1995.269.2.f212 ◽

1995 ◽

Vol 269 (2) ◽

pp. F212-F217 ◽

Cited By ~ 3

Author(s):

K. S. Lau ◽

O. Nakashima ◽

G. R. Aalund ◽

L. Hogarth ◽

K. Ujiie ◽

...

Keyword(s):

Nitric Oxide ◽

Smooth Muscle ◽

Nitric Oxide Synthase ◽

Vascular Smooth Muscle ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Tnf Alpha ◽

No Production ◽

Ifn Gamma ◽

Oxide Synthase

Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription-polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN-gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC.

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Nitric Oxide Promotes Human Hematopoietic Stem Cell Homing and Engraftment Via cGMP-Pkg Signaling

Blood ◽

10.1182/blood-2018-99-112993 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 807-807 ◽

Cited By ~ 1

Author(s):

Xinxin Huang ◽

Bin Guo ◽

Maegan L. Capitano ◽

Hal E. Broxmeyer

Keyword(s):

Guanylyl Cyclase ◽

Pde5 Inhibitor ◽

Soluble Guanylyl Cyclase ◽

Treated Group ◽

Vehicle Control ◽

No Donor ◽

Hematopoietic Stem ◽

Cell Homing ◽

Cd34 Cells ◽

Hsc Transplantation

Abstract The success of hematopoietic stem cell (HSC) transplantation relies on adequate homing and long-term engraftment of HSC into the bone marrow (BM). The free radical nitric oxide (NO) is a gaseous molecule that plays important roles in a variety of physiological regulations. NO can freely diffuse across cellular membranes and activate an enzyme, soluble guanylyl cyclase, to produce cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). cGMP binding activates cGMP-dependent protein kinase (PKG) and other proteins to regulate many biological processes. However, the roles of NO and cGMP in regulating HSC function still remain poorly understood. To explore the importance of NO signaling in HSC, we first evaluated the effects of NO on human cord blood (CB) HSC chemotaxis in an in vitro transwell migration assay. We found that treatment of human CB CD34+ cells for 16 hours with an NO synthesis inhibitor, L-NAME, did not affect chemotaxis towards CXCL12. However, treatment of human CB CD34+ cells 16 hours with NO donor compound sodium nitroprusside (SNP) resulted in 55% more migration toward CXCL12 compared to vehicle control. Enhanced chemotaxis by SNP was also observed in a more primitive HSC cell population (Lin-CD34+CD38-CD45RA-CD49f+CD90+, 71% more migration) suggesting NO donor treatment promote HSC migration. The other two NO donors, SNAP and NOC5 showed similar effects on promoting CD34+ cells and HSC migration. NO activates soluble guanylyl cyclase in target cells, so next we explored the relationship between HSC migration and soluble guanylyl cyclase. By treating human CB CD34+ cells with riociguat, a soluble guanylyl cyclase stimulator, we found that riociguat treatment also resulted in 78% more HSC migration toward CXCL12. The other soluble guanylyl cyclase activator BAY412272 showed a similar effect as riociguat by promoting HSC chemotaxis (68% more migration compared with vehicle control). Inside the cell cGMP is degraded by phosphodiesterase 5 (PDE5), so a PDE5 inhibitor would suppress cGMP breakdown and activate cGMP signaling. Consistently, we found that CD34+ cells with PDE5 inhibitor avanafil or sildenafil treatment showed increased HSC chemotaxis compared with vehicle control (57% and 62% more migration respectively). Next we used PKG inhibitor KT5823 to test whether PKG is involved in HSC migration and found that KT5823 totally blocked the effects of SNP, riociguat and avanafil on enhanced HSC migration, suggesting that NO promote HSC migration through cGMP-PKG signaling. To directly evaluate in vivo homing, vehicle, SNP or riociguat treated CB CD34+ cells were injected into sublethally irradiated NSG (NOD.Cg-PrkdcscidIL2rgtm1Wjl/Sz) mice, and human cell homing to mouse BM, as indicated by human CD45 percentage, was analyzed 24 hours after transplantation. Consistently, SNP or riociguat treatment resulted in a 2.1 fold and 2.3 fold increase of human cell homing in NSG mice respectively compared with vehicle control treatment. Next, we performed a limiting dilution assay to compare the frequency of SCID-repopulating cells (SRCs) in vehicle and riociguat treated CB CD34+ cells. Engraftment of riociguat-treated CB CD34+ cells was significantly increased in primary NSG recipient mice compared with that of vehicle control treated group four months after transplantation (47.6% vs 23.7%). Both human myeloid and lymphoid chimerisms were also increased. Poisson distribution analysis revealed an SRC frequency of 1/2977 in vehicle control treated group and 1/512 in Riociguat treatment, resulting in the presence of 335.9 SRCs and 1953.1 SRCs in 1×106 cells from vehicle control and riociguat-treated cultures. We are currently performing RNA-seq and quantitative proteomic analysis in riociguat-treated CB CD34+ cells to reveal specific downstream targets in regulating HSC homing. Taken together, our study suggests that human HSC homing and engraftment can be enhanced by modulating the NO/cGMP signaling pathway. Some compounds tested in our study, such as SNP, riociguat, avanafil are FDA approved medications broadly used for myocardial infarction, pulmonary hypertension and erectile dysfunction. So utilization of these drugs in HSC transplantation should be practical. Our work offers a new and simple approach to bolster the effectiveness of HSC transplantation. Disclosures No relevant conflicts of interest to declare.

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