A primer for measuring cGMP signaling and cGMP-mediated vascular relaxation

Abstract

Soluble guanylyl cyclase (sGC, also called GC1) is the main receptor for nitric oxide (NO) that catalyzes the production of the second messenger molecule, 3′5′ cyclic guanosine monophosphate (cGMP) leading to vaso-relaxation, and inhibition of leukocyte recruitment and platelet aggregation. Enhancing cGMP levels, through sGC agonism or inhibition of cGMP breakdown via phosphodiesterase inhibition, has yielded FDA approval for several cGMP modifier therapies for treatment of cardiovascular and pulmonary diseases. While basic research continues to improve our understanding of cGMP signaling and as new therapies evolve to elevate cGMP levels, we provide a short methodological primer for measuring cGMP and cGMP-mediated vascular relaxation for investigators.

1. Introduction

Soluble guanylyl cyclase (sGC, also called GC1) is the nitric oxide (^•NO) receptor that generates the second messenger molecule, cyclic guanosine 3′, 5′-monophosphate (cGMP). sGC-mediated cGMP production governs a wide range of physiological functions such as vasodilation, inhibition of platelet aggregation, and gut motility [1–3]. Consisting of homologous α and β subunits, the mammalian sGCs form obligate heterodimers, with α1β1 being abundantly and ubiquitously expressed across tissues [4]. Within the β1 N-terminus of sGC resides the “heme-NO/O₂-binding” domain, which is a requisite for ^•NO binding [5]. Upon ^•NO ligation to the ferrous (Fe²⁺) heme group of sGC β, cleavage of the His-Fe²⁺ bond occurs, resulting in the formation of a 5-coordinate, Fe²⁺ -^•NO complex on the distal side. As a result, a conformational change within the sGC α and β subunits’ coiled-coil domains ensues, aligning the catalytic domain of sGC to form the GTP binding pocket and triggering the catalysis of cGMP [6,7,9,12]. In conditions of oxidative stress, heme oxidation (Fe²⁺ →Fe³⁺) can result, which limits ^•NO affinity for heme, rendering it ^•NO insensitive [8–10].

Over the past 2 decades, small molecule drugs such as sGC stimulators and activators have been developed to increase sGC activity [11]. sGC stimulators require ferrous (Fe²⁺) sGC and synergize with ^•NO to amplify cGMP levels. Conversely, sGC activators increase sGC activity when sGC is oxidized in the ferric state (Fe³⁺) or heme deficient. Importantly, sGC stimulators particularly, Riociguat and Vericiguat, have been FDA approved for pulmonary hypertension and heart failure with reduced ejection fraction, respectively. While numerous basic, translational and clinical studies in this field are ongoing, we provide a general primer for assessing sGC-cGMP signaling pathway in cells and tissue as well as measuring ex vivo ^•NO-sGC-cGMP mediated vascular relaxation.

2. Measuring NO-sGC-cGMP signaling

In the first section, we will review a method that assays, in vitro, cGMP formation. We will describe how to measure soluble guanylyl cyclase (GC1) activity by using a radiolabeled GTP (the substrate), and how to assess NO-cGMP signaling by assaying phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a downstream effector of the NO-sGC-cGMP signaling pathway. In the second section, we described myography methods for measuring NO-sGC-cGMP mediated vascular relaxation in both conduit and resistance arteries.

2.1. Measurement of GC1 activity with the radiolabeled substrate [α-³²P] GTP

2.1.1. General principle

As GC1 enzymatic activity converts GTP into cGMP, the principle of this method is to assay the amount of [α-³²P]cGMP produced from the substrate [α-³²P]GTP. In addition to the radiolabeled substrate, the reaction mixture should contain “cold GTP” in excess (for a steady-state assumption of this Michaelis and Menten enzyme), at least a 2-fold excess of the substrate co-factor Mg²⁺ (as a MgCl₂ solution) because cyclization requires two Mg²⁺ ions/molecule of GTP [12]. The reaction should take place between a minimal temperature of 30 °C and an optimal/maximal temperature of 37 °C and the time of reaction between 5 and 10 min (The catalytic rate should be calculated from the linear portion of a time course). The catalytic reaction is stopped with zinc acetate. The formed [α-³²P]cGMP is then separated from the non-catalyzed [α-³²P]GTP by adding sodium carbonate to co-precipitate non-cyclic nucleotides with zinc acetate followed by elution with a neutral alumina column. Scintillation liquid is added to measure radioactivity in a scintillation counter.

2.1.2. Reagents

• [α-³²P]GTP stock with a specific activity of 3000 Ci/mmol, 250 μCi from PerkinElmer (NEG006H250UC). Dilute [α-³²P]GTP to have 0.8 to 1.2 million cpm/assay tube. Dilute 1 μl of the [α-³²P]GTP stock in 50 μl of distilled water. In duplicate, add 1 μl of the dilution to 5 ml of 100 mM Tris, pH 7.5 (elution buffer) and mix with 10 ml scintillation liquid (National Diagnostics, Cat# LS-272) to measure cpm in a scintillation counter.

• “cold” (unlabeled) GTP (Sigma, Cat# G8877); prepare a 100 mM stock solution; 0.5 mM final concentration.

• MgCl₂ (Sigma, Cat# M8266); prepare a 100 mM stock solution. Final concentration should be 2–4 fold final GTP concentration; i.e. 1–2 mM MgCl₂

• reaction buffer; prepare a stock solution of 250 mM HEPES pH 8.0 or 100 mM Tris pH 8.0. To use at a 50 mM final concentration. A pH of the assay between 7.5 and 8.0 is optimal.

• The use of GC1 activators, stimulators or NO-donors is the experimenter choice.

• 120 mM Zinc acetate (Zn(C₂H₃O₂)₂, Sigma Cat# 383058) to stop the reaction.

• 144 mM Sodium carbonate (Na₂CO₃) (Sigma Cat# 223530) to precipitate the non-cyclic nucleotides.

• 100 mM Tris pH 7.5 (elution buffer)

• [8-³H]cGMP; cGMP-tritiated at C8. Stock is at 14.3 Ci/mmol, 250 μCi from PerkinElmer (NET337250UC). Use as a radiotracer to measure the % recovery of each column. Dilute in water to have 6000 cpm/assay tube.

• neutral Alumina (Sigma Cat# 199974)

• Scintillation liquid (National Diagnostics, Cat# LS-272)

Notes:

1. We do not recommend to use thiol reducing agents as they could interfere with the enzymatic properties of GC1 by modifying its thiol redox state, in particular if one uses TCEP (tris(2-carboxyethyl) phosphine [13]. If there is evidence that the heme is partially oxidized in some GC1 molecules, dithiothreitol (DTT) could be used to stabilize the enzyme. A 50 mM DTT stock solution in water should be prepared fresh. The absorbance of a freshly prepared solution should not be higher than 260 nm. If there is a peak at 280 nm, the solution should be discarded as it indicates oxidized DTT.

2. A radioactive license is usually required from the radioactive safety department and all safety protocols should be followed accordingly.

2.1.3. Enzymatic assay

If the enzyme source is from cells or tissues, it is recommended to add to the lysis buffer a wide-spectrum inhibitor of phosphodiesterases, such as 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM final concentration), and proteases inhibitors (Protease inhibitor cocktail (Sigma, Cat# P8340)). The lysis buffer should not contain EDTA at concentration higher than 0.5 mM as it would chelate the Mg²⁺ necessary for the catalytic reaction. The enzyme source should be kept on ice until the start of the assay. Each assay should be done in duplicate.

The recommended total volume of the reaction assay is 100 μl/tube with 50 μl of reaction buffer and 50 μl of the enzyme source. For 100 reactions (50 assays in duplicate), prepare racks to accommodate 100 12 × 75 mm glass tubes that will fit in a water bath pre-warmed at the desired reaction temperature. Next, prepare 6 ml of reaction buffer containing 2.4 ml of 250 mM HEPES pH 8.0, 60 μl of a 100 mM GTP solution, 120 μl of a 100 mM MgCl₂ solution, 100 μl of [α-³²P]GTP at 0.8–1 million cpm/μl, GC1 activators/stimulators as needed, and distilled water to 6 ml. On ice, add 50 μl of the enzyme source to each tube, transfer the rack to the water bath. To start the reaction, add 50 μl of the reaction buffer to each tube. Stop the reaction by adding 500 μl of 120 mM Zinc acetate. The time of the reaction should be in the linear part of the curve of cGMP production to avoid depletion of the substrate and the half-life of the NO-donor (if used) should be considered as well. Prepare controls in duplicate by adding 500 μl of 120 mM Zinc acetate to tubes containing the enzyme source prior to addition of the reaction buffer. They will be used to estimate the radioactive background of the assay (time 0 control).

Notes: Many NO-donors can be used for stimulation of GC1 activity. However, they exhibit different properties, in particular their half-life can vary from 2 min to several hours [14,15]. Because of their well-known rate of NO release, which is mostly not affected by other reactants, we recommend the use of NONOates, such as diethylamine NONOate (DEA-NO) for in vitro enzymatic assay. The concentration should range from 1 to 10 μM, or up to 100 μM if the GC activity is expected to be low. The pH of the reaction should be between 7.5 and 8.0, as NO release is dependent on the pH and greatly accelerated at pH < 6 while very slow at pH > 9. For this reason, stock solution of DEA-NO prepared in 10 mM NaOH will be stable for at least 24 h. For a longer half-life (20 h), diethylene triamine (DETA-NO) can be used.

We do not recommend the use of low molecular weight S-nitrosothiols such as S-nitrosoglutathione (GSNO) or S-nitroso-N-acetyl-dl-penicillamine (SNAP) because of their potential to induce S-nitrosation, especially considering that the NO-stimulated sGC activity is inhibited by S-nitrosation [16]. The decomposition of S-nitrosothiols is photo-sensitive, enhanced by transition metals (including Mg²⁺ which is present in the reaction buffer) and, conversely, stability is increased by chelating agents such as EDTA.

2.1.4. Purification of cGMP product

The [α-³²P]cGMP produced by the reaction is separated from the [α-³²P]GTP substrate using an alumina column (see Fig. 1 for the setup).

Fig. 1.

Setup for cGMP separation.

1. preparation of the alumina columns. Fill each column (Bio-Rad, Cat# 7311550, 9 cm high, 2 ml bed volume) with neutral alumina to a height of 2.5 cm. Wash the alumina with 8 ml of distilled water by pipetting up and down, let drip and pour 5 ml of 100 mM Tris pH 7.5. Let the buffer drip completely. The columns can be prepared in advance but should not become dry.

2. precipitation of the non-cyclic nucleotides. Add 500 μl of 144 mM Sodium carbonate to the assay tubes containing the Zinc acetate. Add 100 μl of [8-³H]cGMP for a total of 6000 cpm/tube. Vortex and keep in a −20 °C freezer for 20–30 min for precipitation. After thawing, centrifuge at 4 °C for 20 min at 3200 rpm.

3. pour the supernatant of each tube to each alumina column and let it drip completely.

4. elute [α-³²P]cGMP (and [8-³H]cGMP) with 5 ml of 100 mM Tris pH 7.5 directly in scintillation vials adapted to the scintillation counter (Fig. 1; of note the protective frontal shield was removed for the picture). Add 10 ml of scintillation liquid to each vial.

2.1.5. Measurements and calculations

For counting, additional vials (in duplicate) should be included that contain, in addition to 5 ml 100 mM Tris pH 7.5 and 10 ml of scintillation liquid, a) 100 μl of [8-³H]cGMP to assess the recovery of the columns (usually around 60%); b) 5 μl of the reaction buffer with [α-³²P] GTP. Blanks are vials containing 5 ml 100 mM Tris pH 7.5 and 10 ml of scintillation liquid. The scintillation counter is set to measure the ³H isotope within an energy spectrum window of 0–300 and the ³²P isotope in the 300–1000 channel. The spillover factor (cpm from one isotope channel overlapping with the cpm of the other isotope channel) is measured from a/b and b/a after subtracting the blank values. The amount of cGMP produced is a function of the amount of pmol GTP/tube (=50,000 in our example: 100 μl assay volume * 0.5 mM GTP) at ~1,000,000 cpm/assay tube (measured with b vials), hence pmol of cGMP produced/tube = (50,000 * cpm of cGMP)/1,000,000. This activity has to be corrected for % recovery of each column and for the spillover factors and after subtracting the blank values. Likewise, the pmol activity of the time 0 control should be subtracted to account for the radioactivity background. To obtain the guanylyl cyclase specific activity, the pmol activity is divided by the time of the reaction and the amount of protein and is expressed in mol cGMP• min⁻¹ • mg⁻¹.

2.1.6. Conclusions

The above method measures the specific activity of the soluble guanylyl cyclase from tissues, cells culture or purified enzyme. It allows to directly assay the response to various activators, stimulators and NO-donors, establish dose-response curves and defining the potency and efficacy of these drugs. It is also the most reliable way to assay the kinetics parameters such as K_m and V_max under basal or stimulated conditions. The principle is different from ELISA and radio-immuno-assay (RIA), which measures the accumulation of cGMP. ELISA or RIA were not described here as most labs nowadays use ELISA kit. RIA is probably the most sensitive method to measure cGMP in tissues however it does not allow to differentiate between the cGMP produced by GC1 and the cGMP from membrane-bound (particulate) guanylyl cyclases unless one compares cGMP formed under basal and stimulated activity with specific GC1 drugs. In cells culture, some cell types are known to excrete cGMP in the extracellular medium. This cGMP efflux is regulated by multidrug resistance protein (MRP) specifically MPR4 and MRP5 [17,18]. Thus, it is recommended to conduct a pilot study to assess the potential “loss” in cGMP by measuring the total cGMP and compared it to the intracellular cGMP.

2.2. Assessing Ser239 phosphorylation of the vasodilator-stimulated phosphoprotein in vitro

The vasodilator-stimulated phosphoprotein (VASP) was originally identified in platelets as a substrate of both the cAMP protein kinase (PKA) and cGMP protein kinase (PKG) pathway [19,20]. Ser 239 is the preferred site for phosphorylation by PKG, while Ser157 is primarily phosphorylated by PKA. As such, changes in phosphorylation of Ser239 is a reliable marker of the NO-sGC-cGMP signaling pathway by indirect measurement of PKG activity [21]. The method below describes the detection in vitro of Ser239 VASP phosphorylation.

2.2.1. Preparation of lysates from cells or tissues

• Cells are broken down with an ice-cold RIPA-type lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), containing a “cocktail” of proteases and phosphatases (Halt^™ protease and phosphatase single-use cocktail, 100x, ThermoFisher, Cat# 78440), and 0.5 mM final IBMX.

• Tissues are ground first with an Ultra-Turrax dispenser (IKA, Wilmington, NC.) in cold lysis buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 50 mM NaF, 1 mM Na orthovanadate, and protease inhibitor cocktail, containing 1% Triton X100. Lysate is then centrifuged (4 °C, 2 min, 500 g) to remove debris.

• For both types of lysates, measure the protein concentration with BCA protein assay (Pierce^™ BCA Protein Assay Kit, cat # 23225). A concentration of 0.5 mg/ml to 1 mg/ml is optimal.

2.2.2. Electrophoresis and western blot analysis

• Mix 1–50 μg of protein samples with a 5X Laemmli buffer containing 0.5 M DTT and boil for 3 min.

• Subject the samples to electrophoresis on a 7.5% SDS-PAGE. Transfer to a nitrocellulose membrane. Blocked the membrane with 1% BSA in PBS for 1 h.

• Probe O/N at 4 °C with the primary antibody against phosphorylated Ser239 of VASP (p-VASP239 is a polyclonal antibodies raised in rabbit. cell signaling, cat #3114). The secondary antibody against rabbit and the method of detection is the experimenter’ choice.

• Additionally, the membrane can be reprobed with a “total” VASP antibody (t-VASP, BD bioscience, Cat #610448) to assess the ratio of P-VASP vs. t-VASP.

Notes: Smolenski et al. by using mutations at Ser157 (PKA target) and/or Ser239 (PKG targets) showed that only phosphorylation at Ser157 leads to a shift in VASP molecular weight from 46 kDa to 50 kDa. They have developed a mouse monoclonal antibody (16C2), which is specific for VASP Ser239 ²¹ (SigmaAldrich, Cat #05–611).

2.3. Measuring NO-mediated arterial vascular relaxation

2.3.1. Introduction

Along the vascular tree, the arterial vasculature including conduit arteries, resistance arteries and capillaries, contribute to diverse functions. For example, conduit arteries (i.e. aorta) serve as a “central highway” for blood distribution throughout the body. Downstream of conduit arteries are resistance arteries and arterioles (i.e. mesentery), which play an critical role in blood pressure and local blood flow regulation by controlling changes in vascular tone (i.e. constriction and dilation). Lastly, capillaries contribute to gas and nutrient exchange in organs, however, they lack contractile smooth muscle cells.

In order to study the effects of vasoactive molecules that can impact the ^•NO -sGC-cGMP-PKG pathway, it is important to consider the type of artery and/or arteriole and the physiological implications of the results obtained. First, it is essential to note that contraction and dilation primarily occurs at the resistance artery and arteriole (lumen diameter <250 μm) level, and less so in conduit arteries. Conduit arteries have little influence on blood pressure but rather transport blood to resistance arteries to modulate local tissue blood flow. Although vascular function studies are routinely performed in conduit arteries and can serve as an important “ex vivo bioassay” for studying mechanisms regulating the •NO-sGC-cGMP-PKG, translating the physiological implications related to blood flow and pressure effects can be limited. Therefore, in order to translate changes of artery/arteriole contraction and relaxation into a physiological context such as blood flow and pressure, resistance arteries should be used.

2.3.2. Principle

When measuring vascular contraction and relaxation, a pressure or 2-pin (conduit arteries)/wire (resistance arteries) myograph is used. Briefly, pressure myography involves cannulation of arteries/arterioles and pressure is set to a physiological range. A 2-pin/wire myograph involves stretching of the artery and measuring of tension generated by smooth muscle. While both have strengths and weaknesses, we provide an overview for measuring conduit (aorta) and resistance artery (mesenteric) contraction and relaxation using 2-pin and wire myography. Importantly, this protocol should serve as a general primer and guideline for measuring the ^•NO-sGC-cGMP-PKG pathway and accessing the impact of this pathway on vascular contraction and relaxation in a conduit (aorta) and resistance arteries (mesenteric). For more in-depth discussion related to strengths and weaknesses of myograph techniques, selection of vascular beds, and protocol design please see reference. [22].

2.3.3. Protocol

1. Before starting, ensure that proper Institutional Animal Care and Use Committee protocols are approved, and guidelines are followed for animal handling.

2. To isolate the thoracic aorta and mesenteric arteries, sacrifice the animal by asphyxia using CO₂. Use a toe pinch to make sure the animal is dead.

3. Place the animal on its back and make a midline incision along the abdomen to expose the organs (Fig. 2A).

4. For thoracic aorta isolation, remove the diaphragm, lungs and heart. Gently cut away the perivascular fat to expose the thoracic aorta (Fig. 2B).

5. After removing the fat, cut parallel along the aorta (~10 mm) holding one end of the aorta with fine forceps.

6. Place the excised aorta in a petri dish containing room temperature physiological salt solution (PSS) containing (in mM): NaCl 119, KCl 4.7, MgSO₄ 1.17, KH₂ PO₄ 1.18, d-glucose 5.5, NaHCO₃ 25, EDTA 0.027, CaCl₂ 2.5, pH 7.4 when bubbled with 95% O₂ 5% CO₂ at 37°.

7. While keeping the intact aorta bathed in PSS, gently cut the aorta in 2 mm rings. Caution should be taken to limit stretching of the aorta while cutting the rings as this will lead to endothelial cell damage.

8. For mesenteric artery isolation, cut out the whole gut and gently pin out the mesenteric arcade taking care to avoid over stretching the arteries (Fig. 2C–E).

9. Remove the perivascular fat to expose the mesentery arteries and veins. The vein will be larger in diameter and dark red in appearance. The artery will be smaller in diameter, with a light pink and white appearance (Fig. 2F). When removing fat, avoid touching or pinching the artery which will result in artery damage. This can be accomplished by pinching the fat for removal as opposed to the artery.

10. Cut third order mesenteric arteries into 2 mm rings while bathing in PSS and minimize any stretch to artery.

11. Using a DMT 620 M myograph, (Fig. 3A), place the 2 mm aortic rings on a 2-pin chamber (Fig. 3B, upper image). For mesenteric arteries, slide one 25 or 40 μm wire through the lumen of the artery and place artery in between chamber jaws and screw down wire (Fig. 3B, lower image). Using the affixed wire as a guide, feed a second wire through the lumen and screw down to the other side. In both cases care should be taken to handle the arteries as little as possible to avoid damaging the endothelium or smooth muscle layers.

12. Let both arteries equilibrated for 30 min in PSS bubbled with 95% O₂ 5% CO₂ at 37°.

13. After 30 min gradually stretch the aorta, 100 mg of tension every 2 min, until a resting tension of 500 mg is achieved. Whereas mesenteric arteries, using DMT’s normalization tool, are gradually stretched to a tension equivalent to 80 mmHg at a normalization factor of 1.0. For further information on DMT’s normalization tool please reference their normalization guide.

14. Following equilibration and stretch, the PSS buffer is then replaced with a potassium rich buffer KPSS containing (in mM): NaCl 63.7, KCl 60, MgSO₄ 1.17, KH₂ PO₄ 1.18, d-glucose 5.5, NaHCO₃ 25, EDTA 0.027, CaCl₂ 2.5. This step serves as a positive control to ensure artery is alive.

15. After 5 min, aortas and mesenteric arteries are washed 3 times with PSS and allowed to rest for 30 min.

16. A final refresh of PSS is then performed and aortas and mesenteric arteries allowed to rest for an additional 10 min.

17. Preconstrict the aortas with prostaglandin f2α (100 nM-10 μM) or phenylephrine (100 nM-10 μM) to achieve about 50% constriction. For mesenteric arteries, preconstrict with U46619 (100 nM–500 nM) or phenylephrine (100 nM-10 μM).

18. Following a stable pre-constriction, assess NO-dependent and NO-independent mediated cGMP SMC relaxation as shown in Fig. 3D.

19. A cumulative-concentration dose response to the Acetylcholine (10 nM-100 μM), SNP (1 nM-100 μM), BAY 58–2667 (1 pM-1 μM) or BAY 41–2272 (1 pM-1 μM) with 5 min between each dose (Fig. 3E).

20. Upon completion of step 18, PSS is replaced with Ca⁺ free PSS +100μM SNP for 10 min to achieve max dilation. This step serves as a positive control to ensure that the artery can fully relax.

21. Relaxation % is calculated by taking (max dilation value from drug-max constriction from constrictor)/(Ca⁺ free PSS- max constriction from constrictor)*100.

22. Conduct a 2-WAY ANOVA to test for significance between each condition(s) and individual dilator concentration.

Fig. 2. Overview of thoracic aorta and mesenteric vasculature anatomy.

a) Schematic of a mouse showing anatomic location of a mouse thoracic aorta and mesentery arteries. b) Image of a mouse thoracic aorta with heart shown in upper left corner. c) Zoomed in image of anatomic location of mesenteric artery cascade. d) Schematic showing first, second and third order mesenteric arteries. e) Image of a pinned out mesenteric. f) Zoomed image of a mesentery vein (left vessel, dark red) and an artery (right, light pink and white).

Fig. 3. Overview of aorta and mesenteric artery myography setup and standard protocol for assessing cGMP-mediated vascular relaxation.

A) Image of a 4-chamber DMT620 myograph used to assess aortic and mesenteric artery function. B) Image of a single 2-pin setup for an aorta or C) wire setup for a mesenteric artery. Images below show mounted aorta and mesenteric artery. D) Schematic diagram illustrating the NO-sGC-cGMP pathway and agonists used to determine NO-dependent and NO-independent vasodilation. E) A schematic workflow of a myography experiment assessing vasodilatation to sGC modulator compounds.

Notes: One of the most significant challenges with myography is variability which can come from technical and non-technical factors. A major technical challenge is artery isolation which can take weeks to months to perfect. Reducing technical variability can be achieved by 1) constantly maintaining the artery in bathed PSS, 2) reducing over cleaning of the adventitia which can induce damage to the medial layers of the artery and 3) eliminating any stretching during the isolation process. Non-technical variability can arise from the following: 1) animal species, 2) the tissue bed in which the artery was isolated, 3) pressure vs. wire myography technique, 4) the artery caliber and 5) type of pre-constrictor used. Notably, it is recommended that a highly detailed description of methods is reported to enable readers to consider all potential variables particularly when comparing results across studies.

3. Conclusions

sGC is one of the most sought-after targets to treat cardiovascular and pulmonary diseases. Herein we provide the tools to evaluate biochemically and physiologically the impact of GC1 stimulating and activating drugs. The biochemical methods allow one to measure accurately the catalytic activity of GC1, using radiolabeled GTP and the activity of PKG by detecting VASP phosphorylation at Ser239. The physiological method, using wired myography, allows to assess ex vivo vascular contraction/relaxation in resistance and conduit arteries. Applied together, they help to explore the NO-cGMP signaling pathway.