Cardiovascular Research

Cardiovascular Research 2007 76(1):149-159; doi:10.1016/j.cardiores.2007.06.002

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Copyright © 2007, European Society of Cardiology

Functional role of the soluble guanylyl cyclase ₁ subunit in vascular smooth muscle relaxation

Sofie Nimmegeers^a, Patrick Sips^b,c, Emmanuel Buys^b,c,d, Peter Brouckaert^b,c and Johan Van de Voorde^a,*

^aDepartment of Physiology and Physiopathology, Ghent University, Ghent, Belgium
^bDepartment for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent, Belgium
^cDepartment of Molecular Biology, Ghent University, Ghent, Belgium
^dCardiovascular Research Center, Massachusetts General Hospital, 149, 13th street, Charlestown, MA 02129, United States

*Corresponding author. Department of Physiology and Physiopathology, De Pintelaan 185, 9000 Ghent, Belgium. Tel.: +32 9 240 33 42; fax: +32 9 240 30 59. johan.vandevoorde{at}UGent.be

Received 9 January 2007; revised 4 June 2007; accepted 6 June 2007

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Acknowledgments References

 
Objective Soluble guanylyl cyclase (sGC), the predominant receptor for nitric oxide (NO), exists in 2 active isoforms (₂β₁ and ₁β₁). In vascular tissue sGC₁β₁ is believed to be the most important. The aim of our study was to investigate the functional importance of the sGC₁-subunit in vasorelaxation.

Methods Aortic and femoral artery segments from male and/or female sGC₁^–/– mice and wild-type littermates were mounted in a small-vessel myograph for isometric tension recording. This was supplemented with biochemical measurements of the cGMP concentration and sGC enzyme activity.

Results The functional importance of sGC₁β₁ was demonstrated by the significantly decreased relaxing effects of acetylcholine (ACh), sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), NO gas, YC-1, BAY 41-2272 and T-1032 in the sGC₁^–/– mice of both genders. Moreover, the basal and SNP-stimulated cGMP levels and basal sGC activity were significantly lower in the sGC₁^–/– mice. However, the relaxing effects of NO, BAY 41-2272 and YC-1 seen in blood vessels from sGC₁^–/– mice indicate a role for an sGC₁β₁-independent mechanism. The increase in sGC activity after addition of BAY 41-2272 and the inhibition of the ACh-, SNP-, SNAP- and NO gas-induced response by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) in the sGC₁^–/– mice are observations suggesting that the sGC₂β₁ isoform is also functionally active. However, the insignificant increase in cGMP in response to SNP and the non-upregulated sGC₂ expression level in the sGC₁^–/– mice suggest rather the involvement of (an) sGC-independent mechanism(s).

Conclusions We conclude that sGC₁β₁ is involved in the vasorelaxation induced by NO-dependent and NO-independent sGC activators in both genders. However, the remaining relaxation seen in the sGC₁^–/– mice suggests that besides sGC₁β₁ also the minor isoform sGC₂β₁ and/or (an) sGC-independent mechanism(s) play(s) a substantial role.

KEYWORDS Arteries; Nitric oxide; Endothelial function; Second messengers; Vasoconstriction/dilatation

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Acknowledgments References

 
Soluble guanylyl cyclase (sGC) is considered to be the predominant intracellular receptor for nitric oxide (NO) and hence a very important enzyme for the physiological regulation of vascular tone and blood pressure. The heterodimeric hemoprotein is composed of a larger and a smaller β subunit, both necessary for catalytic activity [2]. Two isoforms for each subunit (₁/₂ and β₁/β₂) have been identified in various species [3]. Theoretically, the association of and β subunits could give rise to at least four different isoforms, but only the ₂β₁ and ₁β₁ isoforms are reported to be active [4]. In the brain, the levels of both isoforms are comparable but in all other tissues, including vascular tissue, the ₁β₁ isoform is predominant [5].

The most important endogenous activator of sGC is NO. In addition to endogenous NO, pharmacological NO-donors (e.g. nitroglycerin, isosorbide dinitrate and SNP) and agents such as BAY 41-2272 and YC-1 stimulate sGC, the latter two also in an NO-independent way [6]. The resulting rise in the intracellular cGMP concentration induces vascular smooth muscle relaxation by lowering the intracellular Ca²⁺ concentration and by desensitization of the contractile apparatus to Ca²⁺ [7].

Dysfunction of the endothelial NO/cGMP signaling pathway contributes to the pathophysiology of a variety of cardiovascular disorders including hypertension, thrombosis, atherosclerosis, myocardial infarction and angina pectoris [8]. This makes the different isoforms of sGC, as effector molecule for NO, attractive therapeutic targets for the treatment of the above mentioned conditions and drugs aiming to target sGC isoforms are currently in development [9]. However, due to the lack of sGC isoform-specific inhibitors, little is known about the specific role and relative importance of the sGC isoforms on vascular tissue response to endogenous and exogenous sGC stimulators. The recently developed knockout mice for the sGC₁ gene [10] allow to unravel this. In the present study the functional importance of the sGC₁-subunit in the vascular system was analysed using aortic and femoral artery segments isolated from soluble guanylyl cyclase alpha 1 knockout (sGC₁^–/–) mice. Experiments were performed on preparations from both genders as it was found that male but not female animals develop hypertension [10].

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Acknowledgments References

 
2.1 Animals and tissue collection
All experiments were performed on male and/or female homozygous soluble guanylyl cyclase alpha 1 knockout (sGC₁^–/–) mice, bred in the Department of Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium (age: 10–15 weeks; genetic background: mixed Swiss-129), using sGC₁^+/+ littermates as control [10]. The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The sGC₁^–/– mice lack exon 6 of the sGC₁ gene, which codes for an essential part in the catalytic domain. After cervical dislocation, the thoracic aorta and femoral artery were carefully removed from the animals and transferred to cooled Krebs–Ringer bicarbonate (KRB) solution.

2.2 Tension measurements
2.2.1 Artery preparations and general experimental protocol
Ring segments of the collected arteries (internal diameter: aorta: 978.0±39.5 µm (n=9); femoral artery: 424.7±11.8 µm (n=15)) were mounted in a small-vessel myograph with a tissue chamber filled with 10 ml of KRB solution and were cleansed from adhering tissue. Two stainless steel wires (40 µm diameter) were guided through the lumen of the segments. One wire was fixed to a force-displacement transducer and the other was connected to a micrometer. After mounting, the preparations were allowed to equilibrate for 30 min in the KRB solution bubbled with 95% O₂–5% CO₂ (pH 7.4) at 37 °C. The aortic rings were gradually stretched until a stable preload of 0.5 g was obtained, whereas the femoral arteries were set to their normalized internal diameter [11]. In short, the arteries were stretched in progressive steps. From the passive wall tension-internal circumferences relationship obtained by these measurements, the artery was stretched to a diameter corresponding to 90% of the diameter the vessel would have under a transmural pressure of 100 mm Hg.

After applying the optimal resting tension, the preparations were contracted 3 times with a KRB solution containing 120 mmol/L K⁺ and 5 µmol/L norepinephrine (NOR), washed, and allowed to relax to basal tension before starting the protocol. Precontraction was elicited with 30 µmol/L prostaglandin (PGF₂) or 5 µmol/L NOR. When a stable contraction plateau was obtained, relaxation responses were examined in a cumulative manner by increasing the concentration in log increments, once the response to the previous concentration had stabilized. Segments of sGC₁^–/– and sGC₁^+/+ mice were always tested in parallel.

2.2.2 Specific experimental protocols
First, the relaxation responses to acetylcholine (ACh) (1 nmol/L–10 µmol/L), sodium nitroprusside (SNP) (1 nmol/L–10 µmol/L), S-nitroso-N-acetylpenicillamine (SNAP) (1 nmol/L–10 µmol/L) and NO (1 µmol/L–100 µmol/L) were measured in varying order. These relaxing substances were also tested in the presence of the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (1 µmol/L for ACh, SNP and SNAP or 10 µmol/L for NO gas; 20 minutes preincubation). Besides vasodilators with NO as active metabolite, also NO-independent sGC stimulators such as BAY 41-2272 (1 nmol/L–10 µmol/L) and YC-1 (10 nmol/L–10 µmol/L) were tested. Also the influence of 1 µmol/L ODQ on the BAY 41-2272-induced response was measured. The influence of accumulation of cGMP formed under basal conditions was investigated by addition of the PDE-5 inhibitor, T-1032 (1 nmol/L–10 µmol/L). sGC-independent relaxation was assessed using the K_ATP-channel opener levcromakalim (Lev) (1 nmol/L–10 µmol/L) and the cGMP-analogue 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) (100 nmol/L–100 µmol/L).

2.3 Measurement of cyclic GMP levels in thoracic aortic rings
After 30 min of equilibration in a KRB solution at 37 °C, bubbled with 95% O₂–5% CO₂ (pH 7.4), the thoracic aorta segments were precontracted with 30 µmol/L PGF₂. In the first series of experiments, a single concentration of either SNP (10 µmol/L) or vehicle was added to the preparations 10 min after the addition of PGF₂. 1 min later, the reaction was stopped by snap freezing in liquid nitrogen. In the second series of experiments, 1 µmol/L ODQ or vehicle was added together with PGF₂. After 20 min, the ring segments were exposed to a single concentration of SNP (10 µmol/L) for 1 min, before being rapidly frozen in liquid nitrogen.

The collected segments were kept at –80 °C until further processing. The frozen aortic rings were pulverized, homogenized in 6% w v^–1 trichloroacetic acid, followed by centrifugation at 1500 xg for 10 minutes at 4 °C. The resulting pellets were used for total protein determination according to the method of Bradford [12]. The supernatants were extracted 4 times with 5 volumes of water-saturated diethyl ether before being dried in a Speed-Vac centrifuge. The redissolved samples were acetylated and cyclic GMP concentration was determined using a non-radioactive enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Assays were performed in duplicate and the amount of cGMP in each blood vessel ring was expressed as fmol µg^–1 protein.

2.4 Measurement of sGC activity in femoral artery preparations
sGC enzyme activity was measured as described by Bloch et al. [13]. After collecting, the femoral artery tissues were homogenized in buffer containing 50 mmol/L tris(hydroxymethyl)aminomethane (Tris).HCl (pH 7.6), 1 mmol/L EDTA, 1 mmol/L dithiothreitol, and 2 mmol/L phenylmethylsulfonyl fluoride. After centrifugation at 20,000 xg for 1h at 4 °C, the supernatants were incubated for 10 min at 37 °C in a reaction mixture containing 50 mmol/L Tris HCl (pH 7.5), 4 mmol/L MgCl₂, 0.5 mmol/L 1-methyl-3-isobutylxanthine, 7.5 mmol/L creatine phosphate, 0.2 mg/ml creatine phosphokinase, 1 mmol/L L-NAME and 1 mmol/L GTP with or without 100 µmol/L BAY41-2272. The reaction was terminated by the addition of 0.9 ml of 0.05 mol/L HCl and the cGMP content in the reaction mixture was measured using a commercial radioimmunoassay (Biomedical Technologies, Stoughton, MA). sGC enzyme activity is expressed as pmol of cGMP produced per minute per milligram of protein in femoral artery extract supernatant.

2.5 Drugs
The experiments were performed in a KRB solution of the following composition (mmol/L): NaCl, 135; KCl, 5; NaHCO₃, 20; glucose, 10; CaCl₂, 2.5; MgSO₄, 1.3; KH₂PO₄, 1.2 and EDTA, 0.026 in H₂O. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), acetylcholine chloride, T-1032, YC-1, 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), trichloroacetic acid, norepinephrine bitartrate, dithiothreitol, phenylmethylsulfonyl fluoride, tris(hydroxymethyl)aminomethane (Tris).HCl, 1-methyl-3-isobutylxanthine, creatine phosphate, creatine phosphokinase, L-NAME and GTP were obtained from Sigma-Aldrich (St. Louis, MO); sodium nitroprusside from Merck (Darmstadt, Germany); BAY 41-2272 and S-nitroso-N-acetylpenicillamine from Alexis (San Diego, USA) and prostaglandin F₂ (Dinolytic) from Upjohn (Puurs, Belgium). ODQ, T-1032, SNAP, YC-1 and BAY 41-2272 were dissolved in dimethylsulfoxide and acetylcholine in 50 mmol/L potassium hydrogen phtalate buffer, pH 4.0. The other drugs were dissolved in distilled water. Saturated NO solution was prepared from gas (Air liquide, Belgium) as described by Kelm and Schrader [14]. All concentrations are expressed as final molar concentrations in the organ bath. The final concentration of dimethylsulfoxide in the organ bath never surpassed 0.1%.

2.6 Calculations and statistics
Data are presented as mean values±SEM; n represents the number of arteries (each obtained from a different mouse). Statistical significance was evaluated by using Student's t-test for paired and unpaired observations (SPSS, version 12) or with two-way ANOVA with Bonferroni post hoc test (GraphPad Prism, version 4), when appropriate. P<0.05 was considered as significant.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Acknowledgments References

 
3.1 NO-dependent sGC-induced relaxations
3.1.1 Effect of ACh
Responses to stimulated release of endothelium-derived NO were determined by recording concentration–relaxation curves to ACh in the aorta (Fig. 1A) and femoral artery (Fig. 1B) from male and female sGC₁^–/– and sGC₁^+/+ mice. The results were essentially similar in both genders. ACh-induced concentration–dependent relaxation was nearly abolished in the aortic rings of the sGC₁^–/– mice (10 µmol/L ACh: female: 67.8%±3.5 vs. 16.4%±5.2 (n=6, P<0.05)). Also in the femoral artery segments of the sGC₁^–/– mice, the ACh-induced response was significantly reduced compared to the sGC₁^+/+ mice (10 µmol/L ACh: female: 89.0%±2.9 vs. 43.1%±10.7 (n=6, P<0.05)).

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Relaxation effect of ACh (A, B), SNP (C, D) and NO gas (E, F) on precontracted (30 µmol/L PGF2 (ACh, NO gas,) and 5 µmol/L NOR (SNP)) aortas (A, C, E) and femoral arteries (B, D, F) from male sGC1+/+ and sGC1–/– mice in control conditions ( and ) and in the presence of ODQ ( and ) (1 µmol/L (ACh, SNP) and 10 µmol/L (NO gas)). *(sGC1+/+ vs. sGC1–/–),+(sGC1+/+ ODQ vs. sGC1–/– ODQ): P<0.05, (n=7–15).

 
A second relaxation curve to ACh was established in the presence of the sGC inhibitor ODQ. ODQ inhibited the ACh-induced response in the aorta (Fig. 1A) (10 µmol/L ACh: female: 67.8%±3.5 vs. 28.8%±3.0 (n=6, P<0.05)) and femoral artery (Fig. 1B) (10 µmol/L ACh: female: 89.0%±2.9 vs. 50.3%±10.9 (n=6, P<0.05)) from control mice. As the response to ACh in the sGC₁^–/– aortic rings was very small, the reducing effect of ODQ was also confined (10 µmol/L ACh: female: 16.4%±5.2 vs. 2.6%±1.3 (n=6, P>0.05)). After treatment with ODQ, the maximal relaxation in the femoral arteries of sGC₁^–/– mice, was approximately reduced by 74% and 52% (43.1%±10.7 vs. 20.9%±9.9 (n=6, P<0.05)) respectively in male and female sGC₁^–/– mice.

3.1.2 Effect of SNP
In this series of experiments, the relaxant effects of increasing concentrations of the NO-donor SNP were compared on precontracted aortas (Fig. 1C) and femoral arteries (Figs. 1D and 2A,B) from male and female sGC₁^–/– and sGC₁^+/+ mice. The results were essentially the same in both genders. The cumulative addition of SNP resulted in a concentration–dependent relaxation in the ring segments of both sGC₁^–/– and sGC₁^+/+ mice. However, the relaxing effect of SNP was significantly reduced in the preparations of the sGC₁^–/– mice (10 µmol/L SNP: female: aorta: 77.9%±4.0 vs. 48.6%±3.6 (n=15, P<0.05); femoral artery: 89.9%±1.8 vs. 70.3%±5.1 (n=14, P<0.05)).

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Original tracings showing a concentration–response curve to SNP in the femoral artery of a male sGC1+/+ (A) and sGC1–/– mice (B) and to BAY 41-2272 in the aorta of a male sGC1+/+ (C) and sGC1–/– mice (D).

 
Following preincubation with ODQ, the relaxing effect of SNP was significantly reduced in the aorta and femoral artery of both sGC₁^–/– and sGC₁^+/+ mice (10 µmol/L SNP: female: aorta: 77.9%±4.0 vs. 36.8%±2.6 (n=15, P<0.05); femoral artery: 89.9%±1.8 vs. 49.5%±3.9 (n=14, P<0.05)). The relaxing effect of 10 µmol/L SNP was reduced by approximately 85% in male aortic rings (Fig. 1C) of sGC₁^–/– mice and 80% (48.6%±3.6 vs. 9.8%±1.9 (n=15, P<0.05)) in the corresponding female segments. Also in the femoral artery preparations (Fig. 1D) of the sGC₁^–/– mice, approximately 70% (female: 70.3%±5.1 vs. 20.5%±1.9 (n=14, P<0.05)) of the response to SNP was eliminated by ODQ.

3.1.3 Effect of SNAP
In these experiments, we investigated the relaxant effect of the NO-donor SNAP on aortic rings of female sGC₁^–/– and sGC₁^+/+ mice. The concentration–dependent relaxant effect of SNAP was nearly abolished in the ring segments of the sGC₁^–/– mice compared to the sGC₁^+/+ mice (10 µmol/L SNAP: 64.4%±4.5 vs. 7.0%±3.6 (n=6, P<0.05)).

Treatment of the aortic rings with ODQ, resulted in a large, significant reduction of the SNAP-induced response in the sGC₁^+/+ aortic rings. In those preparations, the 10 µmol/L SNAP-induced relaxation was reduced by approximately 94% (64.4%±4.5 vs. 3.8%±2.1 (n=6, P<0.05)). Since SNAP had a very small effect in the aortic rings of the sGC₁^–/– mice, the influence of ODQ was rather negligible (7.0%±3.6 vs. 0.7%±0.7 (n=6, P>0.05)).

3.1.4 Effect of NO gas
The relaxing effect of exogenous NO delivered as gas was also examined on the aorta (Fig. 1E) and femoral artery (Fig. 1F) from male and female sGC₁^–/– and sGC₁^+/+ mice. The results were essentially similar in both genders. In the ring segments of both sGC₁^+/+ and sGC₁^–/– mice, NO gas showed a concentration–dependent relaxing effect. The response to NO gas was significantly reduced in the aorta (100 µmol/L NO gas: female: 58.0%±3.5 vs. 33.0%±3.0 (n=7, P<0.05)) and femoral artery (100 µmol/L NO gas: female: 86.4%±2.7 vs. 56.35%±6.3 (n=7, P<0.05)) of the sGC₁^–/– mice.

Preincubation with ODQ before adding NO gas caused a rightward shift of the concentration–response curve in the aorta (Fig. 1E) and femoral artery (Fig. 1F) of both sGC₁^–/– and sGC₁^+/+ mice (100 µmol/L: female: aorta: 58.0%±3.5 vs. 36.9%±3.1; femoral artery: 86.4%±2.7 vs. 75.8%±4.7 (n=7, P<0.05)). The relaxation induced by 100 µmol/L NO on male and female sGC₁^–/– aortas was reduced in the presence of ODQ by respectively 68% (Fig. 1E) and 56% (33.0%±3.0 vs. 14.6%±3.4 (n=7, P<0.05)), while 24% (Fig. 1F) and 32% (56.4%±6.3 vs. 38.1%±7.5 (n=7, P<0.05)) for respectively male and female sGC₁^–/– femoral artery segments.

3.1.5 Effect of basal NO
We also analysed the effect of ODQ on the contraction elicited by 5 µmol/L NOR in the aorta (Fig. 3F) and femoral artery (data not shown) of male and female sGC₁^–/– and sGC₁^+/+ mice. The results were similar for both genders. Addition of ODQ elicited a little or no rise in the precontraction level of the femoral arteries, while in the aortic rings the contraction to norepinephrine was substantially increased by ODQ. This ODQ-induced increase in vascular tone was significantly smaller in the aortas of the sGC₁^–/– mice.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Relaxation effect of YC-1 (A,B) and BAY 41-2272 (C,D) on precontracted (30 µmol/L PGF2) aortas (A,C) and femoral arteries (B,D) from male sGC1+/+ () and sGC1–/– mice () (n=7–9). E, influence of ODQ (1 µmol/L) on the BAY 41-2272-induced response in the aorta of female sGC1+/+ mice (n=4). F, effect of ODQ (1 µmol/L) on the contraction elicited by 5 µmol/L NOR in the aorta of male and female sGC1+/+ and sGC1–/– mice (n=13–15). *P<0.05.

 
3.2 NO-independent sGC-induced relaxations
3.2.1 Effect of YC-1
YC-1 stimulates sGC in an NO-independent way and sensitizes it to NO [6]. To find out which sGC isoform is involved in the vasodilating effect of YC-1, cumulative concentration–response curves to YC-1 were obtained in the aorta (Fig. 3A) and femoral artery (Fig. 3B) from male sGC₁^–/– and sGC₁^+/+ mice. YC-1 induces a concentration–dependent relaxing effect in the ring segments of both sGC₁^–/– and sGC₁^+/+ mice. However, the YC-1-induced response was significantly reduced in the preparations of the sGC₁^–/– mice.

3.2.2 Effect of BAY 41-2272
BAY 41-2272, another NO-independent type of sGC stimulator [9] was added to aortic (Figs. 2C,D and 3C) and femoral artery (Fig. 3D) ring segments of male and female sGC₁^–/– and sGC₁^+/+ mice. This resulted in a relaxing response that was concentration–dependent in the femoral arteries of sGC₁^–/– and sGC₁^+/+ mice. In the aorta, however, the BAY 41-2272-induced response was only clearly concentration–dependent in the sGC₁^+/+ preparations. In the aortic rings from sGC₁^–/– mice, a substantial relaxation was only obtained with a concentration of 10 µmol/L (female: 96.3%±1.4 vs. 66.5%±5.6 (n=8, P<0.05)). Also in the femoral arteries of sGC₁^–/– mice, the relaxing effect of BAY 41-2272 was significantly impaired (female: 90.2%±3.6 vs. 61.5%±4.4 (n=8, P<0.05)). The results were similar for both genders.

The treatment of sGC₁^+/+ aortic rings with ODQ (Fig. 3E) shows that ODQ had a strong inhibitory influence on the BAY 41-2272-induced response, except on the highest BAY 41-2272 concentration (10 µmol/L).

3.3 Relaxation induced by PDE-5 inhibition
The influence of the PDE-5 inhibitor T-1032 was explored in the aorta (Fig. 4A) and femoral artery (Fig. 4B) of female sGC₁^–/– and sGC₁^+/+ mice. Increasing concentrations of T-1032 induced a concentration–dependent relaxation which was almost completely abolished in the aortas isolated from sGC₁ knockout mice. Also in the femoral artery segments of the sGC₁^–/– mice, the T-1032-induced response was significantly reduced compared to the control mice.

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Relaxation effect of T-1032 on precontracted (30 µmol/L PGF2) aortas (A) and femoral arteries (B) from female sGC1+/+ () and sGC1–/– mice () (n=6) in control conditions. Relaxation effect of 8-pCPT-cGMP on precontracted (30 µmol/L PGF2) aortas (C) and femoral arteries (D) from male sGC1+/+ () and sGC1–/– mice () (n=5–8) in control conditions. Relaxation effect of Lev on precontracted (30 µmol/L PGF2) aortas (E) and femoral arteries (F) from female sGC1+/+ () and sGC1–/– mice () (n=6–9) in control conditions. *P<0.05.

 
3.4 sGC-independent relaxations
3.4.1 Effect of 8-pCPT-cGMP
Concentration–response curves to 8-pCPT-cGMP, a cell membrane permeable cGMP-analogue, were recorded in the aorta (Fig. 4C) and femoral artery (Fig. 4D) from male sGC₁^–/– and sGC₁^+/+ mice. The concentration–dependent responses to 8-pCPT-cGMP were not significantly altered in the aorta and femoral artery of sGC₁^–/– mice compared to sGC₁^+/+ preparations.

3.4.2 Effect of levcromakalim
In these experiments, relaxation curves were obtained by addition of cumulative concentrations of the K_ATP-channel opener Lev to the aorta (Fig. 4E) and femoral artery (Fig. 4F) of female sGC₁^–/– and sGC₁^+/+ mice. There was no significant difference in the concentration–dependent response to Lev between the preparations of sGC₁^–/– and sGC₁^+/+ mice.

3.5 cGMP-measurements in thoracic aorta rings
Fig. 5A shows that the basal cGMP content in aortic rings isolated from sGC₁^–/– mice was significantly smaller than in rings from sGC₁^+/+ mice. In the sGC₁^+/+ ring segments stimulated with SNP (10 µmol/L), the cGMP levels increased 100-fold above basal values. Those isolated from sGC₁^–/– mice showed only a non-significant two-fold increase upon stimulation with SNP.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 A, Increase of cGMP elicited by SNP (10 µmol/L) in aortic rings of male and female sGC1+/+ and sGC1–/– mice (n=6–9). #(vehicle vs SNP), *(wild type vs knockout): P<0.001. B, Influence of ODQ (1 µmol/L) on the increase in cGMP elicited by SNP (10 µmol/L) in aortic rings of female sGC1+/+ and sGC1–/– mice (n=9). #(SNP vs SNP+ODQ), *(sGC1+/+ vs sGC1–/–): P<0.001. C, Baseline and BAY 41-2272-stimulated sGC enzyme activity in femoral rings from male and female sGC1+/+ and sGC1–/– mice (n=11–14). #(baseline vs BAY 41-2272), *(sGC1+/+ vs sGC1–/–): P<0.001. Data from male and female animals were pooled.

 
In another series of experiments, the effect of ODQ on the SNP-induced cGMP increase was assessed in aortic segments of female sGC₁^–/– and sGC₁^+/+ mice. The results (Fig. 5B) demonstrate that ODQ significantly reduced the cGMP content of the SNP-treated aortic rings in both sGC₁^–/– and sGC₁^+/+ mice.

3.6 sGC enzyme activity levels in femoral artery rings
In the femoral arteries isolated from male and female sGC₁^–/– mice, the basal sGC activity was significantly smaller compared to sGC₁^+/+ mice. Upon stimulation with 100 µmol/L BAY 41-2272, the sGC activity level increased approximately 50-fold above basal values in the ring segments of the sGC₁^+/+ mice. The corresponding sGC₁^–/– preparations showed only a 3-fold increase (Fig. 5C).

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Acknowledgments References

 
The physiological relevance of the sGC isoforms is of great importance to validate sGC subunits as potential pharmacological targets for the treatment of various diseases. In vascular tissue the sGC₁ subunit is predominantly found [5]. However, our results demonstrate that sGC₁β₁ is not the only target of both NO-dependent and NO-independent sGC stimulators.

The importance of the sGC₁ subunit in vasorelaxations induced by endogenous and exogenous NO is illustrated by the significantly reduced responses to ACh, SNP, SNAP and NO gas in the arteries from sGC₁^–/– mice. The relaxant response to ACh (release of endogenous NO from the endothelium) and SNAP (release of exogenous NO upon nonreductive decomposition) was nearly abolished in the aortic rings of sGC₁^–/– mice, whereas SNP (release of exogenous NO upon biotransformation) and NO gas (represents exogenous NO as such) still elicited relaxation. Femoral arteries of sGC₁^–/– mice showed a substantial response to all three NO-delivering substances (SNAP was not tested).

Besides NO, the endothelium may release other relaxing substances including prostacyclin (PGI₂) and the endothelium-derived hyperpolarising factor (EDHF) depending on the vascular bed studied. In mice aorta, Chataigneau et al. demonstrated that the ACh-induced response is completely blocked by N-nitro-L-arginine (L-NA), indicating that NO is the sole endothelium-derived vasodilator [15]. The same conclusion can be made from analogous experiments we performed in the femoral arteries of sGC₁^+/+ mice (own unpublished observations).

The continuous release of basal NO keeps the cardiovascular system in a state of constant active vasodilation and plays a substantial role in regulating blood flow and blood pressure [16]. The increase in vascular tone elicited by the sGC inhibitor ODQ on precontracted preparations was significantly smaller in the aorta from sGC₁^–/– mice. This suggests that basally released NO acts predominantly through activation of sGC₁β₁.

T-1032 causes an accumulation of the basally produced cGMP by inhibiting phosphodiesterase type 5. In comparison to the femoral arteries of the sGC₁^+/+ mice, the corresponding arteries of the sGC₁^–/– mice showed significantly less relaxation in response to T-1032. This is in line with the significantly lower basal sGC activity we found in the sGC₁^–/– femoral artery preparations, using a biochemical technique. In the aorta of sGC₁^–/– mice, the relaxant effect of T-1032 is almost abolished and a significantly lower basal cGMP content is found. All these data suggest a diminished basal influence of sGC in blood vessels of sGC₁^–/– mice.

Because of their sGC-stimulating effect, there is a great interest in molecules such as YC-1 and BAY 41-2272 [6] as potential new drugs for the treatment of cardiovascular pathologies [9]. Our results confirm that BAY 41-2272 is approximately 30-fold more potent as a vasodilator than YC-1 [9]. The finding that the relaxations in response to YC-1 and BAY 41-2272 were significantly diminished in the arteries from male sGC₁^–/– mice, suggests that YC-1 and BAY 41-2272 mainly act through activation of sGC₁β₁. However YC-1 and BAY 41-2272 still induce a response in the sGC₁^–/– mice.

The finding that the arteries from both sGC₁^–/– and sGC₁^+/+ mice responded in a similar way to the cGMP-analogue, 8 pCPT-cGMP, and the K_ATP-channel opener Lev, excludes that mechanisms downstream of the cGMP formation are affected by knocking out sGC₁.

All observations demonstrate the functional importance of the sGC₁ subunit as was expected from its predominant presence in vascular tissue. However, the surprising observation that NO, BAY 41-2272 and YC-1 still elicit a relaxing effect in sGC₁^–/– mice, indicates that activation of sGC₁β₁ is not the sole mechanism responsible for these relaxations. It should however be mentioned that besides their stimulatory effect on sGC, at higher concentrations BAY 41-2272 and YC-1 also increase intravascular cyclic GMP by inhibition of cGMP breakdown through phosphodiesterase type 5, the major cGMP-degrading enzyme in vascular smooth muscle cells [17]. Despite the fact that the NO-donor drugs, SNP and SNAP release the same NO species (NO^., NO⁺ and NO^–) [18], the SNP-induced response in the aortic rings of the sGC₁^–/– mice is far more pronounced. This could be due to the fact that SNP also decomposes to other bioactive compounds, such as cyanide and iron ions [19], leading to an enhanced oxidative stress and vasorelaxation [20].

Recently Mergia et al. [21] also showed the substantial role of an sGC₁-dependent mechanism in NO-related vasorelaxations. However, possible gender and regional differences were not explored in that study. The potential gender difference could be relevant considering that male but not female sGC₁^–/– mice develop hypertension from the age of 14 weeks due to an increase in peripheral resistance [10]. Because of this remarkable gender dependency, we performed experiments on both female and male sGC₁^–/– and sGC₁^+/+ mice. No gender differences in the response to ACh, SNP, NO gas and BAY 41-2272 were observed indicating that this is not the underlying cause for the development of hypertension in male sGC₁^–/– mice. Potential regional variations were also addressed by studying two different types of arteries: the aorta as an elastic artery and the femoral artery as a muscular artery. Overall, similar results were obtained in both vessel types.

The fact that there is still a substantial relaxation in response to several sGC stimulators in the sGC₁^–/– mice, indicates that besides sGC₁β₁ another mechanism is involved; either activation of the sGC₂β₁ isoform or activation of (an) sGC-independent mechanism(s) or a combination of these. Arguments in favour of both hypotheses are obtained in the present study.

Our finding that there is still an increase in sGC activity of sGC₁^–/– femoral arteries after addition of BAY 41-2272 is in line with the contribution of the sGC₂β₁ isoform in the BAY 41-2272-induced response. The possible contribution of the sGC₂β₁ isoform in the NO-induced relaxations seen in sGC₁^–/– mice is also suggested by the observations that ODQ, which inhibits both sGC isoforms, had a strong inhibitory influence on NO-induced relaxations in both the aorta and femoral artery preparations of the sGC₁^–/– mice and that the cGMP production by SNP was significantly reduced in the sGC₁^–/– aortic rings in the presence of ODQ. From their observations Mergia et al. suggest that sGC₁-independent relaxation is mediated by sGC₂ and that the limited activity of sGC₂ is enough to elicit a response in sGC₁^–/– mice [21]. It should however be mentioned that there is an important difference between the transgenic mouse model developed by Mergia et al. and the one used in the present study. We isolated arteries from sGC₁^–/– mice expressing a mutant sGC₁ protein that is catalytically inactive [10]. This avoids alterations in phenotype due to potential enzyme structural functions as has recently been demonstrated for PI3K knockout mice [22]. Conversely, Mergia et al. generated sGC₁-deficient mice with complete abrogation of sGC₁ expression [21]. Therefore, the possible influence on the phenotype of non-catalytic sGC₁ effects, such as complex formation with e.g. AGAP1 [23], can not be ruled out in that model.

Several observations suggest that also (an)other sGC-independent mechanism(s) might be involved in the remaining relaxation response in sGC₁^–/–. If sGC₂β₁ is the sole isoform responsible for the vasorelaxation seen in sGC₁^–/– mice, one would expect a significant increase in cGMP in the sGC₁^–/– mice upon stimulation with NO. However, the sGC₁^–/– aortic ring segments showed only a two-fold, non-significant increase in cGMP in response to SNP. It is questionable that this small increase in cGMP is sufficient to elicit a substantial relaxation (SNP 10 µmol/L: 63.23%±3.38 and 48.59%±3.55 in respectively male and female sGC₁^–/– aortas). Under the assumption that the cGMP-relaxation relation in rat aorta [24] is similar to that of mice aorta, the level of cGMP we observed in the aorta from sGC₁^–/– mice upon stimulation by SNP, would be able to elicit a relaxation of only 10%. This would imply that the rise of cGMP induced by SNP in the sGC₁^–/– aortas is too small to explain a 50% relaxation. This questions a substantial role of the sGC₂β₁ isoform in NO-induced relaxations. It should however be mentioned that this reasoning is purely hypothetical. More convincing are the QPCR-measurements showing no higher expression of the sGC₂ subunit in the ring segments of sGC₁^–/– mice (own observations).

In addition to the sGC₂β₁ isoform, perhaps (an) sGC-independent mechanism(s) contribute(s) to the substantial relaxation seen in the sGC₁^–/– mice. Various sGC/cGMP-independent actions of NO have been described, including the activation of (i) calcium-and voltage-dependent potassium channels in vascular smooth muscle cells [25,26], (ii) sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase [27], and (iii) vascular Na⁺/K⁺-ATPase [28]. They have not only been reported for NO-donors but also for authentic NO [29] and endogenous NO synthesized from inducible NO synthase [30]. Also the observation that ODQ failed to inhibit the relaxant effect of 10 µmol/L BAY 41-2272 in the sGC₁^+/+ aortic ring, suggests the involvement of (a) cGMP-independent mechanism(s) rather than sGC₂ activation. There are reports on cGMP-independent mechanisms underlying BAY 41-2272 and YC-1 induced vasorelaxation, including inhibition of Ca²⁺ entry [31] and activation of K⁺ channels [32] and Na⁺–K⁺-ATPase [33].

In conclusion, we found that the predominant sGC isoform in the aorta and femoral artery, sGC₁β₁, is indeed involved in the vasorelaxations induced by NO-dependent and NO-independent sGC activators in both genders. However the remaining relaxation seen in the sGC₁^–/– mice, may indicate that besides sGC₁β₁ also the less abundantly expressed isoform sGC₂β₁ and/or (an) sGC-independent mechanism(s) play(s) a very important role.

* Preliminary reports of these findings have been presented at the International Conference on cGMP, Potsdam, 2005 [1].

Time for primary review 29 days

	Acknowledgments

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Acknowledgments References

 
The authors would like to thank the DMBR animal caretakers for maintaining the animal facility and Eric Tack for assistance with the experiments.

This work was supported by a grant of FWO-Vlaanderen and the Bijzonder Onderzoeksfonds (BOF-GOA) of Ghent University. E.B. was supported by an award from the Northeast Affiliate Research Committee of the American Heart Association.

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion Acknowledgments References

 

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