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Cardiovascular Research 2003 60(2):431-437; doi:10.1016/S0008-6363(03)00565-0
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Copyright © 2003, European Society of Cardiology

Basal nitric oxide modulates vascular effects of a peptide activating protease-activated receptor 2

Carla Cicala*,a, Silvana Morelloa, Valentina Velleccoa, Beatrice Severinob, Ludovico Sorrentinoa and Giuseppe Cirinoa

aDepartment of Experimental Pharmacology, University of Naples "Federico II", via Domenico Montesano 49, 80131 Naples, Italy
bDepartment of Medicinal Chemistry, University of Naples "Federico II", via Domenico Montesano 49, 80131 Naples, Italy

*Corresponding author. Tel.: +39-81678437; fax: +39-81678403. Email address: cicala{at}unina.it

Received 5 February 2003; revised 15 July 2003; accepted 22 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor proteolytically activated by trypsin, tryptase or factor Xa. Alternatively, PAR2 can be activated by synthetic peptides whose sequence mimics the tethered ligand exposed after receptor cleavage. It is known that PAR2 modulates vascular reactivity, both in vitro and in vivo. The present study was designed to investigate the role of basal nitric oxide and cyclic nucleotides, adenosine 3'5'cyclic monophosphate (cAMP) and guanosine 3'5' cyclic monophosphate (cGMP), in the vasorelaxation induced by a PAR2-activating peptide (PAR2-AP; SLIGRL-NH2) on rat aorta in vitro. Methods: A concentration–response curve to PAR2-AP was performed on rat thoracic aorta with or without a functional endothelium, and the effect of inhibitors was evaluated. The effect of PAR2-AP (10–7–3 x 10–5 M) on endothelium-denuded aorta was also evaluated after tissue incubation with sodium nitroprusside. Results: PAR2-AP (10–7–3 x 10–5 M) caused an endothelium-dependent relaxation abolished by N{omega}-nitro-L-arginine methyl ester, L-NAME, and by the guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), but unaffected by geldanamycin. Vasorelaxant effect of PAR2-AP was only partially inhibited by the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), and it was increased by tissue incubation with the phosphodiesterase inhibitor, rolipram. On tissue without endothelium, PAR2-AP did not cause vasorelaxation, up to a concentration of 10–4 M. However, after tissue incubation with sodium nitroprusside (SNP, 3 x 10–9 M), the vasorelaxant effect of PAR2-AP was restored. Following tissue incubation with PAR2-AP, cAMP levels were significantly increased compared to control values. Conclusions: Our results suggest that vasorelaxation induced by PAR2-AP is modulated by basal nitric oxide with an involvement of both cyclic nucleotides, cGMP and cAMP.

KEYWORDS PAR2; Nitric oxide; cGMP; cAMP; Vasorelaxation

Abbreviations: Ach, acetylcholine • cAMP, adenosine 3'5' cyclic monophosphate • cGMP, guanosine 3'5' cyclic monophosphate • DMSO, dimethyl sulfoxide • L-NAME, N{omega}-nitro-L-arginine methyl ester • ODQ, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one • PAR2, protease-activated receptor 2 • PEG, polyethylene glycol • SNP, sodium nitroprusside • SQ 22,536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Protease-activated receptors (PARs) are members of seven transmembrane G-protein-coupled receptors activated by proteolytic cleavage. Following receptor cleavage, a new N-terminal tethered peptide is exposed that activates the receptor itself. Synthetic peptides, based on the sequence of the newly N-terminus exposed after cleavage, termed protease-activated receptor-activating peptides (PAR-APs), stimulate PARs and reproduce several in vitro and in vivo effects of endogenous proteases, even in the absence of receptor proteolytic cleavage [1].

Protease-activated receptor 2 (PAR2) is proteolytically activated by trypsin [2], tryptase [3], or factor Xa [4]; however, its endogenous ligand has not yet been identified. Among several tissues, PAR2 has been shown to be highly expressed on endothelium and vascular smooth muscle cells under physiological conditions [5], and its expression is increased by inflammatory stimuli, suggesting that it might play a role in the control of vascular reactivity under physiological and/or pathological conditions [6–8]. However, whether PAR2 contributes, together with physiological mechanisms, such as basal nitric oxide/guanosine 3'5' cyclic monophosphate (cGMP) pathway or β-adrenoceptor functionality, to cardiovascular homeostasis is not known. Several studies performed in vitro, on isolated vascular preparations, and in vivo, on experimental animals, have suggested that PAR2 might contribute to the vascular homeostasis [9].

There is evidence that nitric oxide mediates the vasorelaxation induced by PAR2 activation on isolated tissues [10–13]. Conversely, in vivo nitric oxide seems to play only a marginal role by mainly affecting the endurance of hypotension induced by intravenous injection of PAR2-activating peptide (PAR2-AP) to experimental animals [14–16]. The aim of the present study was a pharmacological dissection of PAR2-AP vascular effects. In particular, in the present study, we have evaluated the contribution of basal nitric oxide/cGMP pathway to PAR2-AP vasorelaxant effect.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Functional studies
All animal experiments complied with the Italian DL no. 116 of 27 January 1992 and associates guidelines in the European Communities Council Directive of 24 November 1986 (86/609/ECC).

Male Wistar rats (200–250 g) were sacrificed by decapitation and exanguinated. Thoracic aorta was rapidly removed, gently cleaned, taking care not to damage the endothelium, and cut in rings of about 3 mm in width each. Aortic rings were placed in a 2.5-ml organ bath containing Krebs solution composed of (in mM) NaCl, 115.3; KCl, 4.9; CaCl2, 1.46, MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; and glucose 11.1; warmed at 37 °C, oxygenated (95% O2 and 5% CO2), and connected to an isometric force transducer (model 7004, Ugo Basile) under a resting tension of 0.5 g. Changes in tension were recorded continuously by a polygraph linear recorder (WR 3310 Graphtec). After about 60 min equilibration period, the tissue was contracted with phenylephrine (PE, 10–6 M), and the presence of a functional endothelium was verified by evaluating the relaxation to a single concentration of acetylcholine (Ach, 10–6 M). Tissues that relaxed in response to Ach less than 80% were discarded. On selected tissues, a concentration–response curve to Ach (10–8–10–5 M) was performed. Tissues were then washed, contracted with PE (10–6 M), and a cumulative concentration–response curve to PAR2-AP (10–7–3 x 10–5 M) was performed.

2.2. Effect of inhibitors
In order to investigate on the mediators involved, the vasorelaxant effect of PAR2-AP (10–7–3 x 10–5 M) on rat aortic rings was also evaluated in presence of the nitric oxide synthase inhibitor, N{omega}-nitro-L-arginine methyl ester (L-NAME; 10–4 M; 15 min incubation); the selective inhibitor of the soluble guanylyl cyclase, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; 5 x 10–6 M; 10 min incubation) or the inhibitor of endothelial nitric oxide synthase (eNOS) activation, geldanamycin (10–5 M; 15 min incubation); the inhibitor of adenylate cyclase, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 10–4 M; 15 min incubation) or the phosphodiesterase IV inhibitor, rolipram (10–8 M; 5 min incubation). All drugs were dissolved in Krebs, except for ODQ and geldanamycin, dissolved in H2O:polyethylene glycol (PEG) 400 (1:1 ratio), and rolipram, in dimethyl sulfoxide (DMSO). All control experiments were performed with the appropriate vehicle. The appropriate concentration and incubation time for all inhibitors was determined by preliminary experiments (data not shown).

2.3. Effect of PAR2-AP on endothelium-denuded aorta after nitric oxide supplementation
The effect of PAR2-AP on endothelium-denuded aorta was evaluated before and after nitric oxide supplementation. Rat thoracic aorta was obtained as described above, and the endothelium was removed from the vessel by a gentle scraping of the lumen. The absence of endothelium was ascertained by the lack of tissue response to Ach (10–6 M). A concentration–response curve to PAR2-AP (10–7–10–4 M) was evaluated on PE (10–6 M) precontracted tissue before and 5 min after incubation with sodium nitroprusside (SNP) at a concentration that had no relaxant effect per se (SNP; 3 x 10–9 M). In some cases, tissue was preincubated with haemoglobin (10–5 M; 15 min incubation) or with ODQ (5 x 10–6 M; 10 min incubation) prior to the addition of SNP, and then the vasorelaxant effect of PAR2-AP was evaluated.

2.4. cAMP assay
In another set of experiments, rat thoracic aortic rings with or without endothelium were prepared as described above, weighted, and placed in 24-well plates and incubated for 15 min at 37 °C under 5% CO2 humidified air. IBMX (10–5 M) was added to each well, and 5 min thereafter, tissues with endothelium were stimulated with PAR2-AP at a concentration ranging from 10–6 to 10–5 M. Rings without endothelium were stimulated with PAR2-AP (10–5–10–4 M) either in the absence or in the presence of SNP (3 x 10–9 M). After 5 min incubation with drugs, at 37 °C and 5% CO2 humidified air, reaction was stopped by ice cooling. Tissue levels of adenosine 3'5' cyclic monophosphate (cAMP) were evaluated by an EIA kit Cayman (SpiBio, France) following manufacturer instructions and expressed as picomoles per gram of wet weight tissue.

2.5. Drugs
PAR2-AP (SLIGRL-NH2) was synthesised at the Department of Medicinal Chemistry of the University of Naples "Federico II" as previously described [16].

Acetylcholine; the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536); the guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ); the nitric oxide synthase inhibitor, N{omega}-nitro-L-arginine methyl ester (L-NAME); phenylephrine; the phosphodiesterase inhibitor, rolipram; the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX); sodium nitroprusside (SNP); and haemoglobin were purchased from Sigma-Aldrich (Milano, Italy). All salts were purchased from Carlo Erba. Geldanamycin (GA) was a generous gift of the Drug Synthesis and Chemistry Branch, Development Therapeutics Program, Division of Cancer Treatment at the National Cancer Institute (Bethesda, MD, USA).

2.6. Statistical analysis
All data are expressed as mean±S.E.M. Relaxation is expressed as the percentage relaxation relative to the relaxation achieved by Ach (10–6 M). Results obtained on endothelium-denuded tissues are expressed as absolute percentage relaxation of the tissue precontracted with PE (10–6 M). Nonlinear regressions of all concentration–response curves were performed. Results were analysed by two-way ANOVA, followed by Bonferroni's test for multiple comparisons. A value of p<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effect of drugs on vasorelaxation induced by PAR2-AP
PAR2-AP caused a concentration-dependent relaxation of PE precontracted rat aortic rings in the presence of endothelium (pEC50 5.52±0.27; Emax 80.7±3.9%; n = 7) that was completely inhibited by preincubation with L-NAME (10–4 M) or with the guanylate cyclase inhibitor, ODQ (5 x 10–6 M), but it was unaffected by geldanamycin (10–5 M) at a concentration that inhibited relaxation induced by Ach (Fig. 1). All three inhibitors used caused similar changes in vascular tone, causing an increase of 19.83±3.6%, 15.2±3.7%, and 17.00±3.8%, respectively (n = 7). ODQ abolished Ach-induced relaxation (data not shown).


Figure 1
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  		Fig. 1 (A, B, and C) Effect of L-NAME (10–4 M; A), ODQ (5 x 10–6 M; B), and geldanamycin (10–5 M; C) on vasorelaxation induced by PAR2-AP (10–7–3 x 10–5 M) on rat thoracic aortic rings precontracted with PE (10–6 M). (D) Effect of geldanamycin on Ach (10–8–10–5 M)-induced relaxation (***p<0.001; n = 4–7).

 
Incubation of the tissue with the adenylate cyclase inhibitor, SQ 22,536 (10–4 M), inhibited PAR2-AP-induced relaxation (p<0.01 two-way ANOVA). SQ 22,536 reduced the maximum vasorelaxant effect induced by PAR2-AP (10–5 M) from 70.50±6.65% to 39.25±13.90% (n = 4; p<0.01), without affecting the EC50 value (pEC50 5.00±0.86 vs. 5.00±0.36; n = 4). PAR2-AP vasorelaxant effect was significantly (p<0.001 two-way ANOVA) increased by the phosphodiesterase IV inhibitor, rolipram (10–8 M) (pEC50 6.00±0.12 vs. 5.00±0.08; n = 5; Fig. 2). The two inhibitors caused a reduction of vascular tone of 10.00±2.4% and 26.00±8.00%, respectively. Ach-induced relaxation was neither affected by SQ 22,536 (Emax 77.04±3.35% vs. 78.56±2.66%; n = 4) nor by rolipram (Emax 95.85±10.41% vs. 91.09±3.76%; n = 4).


Figure 2
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  		Fig. 2 Effect of SQ 22,536 (10–4 M; A) and rolipram (10–8 M; B) on vasorelaxation induced by PAR2-AP (10–7–3 x 10–5 M) on rat thoracic aortic rings precontracted with PE (10–6 M). **p<0.01 and ***p<0.001 vs. vehicle; two-way ANOVA, n = 5.

 
3.2. Effect of PAR2-AP on endothelium-denuded aorta incubated with SNP
On rat thoracic aortic rings without endothelium, PAR2-AP was ineffective up to a concentration of 10–4 M. However, after tissue incubation with SNP (3 x 10–9 M), the vasorelaxant effect of PAR2-AP was restored although to a lesser extent when compared to the effect obtained in the presence of endothelium, with the EC50 increasing to about 10 times (pEC50 5.55±0.16 vs. 4.45±0.19; n = 6). Relaxation in the presence of SNP was inhibited either by the guanylate cyclase inhibitor, ODQ (5 x 10–6 M) or by the nitric oxide scavenger, haemoglobin (10–6 M) (Fig. 3).


Figure 3
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  		Fig. 3 (A) Effect of PAR2-AP (10–7–10–4 M) on endothelium-denuded rat thoracic aortic rings precontracted with PE (10–6 M) in the absence ({circ}) and in the presence (bullet) of SNP (3 x 10–9 M); n = 6. (B) Effect of haemoglobin (10–5 M) and ODQ (5 x 10–6 M) on relaxation induced by PAR2-AP (10–7–10–4 M) on endothelium-denuded rat thoracic aortic rings incubated with SNP (3 x 10–9 M) and precontracted with PE (10–6 M); n = 6.

 
3.3. cAMP levels after tissue incubation with PAR2-AP
cAMP levels produced by tissue with endothelium after incubation with PAR2-AP (10–6–10–5 M) were significantly increased over the basal values in a concentration-dependent manner. Conversely, in tissue without endothelium, cAMP levels were not modified by the presence of PAR2-AP (10–5–10–4 M) alone or in association to SNP (3 x 10–9 M) (Fig. 4).


Figure 4
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  		Fig. 4 (A) cAMP levels measured in rat thoracic aortic rings with endothelium incubated with PAR2-AP (10–6–10–5 M). (B) cAMP levels measured in rat thoracic aortic rings without endothelium incubated with PAR2-AP (10–5–10–4 M) alone or (C) in the presence of SNP (3 x 10–9 M). All experiments were performed in presence of IBMX (10–5 M). **p<0.01 vs. vehicle (IBMX alone); n = 6–9.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
It is widely known that PAR2 expressed on vascular districts modulates the vascular reactivity, both in vitro and in vivo, under physiological and pathological conditions in mice and rats [9]. The present study was designated to investigate on the pharmacological modulation of vasorelaxation induced by a peptide-activating PAR2.

In agreement with previous results, our data show that PAR2-AP causes an endothelium-dependent vasorelaxation of rat aortic rings. Inhibition of nitric oxide synthase, as well as endothelial denudation of the tissue, abolished PAR2-AP vasorelaxant effect, implying a key role for nitric oxide. This is further confirmed by the finding that cGMP pathway is involved, as demonstrated by the suppression of vasorelaxation by tissue incubation with ODQ, a potent and selective inhibitor of nitric oxide-dependent guanylyl cyclase [17].

On the basis of the inhibitory effect of L-NAME, several papers report that PAR2-mediated vasorelaxation is dependent upon an activation of nitric oxide synthase [10–13]. However, it is known that L-NAME not only inhibits enzyme activation, but also blocks the basal production of nitric oxide from endothelium. The aim of the present study was to investigate on the role played by basal NO in the vasorelaxation triggered by PAR2 activation. To go into the mechanism(s) involved, we also performed experiments with geldanamycin, an inhibitor of the binding of heat shock protein 90 (HSP90) to endothelial nitric oxide (eNOS), that does not interfere with basal eNOS activity, in contrast to L-NAME, as reported by Garcia-Gardena et al. [18]. We observed that PAR2-AP-induced vasorelaxation was not affected by geldanamycin, ruling out any involvement of HSP90. However, geldanamycin in the same experimental conditions inhibited Ach-induced vasorelaxation in agreement with the literature [18]. Nonetheless, our results suggest that vasorelaxation induced by PAR2-AP requires endothelial integrity and the presence of NO/cGMP pathway. However, recently, it has been shown that besides HSP90, agonist-induced eNOS activation might involve other pathway(s) that are not affected by geldanamycin [19]. Whether PAR2-AP triggers eNOS activation through a different mechanism insensitive to geldanamycin needs to be further investigated. Release of nitric oxide from rat aorta stimulated with PAR2-AP has been previously demonstrated [12], however, only at a concentration of agonist of 300 µM, a concentration 30 times higher than that causing the maximum relaxation in our experimental conditions. Thus, on the basis of our results, we suggest a permissive role for basal nitric oxide/cGMP for the vasorelaxation induced by PAR2-AP, and this is further confirmed by the observation that relaxation induced by the peptide was restored on tissue without endothelium after supplementation of nitric oxide. The effect produced by PAR2-AP in presence of SNP was abolished by the nitric oxide scavenger, haemoglobin, and by ODQ, confirming the involvement of the NO/cGMP pathway. Previous studies have outlined the role of basal nitric oxide for the vasorelaxant effect triggered by several agents. For example, it has been shown that basal nitric oxide plays a role in vasorelaxation induced by arachidonic acid in isolated rat arterioles [20]. Similarly, exogenous nitric oxide has been shown to increase vasorelaxation induced on rat aortic smooth muscle by calcitonin gene-related peptide (CGRP) [21] or by isoprenaline [22], and vasorelaxation induced by prostacyclin on porcine pulmonary arteries [23], suggesting that the simultaneous presence of endothelium and basal nitric oxide is critical for a full response to vasodilatory agents. In this context, it is worth noting that PAR2 is expressed on both endothelial and vascular smooth muscle cells [5]. However, on endothelium-denuded vessels, PAR2 activation does not cause change in tension, as confirmed also by our data, apart from a vasoconstriction described by Moffatt and Cocks [24] on mouse renal arteries. Nonetheless, PAR2 on vascular smooth muscle cells has been shown to be functional [25]. These data taken together suggest that on vascular smooth muscle cells, the mere presence of the receptor is not sufficient to elicit a response such as a change in tissue tension and strongly support the hypothesis that activation of PAR2 needs to be triggered by a molecular mechanism that is critical besides the simple ligand–receptor interaction. In this respect, basal nitric oxide/cGMP pathway might represent a key step of this mechanism. In other words, our hypothesis is that endothelial nitric oxide release is a prerequisite for PAR2 activation.

Of particular interest is the finding that the endothelium-dependent PAR2-AP effect was also inhibited by the adenylate cyclase inhibitor, SQ 22,536, suggesting a contribution of endothelial-derived cAMP to NO/cGMP-dependent relaxation, while any involvement of smooth muscle adenylate cyclase is ruled out by the lack of PAR2-AP effect on endothelium-denuded aorta. The involvement of cAMP in the endothelium-dependent vasorelaxation induced by PAR2-AP is further supported by evidence that tissue incubation with PAR2-AP increased cAMP tissue production over basal values. Conversely, PAR2-AP did not modify cAMP levels of tissue without endothelium, even when incubated with SNP. Furthermore, we found that rolipram, a specific PDE IV inhibitor that has been shown to increase the effect of cAMP-mediated vasorelaxation [26], increased PAR2-AP vasorelaxant effect. Rolipram effect was related to the presence of a functional endothelium, according to the inhibition of phosphodiesterase IV, enzymes present in endothelial cells [27,28]. These results might imply that endothelium-derived cAMP is also crucial to obtain a full response to PAR2-AP. How endothelial-derived cAMP contributes, directly or indirectly, to vasorelaxation induced by PAR2-AP needs further investigation. However, it has been shown that an increase in endothelial cAMP may activate eNOS and contribute to relaxation mediated by β adrenoceptors in rat thoracic aorta [29]. A possible regulation of NO production by endothelial-derived cAMP has also been demonstrated for canine coronary blood vessels [30]. We cannot rule out the possibility that a similar mechanism is involved in PAR2-AP-induced vasorelaxation.

In conclusion, our results further support the hypothesis that PAR2 plays an important role in the control of vascular reactivity and shed a new light on the significance of tonic basal nitric oxide for a full vascular responsiveness to vasodilatory agents. Furthermore, for the first time, we have also shown an involvement of cAMP in the endothelium-dependent vasorelaxation triggered by PAR2 activation.


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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