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Cardiovascular Research 2000 47(3):574-585; doi:10.1016/S0008-6363(00)00123-1
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Evidence for P2-purinoceptors contribution in H2O2-induced contraction of rat aorta in the absence of endothelium

Jian-Zhong Shena, Xiu-Feng Zhenga and Chiu-Yin Kwana,b,*

aDepartment of Pharmacology, School of Medicine, Zhejiang University, Hubin Campus, 353 Yanan Road, Hangzhou, 310031, PR China
bSmooth Muscle Research Program and Department of Medicine, Faculty of Health Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8 N 3Z5, Canada

* Corresponding author kwancy{at}fhs.mcmaster.ca

Received 10 December 1999; accepted 4 May 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objective: H2O2 can contract many arteries, however the underlying mechanisms are not fully understood. This study aims to test whether H2O2-induced vasoconstriction could be functionally attributed to the activation of P2-purinoceptors in rat aorta and to explore its possible signaling mechanisms. Methods: Isometric tension recording of H2O2 and ATP-induced contractions of rat aortic rings were compared in the absence or presence of various pharmacological tools to identify their possible common signaling pathways. Results: Both H2O2 and ATP induced transient phasic contractions in a concentration-dependent manner (1–1000 µM). Removal of endothelium potentiated the contractile responses to H2O2 and to ATP. H2O2 (30 µM)-induced phasic contraction could be abolished by catalase (800 U/ml), but not affected by SOD (150 U/ml), DMSO (5 mM) and apyrase (5 U/ml), suggesting no involvement of O2–, hydroxyl free radicals and ATP release. Also, several receptor antagonists including phentolamine, atropine, methysergide and chlorpheniramine (each 3 µM) were without effect on H2O2 (30 µM)-induced phasic contraction, suggesting no involvement of typical neurotransmitter release. However, both H2O2 (30 µM) and ATP (1 mM)-induced phasic contractions not only presented homologous desensitization, but also showed heterogeneous desensitization. Furthermore, the phasic contractions in response to H2O2 (30 µM) or ATP (100 µM) could be inhibited or abolished in a concentration dependent manner by RB-2 and suramin (10–100 µM), two widely used P2-purinoceptor antagonists, with only partial inhibition by Evans blue (300 µM), a moderately selective P2x receptor blocker, or by {alpha}-β-methylene-ATP (100 µM), a selective P2x receptor desensitizer. On the other hand, both H2O2 (30 µM) and ATP (100 µM)-induced phasic contractions were also attenuated, to different degree, by inhibitors of several enzymes including PLC, PKC, PLA2 and cyclooxygenase. Lastly, removal of extracellular Ca2+ or pretreatment with procaine (10 mM) and dantrolene (30 µM), two putative intracellular Ca2+ release blockers, or with Ni2+ (100 µM) and tetrandrine (5 µM), two Ca2+ channel blockers, all significantly inhibited H2O2 and ATP-induced contractions. However, nifedipine (1 µM), a voltage-dependent L-type Ca2+ channel blocker, was without effect. Conclusions: Our results demonstrate that H2O2-induced phasic contraction of rat aorta involves, at least in part, the activation of P2-purinoceptors in the aortic smooth muscle cells

KEYWORDS Arteries; Endothelial receptors; Free radicals; Smooth muscle; Vasoconstriction/dilation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
It has been demonstrated in recent years, that hydrogen peroxide (H2O2), like NO, acts as either a cellular signaling molecule or a toxic substance depending on its source and concentration [1,2]. Intracellular production of H2O2 can be triggered by various extracellular stimuli including G-protein coupled or uncoupled receptors activation [3,4]. However, the major source of H2O2 may come from its secretion by activated neutrophils or monocytes. In this case, the local concentration of H2O2 in vasculature could be up to millimolar levels [5]. Thus, the regulatory effect of H2O2 on vascular muscle tone has been intensively studied. Indeed, previous studies had shown that H2O2 can cause contraction of many arteries in vitro, such as rat and rabbit aorta [6,7], canine coronary artery [8], rat and bovine pulmonary artery [9,10], canine basilar artery [11] and human placental arteries [12]. Furthermore, several potential mechanisms underlying H2O2-induced contraction have been proposed, including perturbation of Ca2+ regulatory mechanisms, such as intracellular Ca2+ release and extracellular Ca2+ influx in smooth muscle [13] or activation of several enzymes such as phospholipase A2 (PLA2) [14], phospholipase C (PLC) [15], protein kinase C (PKC) [13], cyclooxygenase (COX) [6] and tyrosine kinase [16]. Nevertheless, up to now, a possible unifying or a primary signaling step which could explain such a contractile effect induced by H2O2 is still missing.

Considering the fact that activation of PLC, PLA2 or tyrosine kinase, in most cases, is the subsequent signaling steps resulting from the activation of G-protein coupled or uncoupled receptors and the previous suggestion that H2O2 mobilizes Ca2+ in both smooth muscle and endothelial cells via a receptor-sensitive pathway [17–19], we, therefore, hypothesize that H2O2-induced vasoconstriction, particularly at low concentrations, may be due to its direct or indirect effect on some plasmalemmal receptors, rather than a highly non-specific effect as generally believed for many years. In this study, we focused on the P2-purinoceptors, since several lines of evidence raised such a possibility. First, it was reported that H2O2-activated MAPKs signaling cascades in vascular smooth muscle cells could be inhibited by a non-selective P2-purinoceptor antagonist of suramin [20]; second, Musat et al. [21] have shown that ATP binding to cardiac plasmalemma ATP receptors (presumably P2-purinoceptors) can be concentration- and time-dependently modulated by H2O2, suggesting a possible modification of P2-purinoceptors by H2O2. Lastly, a recent study by Wartenberg et al. [22] indicated that in a human prostate cancer cell line, both H2O2 and ATP, via similar mechanisms, triggered either proliferative or growth inhibition effects depending on their testing concentrations. In view of these findings, the present study was designed to test whether H2O2-induced vasoconstriction could be functionally attributed to the activation of P2-purinoceptors.

It is well known that P2-purinoceptors are further classified into a series of P2x and P2y subtypes, which are based on their agonist selectivity. The fast transmitter function of ATP, including its vasoconstrictor function, is mediated by P2x receptors, which are part of ligand-gated ion channels and present in vascular smooth muscle cells. The P2y receptors are highly expressed in both endothelial cells and vascular smooth muscle cells and are linked to various second messenger systems including PLC, PLA2, PKC and MAPKs via coupling of G-proteins [34,35,41]. In this work, we provided functional evidence to suggest that P2-purinoceptors are involved in H2O2-induced contraction of rat aorta, and this effect, more importantly, cannot be ascribed to possible ATP release due to H2O2 challenge.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Tissue-bath experiments
For this work, 3-month-old Male Sprague–Dawlay rats, weighing 250–300 g were used. Rats were maintained at the University facility for experimental animals under standard conditions and conformed to rules set by Animal Ethic Committee. The rats were killed by cervical dislocation under ether anesthesia. Rat aortas were isolated, and excess fat and connective tissues were removed. Vessels were cut into rings of about 3 mm width. The aortic ring was mounted vertically in a 3-ml organ bath, connected to a force transducer and a pen-recorder. The organ baths contain Krebs’ solutions with the following composition (mM): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgCl2·6 H2O, 1.18; KH2PO4, 1.08; NaHCO3, 25; and glucose, 11 at pH 7.4, maintained at 37°C and bubbled with a 95% O2–5% CO2 gas mixture. Ca2+-free medium was prepared by omitting CaCl2 from the normal Krebs’ solution and replaced with EGTA 100 µM. The solution in the baths was changed every 15 min. The rings were equilibrated for 20 min before stretching them to ~2 g and were allowed to further equilibrate for 90 min. Before data collection, stimulation of the rings with 50 mM KCl was repeated every 20 min until a reproducible contractile response was obtained.

2.2 Experimental
2.2.1 Specimen preparation
To preclude the possible impact of endothelium on the vascular effect induced by H2O2, most of the tests were conducted in endothelium-denuded preparations. So the endothelium was intentionally removed by gently rubbing against the teeth of a pair of forceps. The successful removal of endothelium was assessed by showing that acetylcholine 1 µM failed to relax the rings precontracted with phenylephrine 1 µM.

2.2.2 Preliminary study
In most cases, only one dose of H2O2 or ATP was applied to the resting ring to observe their vasoconstrictor response, since both H2O2 and ATP-induced contractions showed tachyphlaxis phenomenon. Therefore, a cumulative concentration–response curve could not be constructed. To determine whether H2O2-induced contraction as related to endogenous vasoconstrictor release, or the formation of other free radicals, ring preparations were treated with various receptor antagonists for 20 min, or with SOD for 5 min, before addition of H2O2.

2.2.3 Purinoceptor detection
To determine whether P2-purinoceptors are involved in H2O2-induced contraction, aortic rings were pretreated with the putative P2-purinoceptor antagonists suramin, reactive blue 2 (RB-2) or Evans blue with different concentrations for 20 min, or predesensitized against P2x receptors with {alpha}-β-methylene-ATP. Forces developed in response to H2O2 or ATP in the presence or absence of various blockers were compared.

2.2.4 Elucidating post-receptor mechanisms
The post-receptor signaling mechanisms of H2O2 and ATP-induced vasoconstriction, such as the involvement of PLC, PLA2 and PKC, were also investigated by pretreatment of aortic rings with a variety of enzyme inhibitors for 20 min. To test whether H2O2 and ATP-induced contractions are dependent on Ca2+ influx, rat aortic rings incubated in organ baths were washed three times in 5 min with Ca2+-free solution containing EGTA 100 µM [52], or pre-treated with different types of Ca2+ channel blockers for 20 min before addition of ATP or H2O2. To study whether intracellular Ca2+ release also participates in H2O2 and ATP-induced contractions, aortic rings were pretreated with intracellular Ca2+ release blockers before addition of H2O2 or ATP. The concentrations of all the pharmacological tools were used according to their values of IC50 in vascular smooth muscle. In some cases, the contractions induced by phenylephrine (1 µM), KCl (30 mM) or caffeine (10 mM) were also compared in the presence or absence of some blockers to confirm their inhibitory efficacy or selectivity on the aortic rings under our experimental conditions.

2.3 Drugs
The following pharmacological tools were purchased from Sigma (St. Louis, MO, USA): acetylcholine chloride (ACh), phenylephrine hydrochloride (PE), hydrogen peroxide (H2O2), catalase (from bovine liver, 25 000 U/mg protein), SOD (from bovine erythrocytes, 3800 U/mg protein), ethyleneglycol-bis (b-aminoethyl ether) N,N'-tetraacetic acid (EGTA), dantrolene, nifedipine, procaine hydrochloride, NiCl2, neomysin sulfate, 2-nitro-4-carboxypheny(-N,N-diphenylcarbamate (NCDC), indomethacin, mepacrine, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H7), apyrase (from potato, 43 U/mg protein), phentolamine hydrochloride, atropine sulfate, chlorpheniramine, caffeine, ATP, {alpha}-β-methylene-ATP, suramin, reactive blue 2 (cibacron blue 3GA, RB-2), Evans blue, dimethylsulphoxide (DMSO). Methysergide was from Sandoz and tetrandrine was a gift from Zhejiang Jinhua. All other chemicals were obtained from Shanghai No. 3 Chemical Reagents (China) with purity of AR grade.

2.4 Statistical analysis
All of the data are expressed as means±S.E.M. Contractile responses induced by H2O2, ATP, PE, caffeine or low concentration of KCl (30 mM) are expressed as percentage of the 50 mM KCl induced contraction. Students’ t-test was used for comparing two groups. When three or more groups were compared, one-way analysis of variance was used. A P value less than 0.05 was considered significant. At least three rats were used in each set of vascular reactivity experiments.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
3.1 Phasic contractions of rat aortic rings induced by H2O2 and ATP
Fig. 1 shows that in endothelium intact resting rings, both H2O2 and ATP elicited phasic contraction in a dose-related manner. The pattern of contraction induced by H2O2 was similar to that of ATP. Removal of endothelium potentiated the amplitude of phasic contraction in response to ATP or of H2O2. The time duration of the phasic contraction in response to H2O2 (1 mM) is about 10 min, and the maximal tension triggered by H2O2 (1 mM) is 606±77 mg. The same parameters for ATP (1 mM) are approximate 8 min and 482±54 mg. A concentration of H2O2 (30 µM) close to its EC50 was selected for further experiments. Table 1 shows that pretreatment of aortic rings with phentolamine, atropine, methysergide or chlorpheniramine (each 3 µM) did not significantly affect H2O2 (30 µM)-induced contraction.


Figure 1
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  		Fig. 1 Typical trace showing the non-cumulative concentration-dependent phasic contraction induced by ATP (A) and H2O2 (B) in rat aortic rings with (E+) and without (E–) endothelium. Each ring preparation was challenged non-cumulatively with only one concentration of H2O2 or ATP as indicated (in µM). (C) Graph shows the representative concentration–response curves for ATP and H2O2; vertical lines indicate S.E.M. Data in each tracing are representative of experiments using five individual aortic rings taken from at least three rats, and expressed in mg of tension developed.

 

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  		Table 1 Effect of selected inhibitors on the contraction induced by 30 µM H2O2 in rat aortaa

 
3.2 Effects of SOD, DMSO, catalase and apyrase on H2O2- and ATP-induced contractions
Table 1 also shows that pretreatment of the aortic rings with SOD (150 U/ml) or DMSO (3 mM) did not affect H2O2 (30 µM)-induced contraction, indicating no involvement of superoxide anion (O2–) and hydroxyl free radicals. On the other hand, catalase (800 U/ml) pretreatment completely abolished H2O2 (30 µM)-induced contraction, with no effect on ATP (100 µM)-induced contraction. In contrast, pretreatment of the aortic rings with apyrase (5 U/ml), an ATP hydrolase, inhibited ATP (100 µM)-induced contraction by 90%, with no significant effect on H2O2 (30 µM)-induced contraction (Fig. 2A and B). These results clearly indicate that H2O2-induced contractions are independent on a possible release of ATP and ATP-induced contractions are not related to a possible release of H2O2.


Figure 2
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  		Fig. 2 Effects of catalase (800 U/ml) and apyrase (5 U/ml) on the contractions induced by 30 µM H2O2 (A) or 100 µM ATP (B) in endothelium-denuded rat aortic rings. Each preparation was challenged with H2O2 or ATP only once after preincubation with either vehicle (control), catalase or apyrase for 5 min. Each column represents the mean±S.E.M. (n=6–9), expressed as percentage of the tension developed by 50 mM KCl and taken from at least three rats. *, P<0.001 compared with control.

 
3.3 Tachyphylaxis and cross-desensitization for H2O2- and ATP-induced contractions
After the first contraction induced by 30 µM H2O2, which was subsequently washed out for 30 min, a 2nd challenge of 30 µM H2O2 failed to induce a contraction, and totally abolished ATP (1 mM)-induced contraction. Similarly, when aortic rings were first contracted with 1 mM ATP followed by washout for 30 min, a 2nd application of 1 mM ATP triggered only a small transient contraction and also significantly reduced the contraction induced by 30 µM H2O2 (Fig. 3A and B). However, using the same protocols, 30 µM H2O2 or 1 mM ATP did not significantly affect the contraction induced by 30 mM KCl, 1 µM PE or 10 mM caffeine (Fig. 3C–E).


Figure 3
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  		Fig. 3 Tachyphylaxis (A) and cross-desensitization (B) of contractile responses induced by 30 µM H2O2 and 1 mM ATP in endothelium-denuded rat aortic rings. Using the same protocols, H2O2 or ATP did not affect the contractions induced by I µM phenylepherine (PE, C), 30 mM KCl (D) or 10 mM caffeine (E). Test drugs were applied at the points indicated by squares. (//) denotes washout for 30 min. Tracings on the left-hand side and on the right-hand side represent different experiments using different aortic rings. Data in each tracing are representative of experiments using five individual aortic rings taken from at least three rats.

 
3.4 Effects of P2-purinoceptor antagonists or desensitizer on H2O2 and ATP-induced contractions
Fig. 4A and B shows that RB-2 and suramin (10–100 µM), two widely used P2-purinoceptor antagonists, concentration dependently inhibited H2O2 (30 µM)-induced contraction in endothelium-denuded preparations. Similar results were observed for 100 µM ATP (Fig. 4C and D). On the other hand, {alpha}-β-methylene-ATP (100 µM), a selective P2x receptor agonist and desensitizer, also triggered a phasic contraction with a pattern similar to that induced by 100 µM ATP. In addition, after washout of {alpha}-β-methylene-ATP, a second challenge of aortic rings with the same concentration of {alpha}-β-methylene-ATP produced negligible contraction (Fig. 5A). However, {alpha}-β-methylene-ATP, under the same experimental protocol, only slightly depressed the contraction induced by 100 µM ATP or 30 µM H2O2. (Fig. 5B and C). Similarly, evans blue at 300 µM, a moderately selective P2x receptor blocker, completely abolished {alpha}-β-methylene-ATP (100 µM)-induced contraction (Fig. 5A), with only slight inhibition of ATP (100 µM)- and H2O2 (30 µM)-induced contractions (Fig. 5B and C). Neither suramin and RB-2 nor evans blue and {alpha}-β-methylene-ATP at concentrations up to 300 µM could affect KCl (30 mM) or PE (1 µM)-induced contractions in rat aortic rings (data not shown). Fig. 6A and B) further shows that although preincubation of the rat aortic rings with suramin (100 µM) abolished ATP (100 µM)- and H2O2 (30 µM)-induced contractions, after washout of suramin, ATP and H2O2 could still elicit phasic contractions with amplitude comparable to that of controls. However, after pre-incubation with RB-2 (100 µM), both ATP- and H2O2-induced contractions were irreversibly blocked (Fig. 6C and D). This result is consistent with the notion that suramin is a reversible and RB-2 is a irreversible antagonist of P2-purinoceptors.


Figure 4
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  		Fig. 4 Effects of suramin and RB-2 on the contractions induced by 30 µM H2O2 (A,B) or 100 µM ATP (C,D) in endothelium-denuded rat aortic rings. Each preparation was challenged with H2O2 or ATP only once after preincubation with either vehicle (control) or different concentrations of suramin and RB-2 for 20 min. Each column represents the mean±S.E.M. (n=6–9), expressed as percentage of the tension developed by 50 mM KCl and taken from at least three rats. *, P<0.05 and **, P<0.001 compared with control.

 

Figure 5
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  		Fig. 5 Effects of {alpha}-β-methylene-ATP (100 µM) and Evans blue (300 µM) on the contractions induced by 100 µM {alpha}-β-methylene-ATP (A), 100 µM ATP (B) and 30 µM H2O2 (C) in endothelium-denuded rat aortic rings. Each preparation was challenged with {alpha}-β-methylene-ATP or ATP or H2O2 only once after preincubation with either vehicle (control) or Evans blue for 20 min, or after predesensitization of P2x receptors by {alpha}-β-methylene-ATP. Each column represents the mean±S.E.M. (n=6–9), expressed as percentage of the tension developed by 50 mM KCl and taken from at least three rats. *, P<0.05 and **, P<0.001 compared with control.

 

Figure 6
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  		Fig. 6 Effects of P2-purinoceptors inactivation by suramin (100 µM, A and B) and RB-2 (100 µM, C and D) on ATP (100 µM)- and H2O2 (30 µM)-induced contractions of rat aorta. Ring preparations denuded endothelium were pretreated with suramin or RB-2 for 20 min and then challenged with ATP or H2O2. Note that after washout of suramin but not washout of RB-2, a second addition of ATP or H2O2 induced phasic contraction with amplitude comparable to that of controls. Data in each tracing are representative of experiments using five individual aortic rings taken from at least three rats.

 
3.5 Roles of PLC, PLA2, COX and PKC in H2O2 and ATP-induced contractions
In normal Krebs’ solutions, H2O2 (30 µM)-induced contractions were significantly depressed by 10 µM NCDC, a PLC inhibitor [23]; by 5 mM neomycin, a chelator of plasmalemmal PIP2 [24]; by 10 µM H7, a wide-spectrum inhibitor of protein kinases including PKC [25]; by 50 µM mepacrine, a PLA2 inhibitor [26]; or by 3 µM indomethacin, a COX inhibitor (Fig. 7A). The same concentration of NCDC, neomycin, H7, mepacrine and indomethacin also blocked ATP (100 µM)-induced contraction (Fig. 7B), with no influence on KCl (30 mM)-induced contraction (data not shown).


Figure 7
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  		Fig. 7 Effects of NCDC (10 µM), neomycin (5 mM), H7 (10 µM), mepacrine (50 µM) and indomethacin (3 µM) on the contractions induced by 30 µM H2O2 (A) or 100 µM ATP (B). Each preparation was challenged with H2O2 or ATP only once after preincubation with either vehicle (control) or the various inhibitors for 20 min. Each column represents the mean±S.E.M. (n=6–9), expressed as percentage of the tension developed by 50 mM KCl and taken from at least three rats. *, P<0.05 and **, P<0.001 compared with control.

 
3.6 Effects of Ca2+ removal, procaine, dantrolene, Ni2+, nifedipine and tetrandrine on H2O2- and ATP-induced contractions
After pretreatment of the aortic rings with Ca2+-free Krebs’ solution (see Experimental), 30 µM H2O2 still triggered a similar phasic contraction with a smaller magnitude compared to that obtained in Ca2+-containing solution (Fig. 8A). Similar results were obtained when preparations were pretreated with 10 mM procaine and 30 µM dantrolene, two putative intracellular Ca2+ release blockers [27,28]. On the other hand, the contractions induced by 30 µM H2O2 were nearly abolished by 100 µM Ni2+, a non-selective inorganic Ca2+ channel blocker, and also largely inhibited by 30 µM tetrandrine, an organic blocker of Ca2+ channel blocker (for review, see [29,30]). In contrast, 1 µM nifedipine which could inhibit KCl-induced contraction, had no effect on the contraction induced by 30 µM H2O2 (Fig. 8A). Comparable inhibitory effects of the various blockers except for nifedipine on the contraction induced by 100 µM ATP were also observed (Fig. 8B)


Figure 8
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  		Fig. 8 Effects of Ca2+-removal, procaine (10 mM), dantrolene (30 µM), Ni2+ (100 µM), tetrandrine (50 µM) and nifedipine (1 µM) on the contractions induced by 30 µM H2O2 (A) or 100 µM ATP (B). Each preparation was challenged with 30 µM H2O2 only once after preincubation with either vehicle (control) or the various blockers for 20 min. Ca2+-removal denotes washing the tissues with Ca2+-free Krebs’ solution (+100 µM EGTA) for three times in 5 min before addition of H2O2 or ATP. Each column represents the mean±S.E.M. (n=6–9), expressed as a percentage of the tension developed by 50 mM KCl and taken from at least three rats. *, P<0.05 and **, P<0.001 compared with control.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
4.1 H2O2-induced vasoconstriction is pharmacologically selective
In the present study, we found H2O2, a mild endogenous oxidant, can induce an apparent phasic contraction of rat aorta even at a physiological concentration of 30 µM [31]. Such a transient contraction had been reported previously in rat pulmonary arteries [16] and more recently in aorta of WKY and SHR rats [6]. H2O2-induced contraction is not related to the release of typical neurotransmitters, because the response to H2O2 was not affected by phentolamine, an {alpha}-adrenoceptor antagonist; atropine, a muscarinic acetylcholine receptor antagonist; methysergide, a serotonin-receptor antagonist and chlorpheniramine, a histamine-receptor antagonist. In fact, it is widely accepted that rat aorta is not sympathetically innervated. However, as anticipated, catalase pretreatment could abolish H2O2-induced phasic contraction, indicating that the response is truly induced by H2O2. However, superoxide anion (O2–) and hydroxyl free radicals may not be involved, because the O2– scavenger, SOD and the hydroxyl free radical scavenger, DMSO, failed to inhibit H2O2-induced contraction.

It is interesting to note that H2O2-induced contraction is similar to ATP-induced contraction, and that both H2O2- and ATP-induced contractions were potentiated by endothelium removal. These results suggest that both H2O2 and ATP may trigger the release of endothelium-derived vasodilators, presumably as NO and/or PGI2, which may compromise H2O2 and ATP-induced contractions [6,32]. Furthermore, Musat et al. [21] had shown that H2O2 could, in a concentration- and time-dependent manner, modulate ATP binding to plasmalemma ATP receptors (presumably including P2-purinoceptors), suggesting a possible direct modification of P2-purinoceptors by H2O2. The interaction between H2O2 and the ATP-binding site, presumably the P2-purinoceptors is further supported by the finding that H2O2-activated MAPKs signaling cascades in aortic smooth muscle cells could be blocked by suramin, a non-selective antagonist of P2-purinoceptors [20]. Based on these observations, we speculate that H2O2 may contract rat aortic rings via activation of P2-purinoceptors. However, in view of the findings that stress on cells such as mechanical stretch, e.g. as in cell swelling [33] or hypoxia [34] can cause the cell to release ATP, it is possible that our observed contractile responses to H2O2 might be due to the autocrine action of ATP brought about by H2O2. In addition, the lack of inhibitory effect of phentolamine and the poor sympathetic innervation in rat aorta makes the possibility of H2O2-induced release of ATP as a co-transmitter with norepinephrine from the nerve endings highly unlikely. Furthermore, the fact that apyrase, at a concentration that nearly abolished ATP-induced contraction, had no influence on H2O2-induced contraction, would also argue against the pathway due to ATP release.

4.2 H2O2 directly activates P2-purinoceptors
The involvement of P2-purinoceptors activation in response to H2O2 was further assessed. We observed that H2O2- and ATP-induced contractions not only showed homologous functional desensitization, but also heterologous desensitization against each other. Under the same experimental protocols, however, H2O2 and ATP had no significant effect on KCl, PE or caffeine-induced contractions, indicating that brief treatment (10 min) of rat aorta with low dose of H2O2 (30 µM) or high concentration of ATP (1 mM) would not non-selectively impair smooth muscle contractions. This observation is consistent with an early finding by Jin et al. [16] using rat pulmonary arteries. Therefore, the results of cross-desensitization tests lend additional support to our contention that H2O2 and ATP may share common target(s), mediating their contractile responses in rat aorta.

The fact that both suramin and RB-2, two widely used P2-purinoceptor antagonists [35], could concentration-dependently inhibit or abolish H2O2- or ATP-induced contractions, further confirming that P2-purinoceptors activation participates in H2O2-induced contraction of rat aorta. Since P2-purinoceptors can be further classified as P2x and P2y subtypes [35], we further tested which subtype of P2-purinoceptors mediates H2O2-induced phasic contraction. Unlike suramin and RB-2, evans blue which was demonstrated recently to be a moderately selective P2x receptor blocker [35,36], could abolish the selective P2x receptor agonist {alpha}-β-methylene-ATP-induced contraction of rat aorta, with only partial inhibition of ATP or H2O2-induced contractions, suggesting possible involvement of both P2x and P2y receptors in ATP and H2O2 responses. Such a view is further supported by the finding that both ATP- and H2O2-induced contractions are only partially depressed when the preparations are subject to prior desensitization of P2x receptors with {alpha}-β-methylene-ATP. The fact that suramin, which does not readily cross cell membranes due to its large size and highly polar nature [35], could blunt the vascular contractile response induced by H2O2, suggests that despite easy diffusion of exogenous H2O2 to the cell interior, the contractile action of H2O2, at least at the concentration of 30 µM, may be primarily due to its direct effects on the surface membrane. The lack of effect of H2O2 on KCl, and PE-induced contractions also supports the view that H2O2 at concentration of 30 µM, does not elicit non-selective effects on cellular membrane or contractile elements. Although the nature of interaction between H2O2 and P2-purinoceptors is unclear, the fact of reversible inhibition by suramin and irreversible inhibition by RB-2 on H2O2-induced contraction raises the possibility that suramin and RB-2 may have a ‘sealing effect’ on the H2O2-modified sites locating at P2-purinoceptors and possible other plasmalemma proteins. Further study is required to elucidate this question.

4.3 H2O2 and ATP signaling pathways are similar
We also compared the signal transduction pathways which led to contraction in response to H2O2 and ATP. An increase in the production of IP3 by the activation of PLC via P2y receptors has been demonstrated in various tissues, including vascular smooth muscle [34]. NCDC has been shown to inhibit the activity of phosphoinositide-specific PLC [23], and also to inhibit the contractile responses induced by several P2y receptor agonists in rat urinary bladder smooth muscle [37]. Therefore, in this study, we used NCDC to inhibit the activity of PLC in rat aortic smooth muscle cells. At concentration of 10 µM, NCDC inhibited contraction induced by 100 µM ATP and 30 µM H2O2 without affecting the contraction induced by 30 mM KCl, suggesting that PLC activation may be involved in H2O2- and ATP-induced responses. Similar result was also observed in rat pulmonary arteries for H2O2 [15]. It has also been shown in endothelial cells that U73122 [GenBank] , a specific PLC inhibitor, abolished H2O2-induced Ca2+ mobilization [19], and that H2O2 caused hydrolysis of inositol phospholipids [38]. In addition, we have found that neomycin, a chelator of PIP2 [24] or H7, a PKC inhibitor [25], each depressed the peak of the contractions induced by H2O2 or ATP. These results, together with the fact that staurosporine, a selective PKC inhibitor, also attenuates H2O2-induced contraction of rat aorta [13], further confirm that ATP- and H2O2-induced contractions share a common P2y–PLC–PKC signaling cascade.

It is well recognized that P2y receptors are coupled also to PLA2 [34,39], and ATP-induced contraction in smooth muscle could be inhibited by indomethacin [37,40]. Our findings that both H2O2- and ATP-induced contractions could be compromised by mepacrine, a PLA2 inhibitor [26], or by indomethacin, a COX inhibitor, again suggest that activation of vascular contraction by H2O2 utilizes the same signaling pathway as does ATP.

4.4 H2O2- and ATP-induced contractions share common Ca2+ mobilization pathways
Rat aortic smooth muscles express both P2x and P2y receptors [41]. So, it is now generally believed that ATP triggers intracellular Ca2+ release via IP3 signaling pathway owned by P2y receptor activation, and that ATP may also elicit extracellular Ca2+ influx both by direct opening the receptor-operated Ca2+ channels (ROCC) which is now identified as P2x receptors [42] and by indirect opening the putative store-operated Ca2+ channels (STOCC) due to P2y receptor activation. On the other hand, it was demonstrated that both extracellular Ca2+ influx and intracellular Ca2+ release participate in H2O2-induced Ca2+ mobilization in rat aortic smooth muscle cells [13]. The present study also showed that both H2O2- and ATP-induced contractions were inhibited by removal of extracellular Ca2+ or by blockade of intracellular Ca2+ release with procaine [27] and dantrolene [28]. The fact that Ni2+, an inorganic non-selective Ca2+ channel blocker, but not nifedipine, a VDCC blocker, also inhibited H2O2- or ATP-induced contractions, suggests the possible involvement of Ca2+-influx via the ROCC and/or the STOCC pathway(s) in both contractions. This view is further supported by the fact that both H2O2- and ATP-induced contractions were also inhibited by tetrandrine, a blocker of ROCC and STOCC [29,30]. All these functional results are consistent with the previous notion that H2O2 mobilizes cellular Ca2+ via an agonist-sensitive pathway in endothelial cells, which lack VDCC [18]. At present, however, we cannot reconcile the difference that Ca2+-free only partially inhibited and Ni2+ nearly abolished H2O2 and ATP-induced contractions. One likely explanation is that Ni2+, besides blocking multiple types Ca2+ channels, has high affinity for ATP, thus interfering with ATP binding to its receptors. Such an effect of Ni2+ is highly possible, because previous studies had shown that ATP receptors in other cell lines can be inhibited by several multivalent cations, such as La3+ [43], Zn2+ [44] as well as high concentration of Na+ [45].

Oxygen-derived free radicals including H2O2 are known to inhibit the SR Ca2+-pump in coronary smooth muscle [46–48], which may play a role in the regulation of vascular tone. It is therefore possible that H2O2-induced contraction of rat aorta may be the result of SR Ca2+-pump inhibition. However, several lines of evidence suggested that such an effect may play a minor role, if any, in our experimental conditions. First, it was demonstrated that a successful detection of intracellular H2O2 in rat aortic smooth muscle cells required the exogenous H2O2 at least 100 µM [49], indicating that if H2O2-induced contraction is truly due to its direct inhibition of SR Ca2+ pump, then a threshold concentration of 100 µM must be achieved for H2O2-induced contraction. This is contrast with our observation that as low as 3 µM H2O2 could elicit a detectable threshold contraction in rat aorta with an EC50 of 30 µM. Furthermore, our observation that a brief pretreatment with 30 µM H2O2 did not impair PE-induced contraction of rat aorta, would also argue against a significant contribution of SR Ca2+ pump inhibition on H2O2-induced contraction. Second, in isolated cardiac and skeletal muscle SR vesicles, an earlier study demonstrated that it is the hydroxyl free radical rather than H2O2 itself that competitively inhibits the activity of SR Ca2+ pump [50]. In this context, we had already precluded the involvement of hydroxyl free radical in H2O2-induced contraction. Third, unlike H2O2, cyclopiazonic acid (CPA), a selective SR Ca2+ pump inhibitor in rat aortic smooth muscle [51], triggered a slow and sustained contraction of rat aorta, and this response is not significantly affected by suramin and RB-2 (unpublished observations). Despite all above arguments, we still cannot fully rule out the possibility that the SR Ca2+-pump in rat aortic smooth muscles might be somehow impaired if the challenge time and H2O2 concentration were increased to 30 min and 250 µM, respectively, as previously employed in the study of coronary smooth muscles [48].


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
In summary, we have not only shown that exogenously added H2O2 can induce a transient contraction in rat aorta at physiological concentrations, but also for the first time provided pharmacological evidence to suggest that P2-purinoceptors activation may be one of the primary signaling steps for H2O2-induced contraction. These findings also bear potential theoretical significance in the study of the activation state of membrane receptors. Indeed, previous studies have shown that several membrane receptors could be directly activated by UV light irradiation [53], and that β-adrenergic receptors can be functionally activated by dithiothreitol, a disulfide-reducing agent [54]. The molecular mechanism(s) of how a ligand-gated ion channel (such as P2x receptor) and a G-protein coupled receptor (such as P2y receptor) could be activated by an oxidant, such as H2O2, remains to be elucidated.

Time for primary review 36 days.


   Acknowledgements
 
This work is part of the MD thesis of JZS and was supported by grants from the National Natural Science Foundation of China (XYZ) and from the Heart and Stroke Foundation of Ontario (CYK). CYK is a Career Investigator of the Heart and Stroke Foundation of Ontario.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 

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