Received 2001-06-28Accepted 2001-09-04

Corresponding author. Tel: 024-23256666-6264; E-mail address: xueqinding@yahoo.com

Acta Physiologica Sinica

Feb. 2002, 5４ (1), 38～42

Research Paper

Vasodilative action of carbon monoxide on rat pulmonary artery in vitro

DING Xue-Qin*, LIU Gui-Ming, WANG Jun-Ke, SHENG Zhuo-Ren

Department of Anesthesiology, The First Affiliated Hospital, China Medical University, Shenyang 110001

Abstract: The present study investigates the vasodilative action of carbon monoxide on rat pulmonary artery in vitro. After isolation of the pulmonary artery rings (PAR) from Wistar rats, an ACh concentration-response curve was generated; the PARs were incubated with the NOS inhibitor L-NAME (30 μmol/L, n=10) or the heme oxygenase inhibitor ZnPPIX (10 μmol/L)+L-NAME (30 μmol/L, n=10) for 30 min. After that, a second ACh concentration-response curve was elicited. Other isolated PARs were randomly divided into two groups: endothelium-intact group (n=8) and endothelium-denuded group (n=8). The effect of exogenous carbon monoxide (CO) on pulmonary arterial vessel tone was observed. The results showed that ACh induced a concentration-dependent pulmonary vasorelaxation. This relaxation disappeared after endothelium was denuded. The ACh induced relaxation was attenuated after pretreatment with 30 μmol/L L-NAME, and attenuated further after pretreatment with 10 μmol/L ZnPPIX+30 μmol/L L-NAME. Exogenous carbon monoxide relaxed pulmonary artery in both the endothelium-intact group and the endothelium-denuded group. These data suggest that ZnPPIX inhibits ACh induced endothelium-dependent pulmonary artery relaxation and that CO is an endothelium-derived relaxation factor, and exogenous CO can relax pulmonary artery.

Key words: carbon monoxide; nitric oxide; heme oxygenase inhibitor; vasodilation

一氧化碳对大鼠离体肺动脉的舒张作用

丁学琴*, 刘贵明, 王俊科, 盛卓人

中国医科大学第一附属医院麻醉科, 沈阳 110001

摘要: 本研究观察了一氧化碳(CO)对离体大鼠肺动脉的舒张作用。制备Wistar大鼠肺动脉环, 作出ACh浓度效应曲线之后, 肺动脉环用一氧化氮合成酶抑制剂L-NAME 30 μmol/L (n=10)或血红素氧化酶抑制剂ZnPPIX 10 μmol/L+L-NAME 30 μmol/L (n=10) 孵育30 min, 再制备一个ACh的浓度效应曲线, 观察ZnPPIX对ACh的浓度效应曲线的影响。另取一组肺动脉环, 分为内皮完整组和去内皮组, 观察外源性CO对肺动脉环张力的影响。结果表明, 用 L-NAME孵育后, ACh的血管舒张反应受抑, 最大抑制率为50.4±9.2%; 用ZnPPIX+L-NAME孵育后, ACh的血管舒张反应进一步受抑, 最大抑制率为84.4±11.2%。外源性CO无论对内皮完整组还是去内皮组肺动脉都有舒张作用。本研究提示, ZnPPIX可抑制ACh的内皮依赖性肺动脉舒张反应, CO是一个内皮源性的血管舒张因子, 外源性CO可舒张肺动脉。

关键词: 一氧化碳; 一氧化氮; 血红素氧化酶抑制剂; 血管舒张反应

学科分类号: Q463; R331.3＋３

Regulation of blood vessel tone is pivotal for the maintenance of adequate tissue oxygenation and perfusion. The process of this regulation involves a delicate balance between vasodilators and vasoconstrictors. Nitric oxide (NO), a potent vasodilator is an endothelium-derived mediator that helps maintain normal vascular tone by stimulating guanylyl cyclase in vascular smooth muscle cells (SMCs) and elevating cGMP levels. Like NO, carbon monoxide (CO) is another endogenously produced gas molecule and possibly plays arole in regulating the blood vessel tone[1]. CO is produced mainly by oxidation of heme via heme oxygenase (HO)[2]. There is evidence for HO-catalyzed CO formation in rat aortic tissue[3]　and relaxation in response to CO has been demonstrated in rat coronary[4]　and in canine coronary, femoral, and carotid artery preparations[5]. However, whether CO contributes to pulmonary artery relaxation is still not clear.

Therefore, we designed these experiments to investigate the role of endogenous carbon monoxide in pulmonary artery relaxation by observing the effect of heme oxygenase inhibitor zinc protoporphyrin IX (ZnPPIX) on acetylcholine (ACh) concentration-response relationship. Meanwhile, we examined the effect of exogenous carbon monoxide on pulmonary artery to determine whether exogenous carbon monoxide can relax pulmonary artery. As a result, we can testify the role of CO in the regulation of pulmonary artery tone, which is important in preventing pulmonary artery hypertension and in maintaining pulmonary blood flow.

1MATERIALS AND METHODS

1.1 Pulmonary artery ring preparationWistar rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). After decapitation, the heart and lung were removed rapidly and placed into 0℃ Krebs solution containing (in mmol/L): NaCl 118.3, KCl 4.7, CaCl2 1.2, KH2PO4 1.2, NaHCO3 25 and glucose 11.1. The pulmonary arteries were isolated carefully in order to avoid endothelium injury. Once they were cleaned of adventitia, the pulmonary arteries were cut into 3 mm segments. Endothelium was removed from some rings by gently rubbing the lumen with closed forceps tips. The pulmonary artery rings (PARs) were threaded between two 600-μm-diameter hooks and suspended in tissue baths (37℃) filled with 10　ml Krebs solution. The top hook was connected to a TB-611 force-displacement transducer, and the bottom hook was anchored to an immovable support. Tissue baths were continuously bubbled with 95%　O2-5%　CO2. Rings were stretched to resting tension of 1　g and allowed to be equilibrated for 90　min before experimental observation. After equilibration, endothelium-intact rings were challenged with α1-adrenoceptor agonist phenylephrine (PE 1 μmol/L). At the peak of contraction, endothelial integrity was verified by noting the relaxant response to ACh (10 μmol/L). In endothelium-intact rings, at least 50% relaxation was necessary for inclusion in the experiment.

1.2 Effect of ZnPPIX on ACh responsiveness in PARs To determine whether endogenously produces CO contributed to pulmonary artery relaxation, ACh concentration-response curves were generated for PAR from each group of animals before and after treatment with the HO inhibitor ZnPPIX. All experiments were performed in the presence of the NOS inhibitor N-nitro-L-arginine methylester (L-NAME 30 μmol/L) to eliminate any confounding effects of NO. After the initial contraction and endothelial test, a concentration-response curve to ACh was generated. Then the rings were rinsed, followed by a 30 min incubation with either 10 μmol/L ZnPPIX+30 μmol/L L-NAME (n=10) or vehicle+30 μmol/L L-NAME (n=10). Due to the photosensitivity of protoporphyrin compounds, all the rings in the experiments were incubated in the dark. After the incubation was used, a second concentration-response curve was generated. Relaxant responses were expressed as a percentage of the relaxation of the PE-induced contraction. The inhibition rate was expressed as the difference in the relaxation rate between the control group (before administration of L-NAME or ZnPPIX+L-NAME) and the L-NAME or ZnPPIX+L-NAME groups.

1.3 Effect of exogenous carbon monoxide on PARs Experiments were performed using other endothelium-intact PARs (n=8) and endothelium-denuded PARs (n=8) to determine the effect of exogenous carbon monoxide on PAR. After initial contraction and endothelial test, the PARs were challenged with 1 μmol/L PE. At the peak of contraction, muscle bath gas was changed from 95%　O2-5%　CO2 to 10 ppm CO. When contraction reached the platform, 10 ppm CO was changed to 40 ppm CO. The relaxation was calculated as percent reversal of maximum PE-induced contraction.

1.4 Solutions All the drugs were prepared on the day of experimentation. PE and ACh were dissolved in normal saline. L-NAME (Sigma) was dissolved in water. Whereas ZnPPIX (porphyrin products, Logan, Utah, USA) was dissolved in normal saline containing 50　mmol/L Na2CO3.

1.5 Statistics Data are presented as means±SD. The significance of any difference between two groups and within group was determined with Student′s t test. Differences were considered statistically significant at P<0.05.

2RESULTS

2.1 The role of endothelium in ACh induced vasorelaxation

ACh induced concentration-dependent relaxation in endothelium-intact PARs. However, the relaxation in response to ACh disappeared in endothelium-denuded rings.

2.2 Effect of ZnPPIX on ACh responsiveness

PARs treated with L-NAME appeared to exhibit a smaller relaxation than that in untreated rings (control group), ACh in 10－5 mol/L caused a (90.7±1.5)% relaxation in control group and a (40.3±5.1)% relaxation in L-NAME group (Table 1)．

Rings treated with ZnPPIX+L-NAME appeared to exhibit a much less relaxation. ACh in 10－5 mol/L only caused an (11.1±8.4)% relaxation (Table 2).

L-NAME resulted in an inhibition of (50.4±9.2)% maximal response of ACh, while ZnPPIX 10 μmol/L+L-NAME 30 μmol/L inhibited (84.4±11.2)% of ACh maximal response (Table 3).

Table　１.Effect of L-NAME on ACh concentration-response relationship (%±SD)

Group[]n[]10－8〖〗10－7〖〗10－6〖〗10－5Control[]10[]22.1±9.8[]35.2±11.2[]54.6±11.2[]71.6±13.1[]90.7±1.5L-NAME[]10[]7.5±3.9*[]14.3±3.8*[]21.3±4.1*[]31.5±4.2*[]40.3±5.1**P<0.01 compared with control group. Relaxant responses are expressed as a percentage of the relaxation of the PE-induced contraction.

Table　2. Effect of ZnPPIX+L-NAME on ACh concentration-response relationship (%±SD)

Group[]n[]10－8〖〗10－7〖〗10－6〖〗10－5Control[]10[]20.9±8.2[]34.4±8.2[]52.3±9.7[]74.6±4.1[]95.5±5.4ZnPPIX+L-NAME[]10[]0*[]1.3±1.1*[]2.2±3.4*[]4.9±3.2*[]11.1±8.4**P<0.01 compared with control group. Relaxant responses are expressed as a percentage of the relaxation of the PE-induced contraction.

Table　3.Comparison of two groups in inhibition of ACh endothelium-dependent relaxation

(% ±SD)

Group[]n[]10－8〖〗10－7〖〗10－6〖〗10－5L-NAME[]10[]16.1±8.1[]20.9±12.1[]32.3±12.1[]40.1±11.1[]50.4±9.2ZnPPIX＋L-NAME[]10[]20.9±8.2*[]33.1±8.1**[]50.1±11.2**[]69.5±5.1**[]84.4±11.2***P<0.05, **P<0.01 compared with L-NAME group. The inhibition rate is expressed as the difference of the relaxation rate in controlled group (before administration of L-NAME or ZnPPIX+L-NAME) and in L-NAME or ZnPPIX+L-NAME group.

2.3 Effect of exogenous carbon monoxide on pulmonary artery smooth muscle

Figure 1 demonstrates that administration of exogenous CO to PE-contracted rings elicited a dose-dependent relaxation in both endothelium-intact and -denuded rings. There was no significant difference between the two groups (P>0.05).

Fig.1. The pulmonary artery relaxation of exogenous carbon monoxide. PAR exhibited a constriction after pretreatment with 1 μmol/L PE. Value of vessel tone increased to 0.739±0.09 g in endothelium-intact group (n=8), 0.728±0.08 g in endothelium-denuded group (n=8). When 10 ppm CO was administered at the peak of constriction, PARs were relaxed and the relaxation reached the platform within 2 min. Then 40 ppm CO was administered. PARs continued to relax and the relaxation reached the platform within 3 min.

3DISCUSSION

Acetylcholine (ACh) is known to evoke an endothelium-dependent vasodilatation response, which is unaffected by inhibitors of the L-arginine-NO and cyclooxygenase pathways in the hepatic artery, mediated by NO-independent mechanism or NO-dependent mechanism. This NO-independent relaxation was associated with a hyperpolarization of the smooth muscle cells[6]. It has been speculated that carbon monoxide shares many properties with NO, such as the ability to relax blood vessels[7]. In human jejunal smooth muscle cells, CO has been shown to induce a transient hyperpolarization and an increase in the whole cell outward current probably by activating potassium channels[8]. Since it has been suggested that the ACh-induced L-NOARG-resistant relaxation in the rat hepatic artery is caused by hyperpolarization of the smooth muscle cells[６], the possibility was investigated that CO produced by HO may be an endogenous mediator of this response. In our experiments, ACh caused an endothelium-dependent relaxation via NO-dependent and independent mechanism. ZnPPIX inhibited the NO-independent component of relaxation with a potency resembling its inhibition of HO. Moreover, in our studies, the concentration-dependent vasodilatation was only demonstrated in endothelium-intact PAR. However, this reaction disappeared after endothelium was denuded. This suggests that HO product, CO, can function as an endothelium-derived relaxation factor.

Carbon monoxide is now increasingly recognized as a physiologically important substance rather than a merely toxic waste product. There are three known isoforms of HO. HO-1 is inducible, whereas HO-2 and HO-3 are constitutively expressed. A variety of cellular stressor such as heat shock, oxidative stress, heavy metal, and hemoproteins can induce HO-1[9,10]. Not only is CO formation conditioned by these stimuli, but it is also liable to self-regulation and regulation by NO. In fact, these two messenger systems may interact in a varied manner and ultimately, depending on the condition, influence each other synergistically or antagonistically. Whereas CO inhibits its own synthesis and the synthesis of NO, NO promotes the formation of CO[11,12]. CO, on the other hand, can also displace NO from heme-binding site[13]. In brief, CO and NO form an operational unit whose activity and specific arrangement can vary with the functional demands. For example, under hypoxia and the attendant divergent changes taking place in the CO (upregulation) and NO (downregulation) systems, this interaction is expectedly minimal, if present at all. An opposite situation is likely to occur after exposure to pyrogens or hyperoxia when both systems are fully operational.

It has been demonstrated that L-NAME in 100 μmol/L can inhibit 90% maximal relaxation induced by ACh[14]. To study the role of CO in ACh-induced relaxation, we used a smaller dose of L-NAME (30 μmol/L) to inhibit NOS activity. Our results indicate that L-NAME in 30 μmol/L can inhibit 50% maximal relaxation of ACh while 10 μmol/L ZnPPIX+30 μmol/L L-NAME can inhibit 90% maximal relaxation of ACh. But whether CO is stronger than NO needs further study.

Vessels vary in the degree of NO-dependent and -independent response to muscarinic stimulation depending on vessel size, location and species. Some studies indicated that the expression of NOS in remote small vessels was reduced, and that CO was more effective in these small vessels. However, we demonstrated that CO dilated main pulmonary artery. This may be due to the difference of animals and doses used.

To further clarify the role of carbon monoxide, we demonstrated that exogenous carbon monoxide relaxed pulmonary artery both in endothelium-intact group and endothelium-denuded group. It showed that the response to CO indeed resides within the smooth muscle and is not due to the secondary release by a separate endothelium-derived dilator. We speculate that CO, like NO, acts in a paracrine manner to affect the underlying vascular smooth muscle. It suggests that exogenous CO, like exogenous NO, can be used as a pulmonary vasorelaxant to prevent pulmonary artery hypertension.

Our study demonstrated that ZnPPIX inhibited ACh endothelium-dependent relaxation by inhibiting heme oxygenase and reducing the production of CO. Therefore, CO is an endothelium-derived relaxation factor. Exogenous carbon monoxide can relax pulmonary artery and may be applied clinically for preventing pulmonary artery hypertension and ARDS.

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