CHARACTERISTICS OF APYRASE (EC 3.6.1.5) ON
CULTURED BOVINE ENDOCARDIAL ENDOTHELIAL CELLS

YI　FU-XIAN，SUN　PING,HUANG　SHAO-LING,
LIU　WEN-LAN,GUO　ZHAO-GUI^*

ABSTRACT Apyrase activities in some tissues and cells, such as peripheral vascular endothelial cells, have been reported, but these in endocardium endothelial cells have not been reported. The present study was to characterise the properties of bovine endocardium endothelial cells (BEEC)-associated apyrase. Apyrase activity was assayed by inorganic phosphate release, which could be inhibited concentration-dependently by NaN₃, an apyrase inhibitor. NaF (20 mmol/L), another inhibitor of apyrase, also markedly inhibited the activity. EDTA or EGTA (1 mmol/L) could also inhibit the activity completely. However, the inhibitor for Na⁺/K⁺-ATPase, ouabain (3 mmol/L) did not affect the enzyme activity. BEEC apyrase activity was dependent on divalent cations (Ca²⁺ or Mg²⁺) and pH value.
Key words: apyrase; adenosine diphosphate; endocardium; sodium azide

培养牛心内膜内皮细胞的
腺苷三磷酸双磷酸酶的特性研究

易富贤　孙　平　黄韶玲　刘文兰　郭兆贵

摘　　要:包括血管内皮细胞在内的多种细胞和组织存在腺苷三磷酸双磷酸酶(apyrase), 但心内膜内皮细胞是否含有apyrase尚无报道。本文旨在研究牛心内膜内皮细胞apyrase的特性。以无机磷释放法检测培养牛心内膜内皮细胞(BEEC) apyrase的活性。公认的apyrase 抑制剂叠氮钠呈浓度依赖性地抑制apyrase活性; 另一种apyrase 抑制剂氟化钠 (20 mmol/L)也明显抑制apyrase活性。 而Na⁺/K⁺-ATPase的抑制剂哇巴因 (3 mmol/L)却不能抑制该酶活性。 BEEC apyrase活性依赖于钙或镁等二价阳离子以及pH 的改变, EDTA或EGTA(1 mmol/L)均能完全抑制其活性, 在pH值为8.5时活性最高。由此可见, 牛心内膜内皮细胞存在叠氮钠敏感的、依赖于二价阳离子和pH值的腺苷三磷酸双磷酸酶活性。
关键词: 腺苷三磷酸双磷酸酶; 腺苷二磷酸; 心内膜; 叠氮钠
学科分类号: Q556; R331.3

　　Until recently, nucleotides were thought to act mainly as an intracellular energy resource. It was suggested that nucleotides could be released in response to cell activation or injury, and could elicit biological responses, such as the prothrombotic effects of ADP^［1］ and the negative cardiac response of ATP^［2］.
　　Platelet activation and aggregation are important factors in the mediation of endocarditis and rheumatic valvulitis^［3,4］ and are specifically associated with the rejection of discordant xenograft^［5］. Amongst the various agonists of platelet activation, ADP is most important for the recruitment and further amplification of platelet aggregation^［6,7］. Platelets become unresponsive to all agonists when in proximity to vascular endothelial cells. Only a few years ago this unresponsiveness was thought to be due to prostacyclin (PGI₂) alone^［7］. However, until recently at least three separate endothelial “thromboregulatory” systems have been suggested, viz. PGI₂, endothelium-derived relaxing factor (EDRF/NO), and most importantly an endothelial cell ecto-enzymes termed ATP diphosphohydrolases (apyrase, EC 3.6.1.5)^［7］. Apyrase can hydrolyze both extracellular inflammatory mediators ATP and ADP to AMP^［7,8］, which then generates adenosine, an important antiinflammatory and antiaggregatory mediator^［9］. Apyrase is operative in the complete absence of EDRF/NO and PGI₂, indicating that the latter two are dispensable thromboregulators^［1,6］.
　　At present, whether endocardial endothelial cells (EEC) have apyrase activity has not been reported. The present study aimed to characterize EEC-associated apyrase.

1　MATERIALS AND METHODS

1.1　Materials　　 Adenosine 5′-diphosphate (ADP), M199 medium, collagenase (type IA), ethylenediaminotetraacetic acid (EDTA), egtazic acid (EGTA), diadenosine pentaphosphate (Ap₅A), sodium azide (NaN₃), and Ouabain were obtained from Sigma Chemical Co., sodium fluoride (NaF, AR) from Beijing Chemicals Co., the newborn bovine hearts from a local slaughterhouse, and fetal bovine serum (FBS) from Sijiqing Biologic Engineering Co.
1.2　Bovine endocardial endothelial cells (BEEC) culture　　BEEC from bovine hearts were cultured by the method of Mebazaa et al^［10］. Briefly, atria, valve tissue, and chordae tendineae were removed from freshly isolated newborn bovine hearts. The ventricles were filled with 0.1% collagenase (type IA) in phosphate-buffered saline (PBS) and incubated at 37℃ for 45 min. The inside of each ventricle was gently rubbed with a cell scraper to remove the loosely attached cells. The cell suspension was removed, pelleted, and resuspended in M199 medium supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin. Cells were passaged using 0.05% tyrosine/EDTA. The obtained cell population consisted of >98% endocardial endothelial cells as identified by their cobblestone morphology and by electron microscopy. Cells (from passage 4 to 9) were plated for experiments in 24-well culture dishes at a density of 1×10⁵ cells/cm² and incubated at 37℃ under air containing 5% CO₂.
1.3　Apyrase activity assay　　Intact BEEC were plated in 24-well culture dishes, each well was incubated with ADP 500 μmol/L in 1 ml 50 mmol/L Tis/HCl for 30 min and free phosphate release was determined^［11］. One unit of apyrase activity corresponds to the amount of enzyme which catalyzes the liberation of 1 nmol inorganic phosphate/min at 30℃.
1.4　Statistical analysis　　Results were presented as mean±SD and analyzed by t test.

2　RESULTS

2.1　Effect of pH on BEEC apyrase activity
　　Apyrase activity was determined within the range of pH 6.5～9.5 in the presence of 1 mmol/L Ca²⁺ using 0.5 mmol/L ADP as substrate. The enzyme activity was dependent on pH. Maximum activity was achieved at pH 8.5. The enzyme activity increased with the increase of pH at the range of pH below 8.5, while the activity decreased with the increase of pH when pH was above 8.5. The enzyme activity at pH 6.5 was about 42% of that at pH 7.5 (Fig.1).
2.2　Effect of divalent cations on apyrase activity
　　Apyrase activity was determined at pH 7.5. The enzymic activity was dependent on the con～cen～trations of Ca²⁺ or Mg²⁺. Maximum activity was obtained at 0.5 mmol/L CaCl₂ or MgCl₂. No de～tec～table activity was found in the presence of the chelating agents EDTA or EGTA (1 mmol/L). Furthermore, it was shown that Ca²⁺ is a better activator of the enzyme than Mg²⁺ (Fig.2).

Fig.1　Effect of pH on apyrase activity of bovine endocardial endothelial cells
Results are the mean±SD of six independent experiments each in triplicate.

Fig.2　Effect of divalent cations on apyrase activity of bovine endocardial endothelial cells
Results are the mean±SD of six independent experiments each in triplicate. ～^*　P<0.05, ～^*　*　P<0.01, compared with Ca²⁺.

2.3　Effect of inhibitors on apyrase activity
　　The effects of various inhibitors on apyrase activity were examined. The inhibitors added to BEEC just before the addition of ADP. Sodium azide, a known inhibitor of apyrase^［8］, inhibited the activity in a concentration-dependent manner (Fig.3). NaF, another inhibitor of apyrase, also markedly inhibited the activity. Ouabain, an inhibitor for Na⁺/K⁺-ATPase, did not affect apyrase activity. Ap₅A, an inhibitor of adenylate kinase, exhibited lower effect on apyrase activity (Table 1).

Fig.3　Inhibitory effect of NaN₃ on apyrase activity of bovine endocardial endothelial cells
Results are the mean±SD of five independent experiments each in triplicate.

Table　1　Effect of inhibitors on BEEC apyrase activity

Inhibitor	Concentration /mmol^.L^－1	Apyrase activity (U/10^5 cells)
Control		1.69±0.21
Ouabain	3	1.66±0.19
Ap5A	0.25	1.48±0.16^*
NaF	20	1.04±0.16^**
NaN3	10	0.84±0.21^**
EDTA	5	0.04±0.06^**

　 Results are the mean±SD of five independent experiments each in triplicate. ^*P<0.05,^**P<0.01, compared with control.

3　DISCUSSION

　　Like vascular endothelial cells, the results reported here demonstrate for the first time that NaN₃-sensitive apyrase also exists in bovine endocardial endothelial cells.
　　BEEC apyrase activity is dependent in the presence of divalent cations Ca²⁺ or Mg²⁺. This dependency seems to be a common property of all apyrase studied^{［12～14］}. It has been concluded that divalent cations are required for the binding of the substrate to the apyrase active site^［13］. This activation can either be directly mediated by the cations′ binding to the enzyme, which would then be in an appropriate catalytic conformation, or indirectly, by forming a cation-substrate complex which would serve as a better substrate.
　　Studies of the catalytic properties of BEEC apyrase have showed that the activity is optimal at pH 8.5, which is much higher than those reported in other tissues or cells^{［8,12～14］}. This diversity may be due to tissue difference. The enzyme was shown to lose activity in acid environment. When the pH was at 6.5, the enzyme activity was about only 42% of that at pH 7.5 (Fig.1).
　　The physiological function of BEEC apyrase has not been known. One possible function is to prevent thrombotic diseases such as endocarditis and rheumatic valvulitis, because platelet activation and aggregation are important pathogenetic procedures of these diseases^［3］, while ADP is the most important platelet agonist and renders platelet aggregation irreversible^［15］. The study by Carter et al. shows that acid metabolites such as lactic acid accumulated in ischaemic myocardium can diffuse through the endocardium, which are likely to predispose to the mural thrombosis^［4］. This could be further supported by our present study that BEEC loses its anti-thrombotic apyrase activity in acid environment (Fig.1). Another possible function of BEEC apyrase is to protect BEEC from apoptosis, because ATP and ADP could induce vascular endothelial cell apoptosis through the activation of P₂ receptors and NF-kappa B^［16］.

^*Correspondence to Prof. GUO Zhao-Gui. 　Phn: 0086-0731-4474411,ext 2797. 　Fax: 0086-0731-4471339. E-mail: guozg@public.cs.hn.cn
作者单位:Laboratory of Molecular Pharmacology, Hunan Medical University, Changsha 410078
湖南医科大学分子药理研究室, 长沙 410078

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Received 1998-08-18　　Revised 1998-11-02