Han JL et al / Acta Pharmacol Sin 2003 Apr; 24 (4): 327-331

Functional 1-adrenergic receptor subtypes in human right gastroepiploic artery1

HAN Jiang-Li2, ZHANG You-Yi, LÜ Zhi-Zhen, MAO Jie-Ming2, CHEN Ming-Zhe2, HAN Qi-De3

Institute of Vascular Medicine, 2Department of Cardiovasology, Third Hospital, Peking University, Beijing 100083, China

1 Project supported by the Major State Basic Research Development Program of People's Republic of China (G2000056906) and the National Natural Science Foundation of China (No 30070872, 30171083).

3 Correspondence to Prof HAN Qi-De. Phn 86-10-6209-2306. Fax 86-10-6201-5681. E-mail hanqd@bjumu.edu.cn

Received 2002-04-15 Accepted 2002-11-11

KEY WORDS alpha-1 adrenergic receptors; right gastroepiploic artery; vasoconstriction; adrenergic alpha- antagonists; RS17053; 5-MU; BMY7378; WB4101; chlorethylclonidine

ABSTRACT

AIM: To study the functional 1-adrenergic receptor (1-AR) subtypes in human right gastroepiploic artery (RGA). METHODS: The effects of 2-AR, 1-AR, and 1-AR subtype selective antagonists on norepinephrine (NE)-induced vasoconstriction in isolated human RGA were observed by contractile function experiment. RESULTS: Cumulative concentration-response curves for NE were competitively antagonized in RGA by 2-AR selective antagonist yohimbine (pA2 6.82±0.28, slope 1.12±0.40), 1-AR selective antagonist prazosin (pA2 9.77±0.22, slope 0.90±0.22), 1A-AR selective antagonists RS17053 (pA2 8.42±0.20, slope 0.93±0.20) and 5-MU (pA2 8.42±0.22, slope 0.88±0.18), 1D-AR selective antagonist BMY7378 (pA2 6.84±0.32, slope 1.05±0.17), and 1A-, 1B-AR selective antagonist WB4101 (pA2 8.88±0.20, slope 1.15±0.16). The correlation coefficients between these pA2 values of 1-AR selective antagonists with pKi values of which obtained from 1A-, 1B- and 1D-AR cloned cells are 0.95, 0.82, and 0.42. After the vessels were pretreated by chlorethylclonidine (CEC), an 1B- and 1D-AR irreversible alkylating agent, the pD2 values were changed from 5.9±0.5 to 5.6±0.6 and the maximal contraction was changed from (8.9±3.2) g to (8.0±3.2) g, respectively. The difference was not significant. CONCLUSION: In human RGA, the contraction response is mainly mediated by 1-AR, of which 1A-AR plays an important role, whereas1B- and 1D-AR are not involved in the contraction response.

INTRODUCTION

1-Adrenergic receptor (1-AR) is an important mediator, which contributes to the regulation of vascular activity, and is involved in almost all-vascular smooth muscles to cause contraction response induced by norepinephrine (NE). 1-AR is subdivided into three subtypes (1A, 1B, and 1D-AR) by pharmacological techniques with subtype-selective antagonists and molecular biological techniques. The corresponding cDNA coding for these receptors had been cloned by means of molecular biological methods[1,2]. The distribution of 1-AR and 1-AR subtypes has been extensively characterized in rat and rabbit vessels using functional studies and molecular biological methods[3-5]. However, very little is known regarding the distribution of 1-AR in human vessels. Right gastroepiploic artery (RGA) is an alternate conduit used in coronary artery bypass graft (CABG) because of its similarity to internal mammary artery (IMA). The RGA is reported to exhibit better contractility to NE than the IMA, but comparable endothelium-dependent relaxation[6]. However, it is unknown which subtype of 1-AR mediates NE-induced contraction. In this study the functional role of 1-AR and its 3 subtypes in human RGA was investigated.

MATERIALS AND METHODS

Norepinephrine, yohimbine, propranolol, prazosin, desmethylimipramine, normetanephrine, and acetylcholine were purchased from Sigma. 8-(2-(4-(2-Methoxyphenyl)-1-piperazinyl)-8-azaspiro (4,5) decane-7-dionedihydrochloride) (BMY7378), 2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1, 4-benzo-dioxane (WB4101), 5-methyl-urapidil (5-MU), and chlorethylclonidine (CEC) were purchased from Research Biochemicals. N-(2 -(2-cyclopropylmethoxy) ethyl) 5-choro--dimethyl-1H-indole-3-thylamine (RS17053) was provided by Roche Bioscience.

Organ bath experiments Human undistended RGA from patients (mean age, 61 a±3 a; 14 male and 2 female; range 40 to 74 years old) were obtained during gastrectomy, respectively. The vessels were immediately placed in Krebs' solution of the following composition (mmol/L): NaCl 120, NaHCO3 20, KCl 5.45, NaH2PO41.2, MgCl2 1.2, CaCl2 2.5, glucose 10, and disodium edetic acid 0.04 (saturated with 95 % O2 and 5 % CO2). The vessels were then dissected free of connective tissue, cut into 2-mm ring segments for in vitro contractile studies. They were carefully denuded of endothelium by inserting a small forceps into the lumen and gently rolling the ring backwards and forwards in the dissecting chamber. The lack of functional endothelium was confirmed by the absence of endothelium-dependent relaxation of acetylcholine (10 mmol/L) in raised tone preparations induced by NE.

Desmethylimipramine 0.1 mol/L, normetane-phrine 1 mol/L(to block neuronal and extraneuronal uptake of NE, respectively) and propranolol 10 mol/L(to block -adrenoceptors) were included in the incubation solution (yohimbine 0.1 mol/L was added into the incubation solution when needed). The preparations were mounted at 37 ºC in 10-mL organ baths containing Krebs' solution saturated with 95 % O2 and 5 % CO2, and attached to force displacement transducer. The preparations were equilibrated at an optimal resting tension of 3.0 g for 1 h, and primed twice with 10 µmol/L NE. After thorough washing, NE-cumulative concentration-response curve (CRC) was generated, followed by another 30-min washing. RGA were then incubated in turn with increasing amounts of yohimbine, prazosin, WB4101, 5-MU, RS17053, and BMY7378 for 40 min, after that 3 NE-CRC were generated in the presence of the above antagonists, EC50 values and 95 % confidence limits were calculated for all CRC. The pA2 values were calculated by Schild plot. RGA were incubated with CEC 50 µmol/L for 30 min; after 40-min washing (the solution without CEC), pD2 values of NE and maximal contraction were determined.

Statistics Data were presented as mean±SD. Statistical analysis was performed by Student't test.

RESULTS

Effects of -AR selective antagonists on blood vessel contraction induced with NE In RGA NE caused the vasoconstriction in a concentration-dependent manner. Cumulative concentration-contractile response curves for NE (NE-CRC) were obtained with pD2 value (5.9±0.5) and the maximal contraction (8.9 g±3.2 g). Prazosin (1, 3, and 10 nmol/L) and yohimbine (0.1, 0.3, and 1 µmol/L) competitively inhibited NE-induced contraction in a concentration-dependent manner. The pA2 values and slopes for antagonists were shown in Tab 1 and Fig 1.

Tab 1. The pA2 values and slopes of -AR selective antagonists inhibiting NE-induced contraction in RGA. Mean±SD. aP>0.05 vs 1.

Fig 1. Concentration-contractile response curves and Schild plot for antagonism of norepinephrine-induced contraction by -AR and 1-AR subtype selective antagonists in human RGA. Yohimbine (0.1, 0.3, and 1 mol/L); prazosin (1, 3, and 10 nmol/L); WB4101 (3, 10, and 30 nmol/L); 5-MU (30, 100, and 300 nmol/L); RS17053 (10, 30, and 100 nmol/L); BMY7378 (0.3, 1, and 3 mol/L).

Effects of 1-AR subtype selective antagonists on blood vessel contraction induced with NE WB4101 (3, 10, and 30 nmol/L), 5-MU (30, 100, and 300 nmol/L), RS17053 (10, 30, and 100 nmol/L) and BMY7378 (0.3, 1, and 3 µmol/L) also competitively inhibited NE-induced contraction in a concentration-dependent manner in RGA. The pA2 values and slopes for antagonists were shown in Tab 2 and Fig 1.

Tab 2. pA2 values and slopes of 1-AR selective antagonists inhibiting NE-induced contraction in RGA. Mean±SD. aP>0.05 vs 1.

Effect of CEC on NE-CRC Before and after CEC 50 mmol/L pretreatment, the pD2 values (5.9±0.5 vs 5.6±0.6, n=4) and the maximal contraction (8.9 g± 3.2 g vs 8.0 g±3.2 g, n=4) of NE-CRC had no change (P>0.05, Fig 2).

Fig 2. Concentration-contractile response curve for antagonism of norepinephrine-induced contraction by CEC 50 mol/L in human RGA.

DISCUSSION

The use of arterial grafts instead of vein grafts in CABG has been demonstrated through the years to improve survival and to reduce the recurrence of myocardial ischemia and the occurrence of late myocardial infarction. The current trend in CABG is toward complete arterial revascularization. RGA is one of the arteries used for CABG with better clinical results[7,8], because it is related to the better contraction and dilation functions[9]. The good endothelial function of the RGA might be important for graft function and patency, whereas the enhanced contractility may facilitate vasospasm, especially in the presence of high circulating level of catecholamines. Therefore it is important to investigate the primary functional 1-AR subtype, which contributes to NE-induced vessel contraction. In the present studies, we used 1-AR and 1-AR subtype selective antagonists as tools to assess the function of 1-AR subtypes, which contribute to NE-induced contraction in RGA.

In functional experiments, 1 -AR selective antagonist prazosin antagonized NE-induced contraction with higher affinity than that of 2-AR selective antagonist yohimbine (pA2 values were 9.77±0.22 vs 6.82±0.28). Because the slopes of prazosin and yohimbine were not significantly different from unity, it indicated that there was one site binding of -AR in RGA and the contraction response was mainly mediated by 1-AR.

Further more, we studied the effect of 1-AR subtype selective antagonist on the NE-induced contraction response of RGA. 1A-AR subtype selective antagonists, including 5-MU and RS17053, antagonized NE-induced contraction with higher affinity (pA2 values were 8.42 respectively), whereas BMY7378, an 1D-AR subtype selective antagonist, antagonized NE-induced contraction with lower affinity (pA2 values was 6.84). In addition, the slopes for these drugs in Schild plot were not significantly different from unity. It indicates that 1A-AR might be the primary subtype, which contributes to NE-induced contraction. The further correlation analysis was performed. The pAi values for 1-AR subtype selective antagonists obtained from functional study were correlated with their pKi values measured in radioligand binding competition assays with membranes from HEK293 cells expressing 1A, 1B, and 1D-AR in our previous study[10]. The values for the correlation coefficient r were 0.95, 0.82, and 0.42. And the best correlation is with 1A-AR (P<0.05). It confirms that 1A-AR is a main subtype, which contributes to NE-induced contraction. Because there has no 1B-AR subtype selective antagonist now, we used CEC, an 1B and 1D-AR irreversible alkylating agent in functional study. Before and after RGA were pretreated by CEC, the pD2 values and the maximal contractile response for NE were unchanged. These results indicated strongly that in human RGA, NE-induced contraction was mainly mediated by 1A-AR subtype.

In conclusion, 1A-AR mainly contributes to the NE-induced contraction in human RGA. This result implies that 1A-AR antagonist can be used as an antispastic drug when RGA is employed for CABG.

REFERENCES