Acta Physiologica Sinica,   June   25, 2003, 55(3): 255£­259

Received 2002-08-22  Accepted 2002-11-20

Corresponding author. Tel: 86-311-606-2490;  Fax: 86-311-606-2490;    E-mail: syho@Hebmu.edu.cn

 Research  Paper

Responses of regional vascular beds to local injection of genistein in rats

JI En-Sheng,  ZHANG Li-Hua,  WANG Yi-He,  YUE Hua,  HE Rui-Rong*

Department of Physiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, China

 

Abstract:  The effects of local injection of genistein on femoral, renal, and mesenteric vascular beds were investigated respectively by constant flow perfusion method in 72 anaesthetized rats. The results are as follows: (1) genistein (0.4, 0.8, 1.2  mg/kg) decreased the perfusion pressure (PP) of femoral vascular bed in a dose-dependent manner.  The effect of genistein (0.8  mg/kg) was partially inhibited by L-NAME, or by sodium orthovanadate (50   ¦Ìg/kg), a potent inhibitor of protein tyrosine phosphatase; (2) genistein also decreased the PP of renal vascular bed in a dose-dependent manner  and the effect of genistein was completely inhibited by pretreatment with sodium orthovanadate, but unaffected by L-NAME; and (3) genistein decreased the PP of mesenteric vascular bed in a dose-dependent manner, an effect which was partially inhibited by sodium orthovanadate, but unaffected by L-NAME.  From the results obtained, it is concluded that genistein can decrease the vascular tone in the femoral, renal, and mesenteric vascular beds with the underlying mechanism that involves tyrosine kinase inhibition, while in femoral arterial beds, it also involves NO release. 

 

Key words: genistein; perfusion pressure; vascular bed; L-NAME; sodium orthovanadate

 

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 Phytoestrogens are plant-derived dipheonolic compounds, which are structurally and functionally similar to estradiol. A growing number of reports have documented that phytoestrogens may confer cardiovascular protection[1£­3]. Genistein (GST), one of the most well-known phytoestrogens, is an isoflavone which is also proved to be a specific inhibitor of protein tyrosine kinase (PTK)[4]. Recently, we found that GST relaxed femoral arterial rings in a concentration-dependent manner under the condition of precontraction induced by phenylephrine, and that this effect was only partially endothelium-dependent. Little is known, however, about the effect of GST on femoral, renal, and mesenteric vascular beds. The purpose of this study was to identify the effects of GST on these vascular beds and their possible mechanisms.

 

1  MATERIALS AND METHODS

1.1 Animals.    Sprague Dawley rats  (male, 300£­350 g, n=72, Grade ¢ò) were obtained from the Experimental Animal Center of Hebei Province.

1.2 Surgical procedure.      Rats were anesthetized with urethane (1.0 g/kg) intraperitoneally.  Supplemental doses of anesthetics were given as necessary. The constant flow perfusion method was adapted to measure the tone of left femoral, the left renal and the superior mesenteric arteries. This perfusion method was described previously by Zhao et al[5]. Briefly, a polyethylene cannula containing heparinized (20 U/ml) saline was introduced into the left common carotid artery, and the arterial blood passed through a peristaltic pump (1210 A, Harvard). The outlet of the pump was connected to a T-shaped tube, one end of which was fed to a pressure transducer (MPU-0.5, Nihon Kohden) and a carrier amplifier (AP-620, Nihon Kohden)  for  monitoring blood pressure, the other end was connected with a long polyethylene tube passing through a water bath (with constant temperature of 37¡æ) and then connected to  another T-shaped tube. One end of the second T-shaped tube was inserted into an artery (the left femoral, or left renal, or superior mesenteric artery); the other end was fed to a pressure transducer and a carrier amplifier for monitoring the perfusion pressure of the vascular bed. Thus the arterial blood coming from the left common carotid artery was introduced into the left femoral, the left renal or the superior mesenteric artery via a peristaltic pump. The flow rate of the peristaltic pump was adjusted so that the perfusion pressure was equal to blood pressure. Body temperature was maintained at 37£­38¡æ throughout the experiment.

1.3  Experimental protocols.    After operation, the rat was allowed to stabilize for more than 30 min.  A stable recording was obtained for more than 5 min, the drug (a fixed volume of 10  ¦Ìl over a period of 10 s at a constant speed) was injected into the perfusion circuit directly.  Before application of drug, a  vehicle (DMSO) was used as control.  The changes in BP, HR, and the perfusion pressure(PP) were recorded on a polygraph (RM-6000, Nihon Kohden).  The experimental protocols were:  (1) different doses of genistein (0.4, 0.8, 1.2  mg/kg) were injected into the femoral (n=6), renal (n=6), and mesenteric (n=6) arteries, respectively.  BP, HR, and perfusion pressure was examined; and   (2) L-NAME (1.2  mg/kg), sodium orthovanadate (50  ¦Ìg/kg), or sodium orthovanadate plus L-NAME was injected into the perfusion circuit for 15 min followed by an injection of genistein (0.8 mg/kg).  BP, HR, and perfusion pressure were recorded in femoral (n=6), renal (n=6), and mesenteric (n=6) vascular beds, respectively.

1.4  Drugs.    Genistein, L-NAME, and sodium orthovanadate were purchased from  Sigma Chemical Co. Genistein were dissolved in DMSO. The drugs were freshly prepared before use.

1.5  Statistics.    Data were expressed as mean¡ÀSE and analyzed by ANOVA.  Dunnett's    t test is used as appropriate.  P<0.05 was considered significant.

 

2 RESULTS

2.1  Effects of genistein on femoral vascular bed

 Intrafemoral arterial injection of genistein at the doses of 0.4, 0.8, 1.2  mg/kg decreased the perfusion pressure of femoral vascular bed in a dose-dependent manner (Fig.1A; Table  1). The effect of genistein (0.8 mg/kg) was partially inhibited by L-NAME (1.2 mg/kg), sodium orthovanadate (50 ¦Ìg/kg), or sodium orthovanadate plus L-NAME (Fig.2).

2.2  Effects of genistein on the renal vascular bed

 Intrarenal arterial injection of genistein at the dose of 0.4, 0.8, 1.2  mg/kg decreased the perfusion pressure of renal vascular bed in a dose-dependent manner (Fig.1B; Table 1). This effect of genistein (0.8  mg/kg) was unaffected by pretreatment with L-NAME and was completely inhibited by sodium orthovanadate (50 ¦Ìg/kg) (Fig.2).

2.3  Effect of genistein on mesenteric vascular bed

 Intramesenteric arterial injection of genistein at the doses of 0.4, 0.8, 1.2  mg/kg decreased the perfusion pressure of mesenteric vascular bed in a dose-dependent manner (Fig.1C, Table 1).  The effect of genistein (0.8  mg/kg) was partially inhibited by sodium orthovanadate, but was unaffected by L-NAME (Fig.2).

 

3 DISCUSSION

 Genistein is a naturally occurring plant-derived estrogen-like compound. The precursors of this biologically active compound originate in soybean products and are converted by intestinal bacteria into hormone-like  compounds  with weak antioxidative and estrogenic activity[6]. In the present study, the effects of GST on the regional vascular beds have been characterized. The results showed that GST decreased the perfusion pressure (PP) of femoral, renal, and mesenteric vascular beds in a dose-dependent manner.

 

Fig.1.Effects of genistein (0.4, 0.8, 1.2  mg/kg) on HR, BP, and the perfusion pressure (PP) in femoral, renal, and mesenteric vascular beds.  HR, heart rate; BP,  blood pressure; PP,  perfusion pressure.  A: Intrafemoral arterial injection of genistein.  B: Intrarenal arterial injection of genistein.   C:  Intramesenteric arterial injection of genistein. ¡ø,  local injection of genistein.

Fig.2.Effects of L-NAME (1.2  mg/kg), sodium orthovanadate (50 ¦Ìg/kg) on the genistein (0.8 mg/kg)   induced changes in  PP of  femoral (n=6), renal (n=6), and mesenteric  (n=6) vascular beds.  ##P<0.01 vs control; *P<0.05; **P<0.01 vs GST group.     ¦¤PP,  ¦¤ value of perfusion pressure.

 

Table¡¡£±.Effects of genistein on HR, MAP, and PP in femoral, renal, and mesenteric vessels (n=6)

	HR (bpm)		MAP (mmHg)		PP (mmHg)
	Pretreatment	Posttreatment	Pretreatment	Posttreatment	Pretreatment	Posttreatment
Femoral
Control	377¡À6	375¡À6	108¡À1.4	106.5¡À0.8	107.3¡À1.4	106¡À1
Genistein
0.4 mg/kg	368¡À8	372¡À8	108.7¡À1.2	108.7¡À1.2	108.7¡À1.2	94.7¡À1.3**¢k¢k
0.8 mg/kg	376¡À7	375¡À7	108.7¡À1.2	106.2¡À1.3	108.7¡À1.2	89.7¡À1.3**¢k¢k
1.2 mg/kg	380¡À7	374¡À6	108.7¡À1.2	99.5¡À1.7*¢k	108.7¡À1.2	83.7¡À2.2**¢k¢k
Renal
Control	373¡À13	370¡À11	111.2¡À3.8	110.3¡À2.8	107¡À3.7	106.7¡À3.5
Genistein
0.4 mg/kg	361¡À13	362¡À12	111.7¡À4.8	109.2¡À5.8	111.7¡À4.8	101¡À4.7**¢k
0.8 mg/kg	369¡À12	368¡À11	111.7¡À4.8	105.5¡À5.7	111.7¡À4.8	94.5¡À4.8**¢k¢k
1.2 mg/kg	372¡À12	370¡À10	111.7¡À4.8	92.8¡À4.5**¢k	111.7¡À4.8	87.7¡À3.9**¢k¢k
Mesenteric
Control	388¡À11	386¡À8	99.7¡À3.7	98.7¡À3.3	99.2¡À2.9	98.2¡À2.5
Genistein
0.4 mg/kg	376¡À3	374¡À3	98.7¡À4.2	98.2¡À4.2	98.7¡À4.2	92.3¡À4.9**¢k
0.8 mg/kg	379¡À4	377¡À3	99.2¡À4.4	96.8¡À4.7	99.2¡À4.4	89.8¡À5.0**¢k¢k
1.2 mg/kg	377¡À3	372¡À2	101¡À5.1	92.2¡À5.9*	101¡À5.1	87.2¡À5.4**¢k¢k

 

P<0.05,  **P<0.01 vs pretreatment;   ¢kP<0.05, ¢k¢kP<0.01 vs control.

 

Evidence has been shown that NO release induced by estrogen is in part responsible for its non-genomic actions on the cardiovascular system[7]. Our previous study demonstrated that GST relaxed femoral arterial rings and that the relaxation was partially endothelium-dependent. Tyrosine kinase is possibly involved in the mechanism of  the relaxation by GST in femoral arterial rings[8]. In the present experiments, L-NAME, an inhibitor of NO synthase, also significantly inhibited the effect of GST on femoral arterial bed, suggesting that NO release was involved. GST is also proved to be a specific inhibitor of PTK. It is reported that higher doses of GST have multiple cellular effects such as inhibition of protein kinase[4]. Evidence has been presented to suggest that enhanced tyrosine phosphorylation participates in the mechanisms that regulate the contraction of smooth muscle[9]. Vanadate, an inhibitor of tyrosine phosphatase, can enhance protein tyrosine phosphorylation[10]. Our present study showed that the effect of GST on the femoral arterial bed was inhibited by pretreatment with sodium orthovanadate or sodium orthovanadate plus L-NAME, suggesting that tyrosine kinase pathway was involved. However, either L-NAME or sodium orthovanadate only partially inhibited the effect of GST, implying the possibility of the involvement of other agents such as calcium antagonistic mechanism[8].

 In the mesenteric vascular bed, L-NAME failed to inhibit the effect of GST, suggesting that NO release might not be involved. This result is in accordance with that of  other authors[11]. Our present study also showed that the action of GST on mesenteric vascular bed was partially inhibited by sodium orthovanadate.

 The effect of GST (0.8 mg/kg) on renal vascular bed was unaffected by pretreatment with L-NAME, but was completely inhibited by sodium orthovanadate. These results suggest that the effect of GST on the renal vascular bed is exclusively due to PTK inhibition.

 In summary, the present study provides evidence that GST decreases the perfusion pressure of femoral, renal, and mesenteric vascular beds, involving a tyrosine kinase pathway, while for the femoral vascular bed the NO release also participates in.

 

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