诱导型一氧化氮合酶与Dahl-盐敏感大鼠动脉血压的调节

1、    诱导型一氧化氮合酶与Dahl-盐敏感大鼠动脉血压的调节        摘要目的和方法：为探讨诱导型一氧化氮合酶(iNOS)在血液动力学调控中的作用，本研究通过慢性血液动力学实验观察了静脉输入诱导型一氧化氮合酶（iNOS）抑制剂aminogunidine(AG)对大鼠平均动脉压的影响，并测定了一氧化氮（NO）终产物NO2及NO3含量(UNOx)以及iNOS活性。结果：1. iNOS特异抑制剂AG能明显放大高钠引起的Dahl盐敏感大鼠（DS）的血压上升效应；2. 高NaC

2、l输入在导致的DS大鼠血压升高的同时，能引起iNOS活性及UNOx的明显升高。结论：iNOS对DS大鼠具有重要的血液动力学调节作用。主题词一氧化氮； 血压；大鼠 Article ID1000-4718(2000)03-0207-04 The role of inducible nitric oxide synthase in the regulation of arterial pressure in Dahl salt-sensitive ratsTAN Dun-yong,CHEN Xiao-lin,Dong Jun(Department of Pathophysiology, Jinan

3、University Medical College, Guangzhou 510632,China;)C.Caramelo(Universidad Autonoma de Madrid Facultad de Medicina, 28040 Madrid, Spain)AbstractAIM AND METHODS: To clarify the role of inducible nitric oxide synthase (iNOS) in the hemodynamic regulation of Dahl salt-sensitive (DS) rats，in the present

4、 study, we examined the effect of aminoguanidine (AG), a selective inhibitor of iNOS on the pressor response of DS rats to high sodium chloride (NaCl) intake by chronical in vivo hemodynamic experiment, and the effect of NaCl or NaCl plus AG infusion on urinary nitrate (NO-3)/nitrite (NO-2), the end

5、 product of nitric oxide (NO), excretion by Greiss Reaction. Furthermore, an iNOS activity assay was also made. RESULTS: The hypertensive effect of high NaCl (8%) infusion was greatly amplified by co-infusion of AG. Administration of high NaCl significantly elevated the iNOS activity of renal tissue

6、 including cortex, inner medulla and outer medulla, and greatly increased urinary NO-3/NO-2 excretion. CONCLUSION: Inducible NOS is an important modulator of blood pressure in DS rats under the condition of NaCl-induced hypertension.MeSHNitric oxide; Blood pressure; Rats CLC numberR544.1Document cod

7、eAINTRODUCTIONNitric oxide (NO), enzymatically synthesized from L-arginine, is an important physiological regulator of vascular tone1，2. At least three nitric oxide synthases (NOS) including endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) have been clarified so far. Although num

8、erial studies have evidenced that NO derived from eNOS played a key role in the regulation of arterial pressure since NO, as an endothelium derived relaxing factor (EDRF), was firstly reported by Furchgott et al in 19801，3，4, little has been known about the hemodynamic effect of inducible NOS/NO pat

9、hway on blood pressure regulation. A lot of reports suggested that the inducible isoenzyme has a very different nature from constitutive NOS including eNOS and nNOS. When exposed to cytokines or bacterial products, iNOS could be greatly activated and produces hundreds or thousands fold NO than it do

10、es usually57. Our previous work showed that the urinary nitrite/nitrate (NO-2/NO-3) excretion (UNOx) and the iNOS expression significantly increased in Sprague-Dawley (SD) rats when feeded with high sodium diet and this results led us suggest that iNOS could play a certain role in the regulation of

11、blood pressure in NaCl-induced hypertension.In this study, we examined the effect of high sodium chloride (NaCl) infusion on NO production and iNOS activity in renal tissue and the effect of co-infusion of aminoguanidine (AG), a selective inhibitor of iNOS, on hypertensive effect of high NaCl admini

12、stration in Dahl salt-sensitive rats to clarify the role of iNOS in the regulation of blood pressure.MATERIALS AND METHODSIn vivo chronical hemodynamic experiment Conscious 78 weeks old, male, Dahl salt-sensitive (DS) rats were received when they were 56 weeks old, and surgery was done when the rats

13、 reached a weight of200 g, and experiments were begun 1 week later when the rats had a weight of220 g. Aortic and vena cava catheters were implanted as we have done before3, and 15 mL/d of either hypotonic or hypertonic saline was infused iv containing the following antibiotics: Mezlin, 30 mg/d, (Mi

14、les, Westhaven CT) and penicillin G, 5000 U/d. Rats were placed in a temperature controlled room with a 12h light/dark cycle. Both catheters were exteriorized at the dorsal nape of the neck, and were connected to a dual channel infusion swivel (Instech Laboratories Inc., Plymouth Meeting, PA). Salin

15、e solutions were infused with a syringe pump (AJ5803) through a 0.22 m filter (Cathivex, Millipore Corporation, Bedford, MA). The arterial catheter was filled with 1 000 U/mL heparin and connected to a Cobe pressure transducer (Lakewood CO) and in turn to a pressure amplifier. Pulsatile arterial pre

16、ssure signals from the amplifier were sent to a digital computer through an analog-to-digital converter and were sampled 500 Hz for 4 seconds of each minute throughout the entire 24h period. Arterial pressure were determined from these data samples.Measurement of nitrate/nitrite (UNOx) excretion Uri

17、ne samples were collected and were frozen at -70 until assayed. Urinary nitrate (NO-3) plus nitrite (NO-2) excretion (UNOx), as an index of whole body NO production, was determined using the Greiss reaction7 and nitrate reductase from Escheria coli as we have done before3. Escherichia coli nitrate r

18、eductase was prepared from bacteria grown under anaerobic conditions. Urine samples were incubated with E.coli nitrate reductase at 37 for 90 min. The incubation solution was then centrifugated for 5 min. Known concentrations of NaNO2 were used as standards in each assay. NaNO3 was used to verify th

19、e conversion. Each sample was run in duplicate. Supernatant was collected and reacted with Greiss reagent at room temperature for 10 min. NO2 in the sample was then quantitated using a spectrometer as previously described. The NO-2 measured in this way reflects the total amounts of NO-2 and NO-3 in

20、the original sample. Urinary excretion of NO-2 plus NO-3 (UNOx) was calculated by the 24h urinary volume.Inducible NOS activity assay After the in vivo chronical hemodynamic experiment, the rats were euthanized with an overdose of sodium pentobarbital, and the renal medulla was rapidly removed and f

21、rozen on dry ice. The whole tissue was homogenized using a Potter-Elvenhjem tissue grinder at 3 000 r/min in a solution containing 250 mmol/L sucrose, 1 mmol/L EDTA, 0.1 mmol/L PMSF, and 5 mmol/L potasium phosphate, pH 7.7. The total tissue homogenate was incubated with 1 mmol/L NADPH, 25mol/L FAD,

22、0.5mmol/L EDTA , 10mol/L tetrahydrobiopterin, and 3H-arginine (approximately 300 000 counts/min，2.52×1012Bq/mmol) in 20 mmol/L HEPES buffer, pH 7.2, at 37 for 5 min. The arginine and converted citrulline were seperated by column of Dowex AG 50-X8 (Na form). The amount of converted citrulline an

23、d the total counts were quantified by radiochemical detection (Packard). Experimental protocols The following 4 groups (n=8) of rats were studied: Low NaCl alone, low NaCl+AG , high NaCl and high NaCl+AG. During a 7d surgical recovery period, all rats were infused with 0.3% NaCl solution (15 mL/d) a

24、nd feeded on low NaCl food. In 10d experimental period, the rats were infused with NaCl solution alone (0.3% for low NaCl and 8% for high NaCl alone groups) or NaCl solution (0.3% for low NaCl+AG and 8% for high NaCl+AG) plus AG (12.3 mg.kg-1.h-1). The urine was colleted on the last day for measurem

25、ent of nitrate/nitrite. The renal cortex and medulla were taken for iNOS activity assay after hemodynamic experiment.RESULTSThe effect of chronical infusion of NaCl and AG on MAP As shown in Figure 1, The mean arterial pressure (MAP) did not responde to low NaCl infusion, also there was not signific

26、ant difference in MAP between NaCl alone and NaCl+AG infused rats. Albeit, intravenous infusion of high (8%) NaCl solution significantly increased the blood pressure. After 10d infusion, the MAP were 12.43±0.41 and 18.83±0.13 kPa respectively in low and high NaCl-infused rats (P0.05). Also

27、, the pressor effect of high NaCl infusion was significantly amplified by co-administration of AG (12.3mg.kg-1.h-1). The MAP of high NaCl+AG infused rats was increased to 21.49±0.23 kPa (P0.05,vs that of the rats infused with high NaCl alone).Effect of NaCl and AG on UNOx excretionAs showed in

28、figure 2, a significant increase in urinary nitrite/nitrate (UNOx) excretion was observed when infused with high NaCl (8%) solution. The UNOx excretion were 13 298±432 nmol/24 h and 3 543±231 nmol/24h in high NaCl and low NaCl infused rats respectively (P0.01). Furthermore, the stimulating

29、 effect of high NaCl administration on nitrite/nitrate excretion was almost completely abolished by co-infusion of AG (12.3mg.kg-1.h-1). The UNOx of the rats infused with 8% NaCl+AG were decreased to 4 098±342 nmol/24h (P0.01, compared with that of the rats treated with 8% NaCl alone). 

30、60;        Effect of NaCl and AG on iNOS activity High NaCl infusion induced a significant increase in iNOS activity in renal tissue. The radioactivity of cortex, outer medulla and inner medulla of low NaCl infused rats are 6 876±1 011,5 689±342 and 4 233±

31、;554 counts/min respectively, while they are 16 985±2 212, 49 876±3 012 and 75 698±1 380 counts/min respectively in high NaCl infused rats. Also, the high NaCl-induced iNOS activity were significantly decreased by co-infusion of AG (Tab 1). Low NaClLow NaCl+AGHigh NaClHigh NaCl+AGCort

32、ex6 876±1 0115 987±1 78616 985±2 212*9 054±2 098#Outer medulla5 689±3425 177±29849 876±3 012* 8 998±2 643#Inner Medulla4 233±5544 110±19875 698±1 380*10 985±2 345#     *P0.05,*P0.01, vs low NaCl alone; #P0.05, #P0.01, vs

33、 high NaCl alone DISCUSSIONTo elucidate the possible role of iNOS in the regulation of arterial pressure, in the present study, we examined the effect of intravenously administration of aminoguanidine (AG), an iNOS selective inhibitor8，9, on the mean arterial pressure of Dahl salt-sensitive rats. Fr

34、om figure 1 , we can see that in the Dahl salt-sensitive rats, intravenous infusion of high (8%) NaCl solution for 10 d significantly increased the blood pressure. The MAP were 12.43±0.40 and 18.83±0.13 kPa respectively in low and high NaCl-infused rats. The co-administration of AG (12.3 m

35、g.kg-1.h-1 ) significantly amplified the hypertensive effect of high sodium infusion to the Dahl salt-sensitive rats. The MAP of high NaCl + AG infused rats was increased to 21.49±0.22 kPa (P0.05, compared with that of the rats infused with high sodium alone). This results suggested that NO pro

36、duced by iNOS could play an important depressor role in case of hypertension, and iNOS could be activated through an unknown feed-back mechanism, perhaps a pressure-dependent mechanism, when blood pressure was elevated by certain hypertensive factors such as high sodium intake,etc. And this is stron

37、gly supported by iNOS activity assay which showed that in low NaCl infusion, iNOS activity was very low (table 1), while iNOS activity was increased significantly when infused with high NaCl solution.Different from constitutive NOS including both nNOS and eNOS, inducible NOS, under certain circumsta

38、nces such as bacteria infection, being exposed to cytokine, could produce hundreds even thousands fold NO than constitutive NOS, including eNOS does57. Also, numerous studies suggested that iNOS exists not only in rodent macrophages, where iNOS was firstly clarified, but also in the blood vessel wal

39、l, renal cortex and medulla, etc8，9. This provided the structural and functional basis for iNOS to play an important role in the regulation of blood pressure and vasular tone in case of hypertension. The above results and analysis also lead us suggest that iNOS and eNOS could play different role in

40、the hemodynamic regulation. NO produced by eNOS could be an important determinant of basal vasular tone and could counteract the actions of vasoconstractors such as angiotensin II, endothelins, etc., however, NO produced by iNOS could play a much more important regulatory role in case of high blood

41、pressure, for instance, the high NaCl-induced hypertension in this study. In summary, the results of the present study indicated that iNOS participates in the regulation of arterial pressure of Dahl salt sensitive rats when infused with high NaCl solution. Systemic AG infusion led to the development

42、 of high NaCl-induced hypertension. Further studies need to be performed to elucidate the detail mechanisms under which iNOS is activated and plays regulatory role in the control of arterial pressure.Foundation item National Natural Science Foundation of China (No. 39870359); The project sponsered b

43、y SRF for ROCS,SEM谭敦勇（暨南大学医学院病理生理教研室，广东 广州 510632;Universidad Autonoma de Madrid Facutad de Medicina, 28040 Madrid, Spain）陈小琳（暨南大学医学院病理生理教研室，广东 广州 510632）董军（暨南大学医学院病理生理教研室，广东 广州 510632）C.Caramelo（Universidad Autonoma de Madrid Facutad de Medicina, 28040 Madrid, Spain）REFERENCES1Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscl