［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NaCl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。

［Article ID］1000-4718(2000)03-0207-04

The role of inducible nitric oxide synthase in the regulation of arterial pressure in Dahl salt-sensitive rats

TAN Dun-yong,CHEN Xiao-lin,Dong Jun

(Department of Pathophysiology, Jinan University Medical College, Guangzhou 510632,China;)

C.Caramelo

(Universidad Autonoma de Madrid Facultad de Medicina, 28040 Madrid, Spain)

［Abstract］AIM 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 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 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 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.

［MeSH］Nitric oxide; Blood pressure; Rats

［CLC number］R544.1［Document code］A

INTRODUCTION

Nitric oxide (NO), enzymatically synthesized from L-arginine, is an important physiological regulator of vascular tone^［1，2］. At least three nitric oxide synthases (NOS) including endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) have been clarified so far. Although numerial 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 1980^{［1，3，4］}, little has been known about the hemodynamic effect of inducible NOS/NO pathway 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 does usually^［5～7］. 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 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 administration in Dahl salt-sensitive rats to clarify the role of iNOS in the regulation of blood pressure.

MATERIALS AND METHODS

In vivo chronical hemodynamic experiment Conscious 7～8 weeks old, male, Dahl salt-sensitive (DS) rats were received when they were 5～6 weeks old, and surgery was done when the rats reached a weight of～200 g, and experiments were begun 1 week later when the rats had a weight of～220 g. Aortic and vena cava catheters were implanted as we have done before^［3］, and 15 mL/d of either hypotonic or hypertonic saline was infused iv containing the following antibiotics: Mezlin, 30 mg/d, (Miles, 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). Saline solutions were infused with a syringe pump (AJ5803) through a 0.22 μm 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 pressure 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 Urine 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 reaction^［7］ and nitrate reductase from Escheria coli as we have done before^［3］. Escherichia coli nitrate reductase 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 the 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 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 frozen 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, 25μmol/L FAD, 0.5mmol/L EDTA , 10μmol/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 and the total counts were quantified by radiochemical detection (Packard).

Experimental protocols The following 4 groups (n=8)<