Effect of nitric oxide synthase inhibitor N^G-nitro-L-arginine on the content of amino acid in ischemic brain tissues of rats

BACKGROUND： Nitric oxide synthase （NOS） inhibrtors have been widely used to investigate the role of NO on cerebral ischemic injury, but the results are controversial. Moreover, it has been considered to aggravate the ischemic neuronal damage with the release of excessively excitatory amino acids （EAA） during cerebral ischemia. On the other hand, some inhibitory amino acid is suggested to be important for the neuronal protection against ischemic brain damage. Our study has recently showed that treatment with the NOS inhibitor NG-nitro-L-arginine （L-NA） reduced focal cerebral ischemic damage. The effect of L-NA on the contents of excitatory and inhibitory amino acid in the rat brain following cerebral ischemia is still unclear. OBJECTIVE： By evaluating the effect of NOS inhibitor, L-NA on the contents of aspartate, glutamate, glycine and γ-aminobutyric acid （GABA） in striatum, hippocampus and cortex in the rat brain following cerebral ischemia respectively, to investigate the beneficial effect of L-NA on cerebral ischemic injury and the possible mechanism. DESIGN： A randomized and controlled experiment SETTING ： Department of Pharmacology, Hebei Academy of Medical Sciences MATERIALS： A total of 42 male healthy SD rats （grade Ⅱ, weighting 250-300 g） were provided by the Experimental Animal Center of Hebei Province （Certification： 04036）. Aspartate, glutamate, glycine, GABA, L-NA and 2,3,5-triphenyltetrazolium chloride （TTC） were obtained from Sigma Chemicals Co, St Louis, MO, USA. HPLC-ultraviolet detector system consisted of Agilent 1100 HPLC. METHODS： The experiment was carried out in Department of Pharmrcology, Hebei Academy of Medical Sciences from June 2005 to June 2006. Rats were randomly divided into three groups： sham-operated group （n = 6）, ischemic group （n = 18）, L-NA group （n = 18）. The model of focal cerebral ischemia in rat was prepared with intraluminal line occlusion methods. In sham-operated rats, the external carotid artery was surgically prepared, but the filament was not inserted. Each group was further divided into 3 subgroups （n = 6 for each）： drugs were administrated at 2, 6 and 12 hours after the middle cerebral artery occlusion （MCAO） respectively. L-NA （20 mg/kg, ip） was administrated, twice a day, for 3 consecutive days. Same volume of normal saline was administrated in ischemic and sham operation groups. The changes of infarcted volume and the contents of amino acids were respectively assayed. Image analysis software was used for the measurement of the infarcted area. The results were expressed as a percentage of the infarcted volume of cerebral/volume of whole brain （IV%） in order to control for edema formation. The contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in the rat brain following cerebral ischemia were respectively measured by HPLC method. All data were analyzed with one-way ANOVA and Dunnett＇s test. MAIN OUTCOME MEASURES： （1） The volume of cerebral infarction; （2） The contents of aspartate, glutamate glycine and GABA in brain tissue after cerebral ischemia. RESULTS ： All 42 rats were involved in the final analysis. （1） Infarcted volume： Volume was 0 in sham-operated group. When L-NA was administrated at 2 and 6 hours after MCAO, the infarcted volume was （20.13±3.59）% and （23.12±5.84）% in L-NA group, which was not similar to that in ischemic group [（22.10±3.98）%, （25.38± 5.37）%, P〉 0.05]. However, the infarcted volume was markedly decreased compared with that of ischemic group when L-NA was administrated at 12 hours after MCAO [（26.11±3.55）% and （37.15±3.58）%, P 〈 0.01]. Changes of amino acid content： At 2 and 6 hours after ischemia, the contents of aspartate, glutamate, glycine and GABA in striatum, hippocampus and cortex in ischemic group were significantly increased compared with those in sham-operated group （ P〈 0.05-0.01）. However, contents in L-NA group were similar to those in ischemic group （P 〉 0.05）. At 12 hours after ischemia, the contents of aspartate [（0.21 ±0.06）, （0.36±0.05）, （0.29±0.12） mg/g] and glutamate [（0.55±0.06）, （0.78±0.10）, （0.52±0.10） mg/g] in striatum, hippocampus and cortex in L-NA group were significantly decreased compared with those in ischemic group [（0.49±0.17）, （0.63± 0.03）, （0.51±0.15） mg/g; （0.98±0.30）, （1.15±0.15）, （0.93±0.15） mg/g, P〈 0.05-0.01]. Glycine in hippocampus was （0.40±0.07） mg/g, which was higher than that in ischemic group [（0.21±0.07） mg/g, P 〈 0.05]. GABA in striatum, hippocampus and cortex was （0.93±0.10）, （0.62±0.12） and （0.81 ±0.10） mg/g, respectively, which was higher than that in ischemic group [（0.60±0.08）, （0.37±0.17）, （0.59±0.10） mg/g, P 〈 0.05-0.01]. CONCLUSION ： It may be concluded that L-NA have beneficial effect on ischemic cerebral injury in ischemic later stage in rats. The possible mechanism is that L-NA can decrease the contents of aspartate and glutamate, increase the contents of glycine and GABA.

Studies on the Compounds of d4T Combined with Nitric Oxide Donors and Nitric Oxide Synthase Inhibitors and their Anti-HIV and AIDS Activity

Stavudine, a potent anti HIV and AIDS related complex, is one of the Nucleoside Analogue Reverse Transcriptase Inhibitors (NARTIs). It is phosphorylated intracellularly and then inhibits the viral reverse transcriptase by acting as a false substrate. Modifications made on the hydrogen labile at the 5' position on the sugar is an interesting template for the elaboration of new potent anti HIV and AIDS drugs. The expected advantages of the modified stavudine prodrugs can be multiple: synergistic drug activities, enhancement of stavudine intracellular uptake, increase of stavudine brain delivery, and bypass of the first stavudine phosphorylation step into the cells. Nitric oxide synthase inhibitors of stavudine and nitric oxide donors of stavudine may hold significant promise for the treatment of HIV and AIDS.

Effect of endogenous nitric oxide synthase inhibitors on cardiovascular function:Insight obtained from tissue specific dimethylarginine dimethylaminohydrolase 1 deficient mice

Nitric oxide (NO) produced by nitric oxide synthases (NOS) exerts many important biological functions. Asymmetric dimethylarginine (ADMA) and

Synthesis of novel methotrexate derivatives with inhibition activity of nitric oxide synthase

Seventeen 4-alkylamino/arylamino-substituted methotrexate （MTX） derivatives 6a-14a were designed and synthesized. Their inhibition activities against inducible nitric oxide synthase （iNOS） were evaluated in vitro. The pharmacological results showed that most of the prepared compounds displayed the potent inhibitory effects on iNOS.

Lovastatin increases nitric oxide synthesis in IL-1β-stimulated smooth muscle cells

Objective Nitric oxide(NO)production by inducible NO synthase(Inos)may play an important role in the pathogenesis of atherosclerosis.Although lovastatin has been shown to reduce the progression of atherosclerosis,it is not known whether it regulates NO production.We investigated the effects of lovastatin on NO synthesis and the mechanisms by which lovastatin exerts its effects in rat vascular smooth muscle cells.Methods Primary cultures of the vascular smooth muscle cells were obtained from the media of the thoracic aorta of Sprague Dawley rats(200 - 250 g).Nitrite levels in the culture medium of rat vascular smooth muscle cells were determined colorimetrically.Results Lovastatin(10-5 mol/L)significantly increased interieukin-1β(IL-1β,10 ng/Ml)-induced nitrite accumulation in a time(0- 24 hours)-dependent manner.Exogenous mevalonate and geranylgeranylpyrophosphate completely reversed the stimulatory effects of lovastatin on nitrite production.Furthermore,inhibition of Rho by C3 exoenzyme mimicked the increase in IL-1β-induced nitrite accumulation induced by lovastatin in the vascular smooth muscle cells.Conclusion These results demonstrate that lovastatin up-regulates NO formation in rat vascular smooth muscle cells stimulated by IL-1β,and the effect may be associated with the inhibition of Rho activity.

Effects of aminoguanidine on retinal apoptosis in mice with oxygen-induced retinopathy

AIM:To explore the protective effects of amino-guanidine(AG) on retinal apoptosis in mice with oxygeninduced retinopathy(OIR).·METHODS:A total of 80 C57BL/6J mice,aged 7 days,were randomly divided into four groups:normal,high oxygen,high oxygen saline and high oxygen treated with AG.In the normal group,mice were housed in normoxic conditions from postnatal day P7 to P17.Mice in the other 3 groups were placed under hyperoxic conditions(75 ±2% O2) in an oxygen-regulated chamber for 5 days and subsequently placed in normoxic conditions for 5days.Mice in the AG group were treated once daily,from P12 to P17,with AG hemisulfate(100mg/kg body weight,intraperitoneally) dissolved in physiological saline.An equivalent amount of 0.9% physiological saline was administered,as above,to mice in the high oxygen saline group.Ten mice were randomly selected from each group on P14 and on P17,euthanized and the retinas examined.Apoptotic cells in the retina were detected using the terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) method.The expression of nitric oxide synthase(iNOS) in the retina was detected by immunohistochemistry and changes in rod cells were observed using electron microscopy.·RESULTS:TUNEL-positive cells and iNOS immunoreactive neurons were present in the inner nuclear and ganglion cell retinal layers of mice in the high oxygen group.The number of TUNEL-positive cells was significantly greater in the high oxygen group compared with the normal group(t =-20.81,P14d【0.05;t =-15.05,P17d【0.05).However,the number of TUNEL-positive cells in the AG treatment group was significantly lower(t =-13.21,P14d【0.05;t =-6.61,P17d【0.05) compared with thehigh oxygen group.The expression of iNOS was significantly higher in the high oxygen group compared with the normal group(t =-21.95,P14d【0.05;t =-17.30,P17d【0.05).However,the expression of iNOS in the AG treatment group was significantly lower(t =-12.17,P14d【0.05;t =-10.30,P17d【0.05) compared with the high oxygen group.The outer segments of the rods were disorganized and short in the high oxygen group.Rod morphology appeared to be slightly improved in the AG group.·CONCLUSION:AG may protect retinal neurons in OIR by inhibiting apoptosis.The mechanism may be related to iNOS.