Administration of Folium mori extract decreases nitric oxide synthase expression in the hypothalamus of streptozotocin-induced diabetic rats

Folium mori, the leaves of Morus alba L., has traditionally been used for the treatment of diabetic hyperglycemia. It has been shown to induce enhanced NOS expression in the hypothalamus of rats with streptozotocin (STZ)-induced diabetes. In the present study, the effect of Folium mori on the expression of nitric oxide synthase (NOS) in the hypothalamus of STZ-induced diabetic rats was investigated via nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Enhanced NAPDH-d expression was detected in the paraventricular nucleus, ventromedial hypothalamic nucleus, and lateral hypothalamic area of the hypothalamus in the STZ-induced diabetes group. Administration of the aqueous extract of Folium mori to rats with STZ-induced diabetes resulted in decreased NADPH-d positivity. These results suggest that Folium mori treatment is effective in curbing the desire for food under diabetic conditions via modulation of NO expression in the hypothalamus.     

Altered intestinal P-glycoprotein expression levels affect pharmacodynamics under diabetic condition

P-glycoprotein (P-gp), one of the important drug-efflux pumps, is known to affect pharmacokinetics and pharmacodynamics of P-gp substrate drugs. We have previously reported that intestinal P-gp expression levels are transiently decreased in streptozotocin (STZ)-induced type 1 diabetic mouse model. Herein, we examined the analgesic effects of orally administered morphine and its pharmacokinetic properties under diabetic conditions, specifically focusing on the involvement of intestinal P-gp in a type 1 diabetic mouse model. Type 1 diabetes was induced in male ddY mice by an i.p. injection of STZ (230 mg/kg). We assessed the oral morphine analgesia using the tail-flick test. Serum and brain morphine content were determined on a HPLC-ECD system. Intestinal P-gp expression levels were significantly decreased on day 9 after STZ administration. On the other hands, oral morphine analgesia, and serum and brain morphine content were significantly increased on day 9 after STZ administration. The decrease in the intestinal P-gp expression levels were suppressed by aminoguanidine, a specific iNOS inhibitor. Interestingly, the increase in the analgesic effect of morphine, as well as serum and brain morphine content, was suppressed by aminoguanidine. Conversely, there was no change in the analgesic effect obtained with subcutaneous morphine in STZ-treated mice. In conclusions, our findings suggest that the oral morphine analgesia is dependent on intestinal P-gp expression, and that may be one of the problems against obtaining stable pharmacological effects of morphine in diabetic patients.     

Inhibition of nitric oxide synthase uncoupling by sepiapterin improves left ventricular function in streptozotocin-induced diabetic mice

1. Uncoupling of nitric oxide synthase (NOS) has been implicated in the pathogenesis of left ventricular (LV) dysfunction in diabetes mellitus. In the present study, we investigated the role of NOS uncoupling in oxidative/nitrosative stress and LV dysfunction in the diabetic mouse heart. 2. Diabetes was induced in wild-type (WT), endothelial (e) NOS knockout (eNOS(-/-)), inducible (i) NOS knockout (iNOS(-/-)) and neuronal (n) NOS knockout (nNOS(-/-)) mice by streptozotocin (STZ) treatment. 3. In the diabetic heart, iNOS, but not eNOS or nNOS, expression was increased. Levels of malondialdehyde (MDA), 4-hydroxy-noneal (HNE) and nitrotyrosine (NT), as markers of oxidative/nitrosative stress, were increased in the diabetic mouse heart, but the increase in oxidative/nitrosative stress was significantly repressed in the iNOS(-/-) diabetic mouse heart. Levels of nitrite and nitrate (NO(x)), as an index of nitric oxide, bioavailability were significantly decreased in the iNOS(-/-) diabetic mouse heart. 4. Oral administration of sepiapterin (10 mg/kg per day), a precursor of tetrahydrobiopterin (BH(4)), significantly increased BH(4) and the BH(4)/BH(2) ratio in diabetic mouse heart. Similarly, sepiapterin inhibited the formation of HNE, MDA and NT in diabetic hearts from all three genotypes, but the increase in NO(x) following sepiapterin treatment was significantly attenuated in the iNOS(-/-) diabetic mouse heart. Percentage fractional shortening (FS), evaluated by echocardiography, decreased significantly in all genotypes of diabetic mice. Sepiapterin significantly increased percentage FS in diabetic mice, except in iNOS(-/-) mice. 5. These results suggest that sepiapterin inhibits uncoupling of NOS and improves LV function presumably by increasing iNOS-derived nitric oxide in the diabetic heart.     

Effect of cyclooxygenase and nitric oxide synthase inhibitors on streptozotocin-induced hyperalgesia in rats

We examined a possible involvement of cyclooxygenase (COX) and nitric oxide synthase (NOS) products in hyperalgesia occurring during streptozotocin (STZ)-induced diabetes. Indomethacin and celecoxib were used as relatively selective inhibitors of COX-1 and COX-2, respectively. NOS inhibitors included: non-specific inhibitor N(G)-nitro-L-arginine and L-N(6)-(1-iminoethyl)lysine preferentially acting on inducible NOS (iNOS) as well as 7-nitroindazole relatively specific inhibitor neuronal NOS (nNOS). The above-mentioned agents, except 7-nitroindazole, suppressed hyperalgesia occurring after administration of STZ. The results of the study suggest participation of COX-1, COX-2 and iNOS, but not nNOS, in transmission of pain stimuli in STZ-induced diabetic hyperalgesia.     

Tissue-specific alterations in expression and function of P-glycoprotein in streptozotocin-induced diabetic rats

Aim:

To investigate the changes of expression and function of P-glycoprotein (P-GP) in cerebral cortex, hippocampus, liver, intestinal mucosa and kidney of streptozocin-induced diabetic rats.

Methods:

Diabetic rats were prepared via a single dose of streptozocin (65 mg/kg, ip). Abcb1/P-GP mRNA and protein expression levels in tissues were evaluated using quantitative real time polymerase chain reaction (QRT-PCR) analysis and Western blot, respectively. P-GP function was investigated via measuring tissue-to-plasma concentration ratios and body fluid excretion percentages of rhodamine 123.

Results:

In 5- and 8-week diabetic rats, Abcb1a mRNA levels were significantly decreased in cerebral cortices and intestinal mucosa, but dramatically increased in hippocampus and kidney. In liver, the level was increased in 5-week diabetic rats, and decreased in 8-week diabetic rats. Abcb1b mRNA levels were increased in cerebral cortex, hippocampus and kidney, but reduced in liver and intestinal mucosa in the diabetic rats. Western blot results were in accordance with the alterations of Abcb1a mRNA levels in most tissues examined. P-GP activity was markedly decreased in most tissues of diabetic rats, except kidney tissues.

Conclusion:

Alterations in the expression and function of Abcb1/P-GP under diabetic conditions are tissue specific, Abcb1 specific and diabetic duration-dependent.     

Inducible nitric oxide synthase activity and expression in liver and hepatocytes of diabetic rats

Inducible nitric oxide synthase (iNOS) is expressed by the liver in a number of physiological and pathophysiological conditions. The aim of this study was to investigate the relationship between the diabetic state, iNOS and oxidative stress in the rat liver and isolated hepatocytes. Hepatic iNOS expression and activity was measured in both healthy and streptozotocin-induced diabetic rats and determined in hepatocytes in the presence and absence of insulin. Cu/Zn superoxide dismutase (SOD) and phosphatidylinositol-3-kinase (PI3K) were also measured. In a separate experiment lasting 3 weeks, diabetic rats received either no treatment, two daily injections of insulin or aminoguanidine in the drinking water. Diabetes led to increased activity (45%) and expression (70%) of liver iNOS, an effect that was attenuated by insulin treatment both in vitro and in whole animals. Hepatocyte iNOS expression increased by 56%. Hepatic SOD expression was elevated in the diabetic state, but activity levels were similar to healthy controls. Insulin treatment in vivo led to increased enzyme activity but expression was not modified. Levels of PI3K protein were significantly lower in diabetic rats while insulin treatment markedly increased expression. Aminoguanidine did not inhibit hepatic iNOS in this study. Glycemic control via insulin administration was able to downregulate enhanced hepatic iNOS activity and expression in the liver observed in the diabetic state and improve SOD activity, responses that can potentially reduce the free radical damage associated with diabetes.     

Effect of isosorbide dinitrate on nitric oxide synthase under hypoxia

Nitric oxide synthase (NOS) catalyzes nitric oxide (NO) formation from L-arginine in the presence of molecular oxygen and NADPH. NO is involved in the regulation of microvasculature. Isosorbide dinitrate (ISDN) and glyceryl trinitrate (GTN) have been widely used as vasodilators to treat acute myocardial ischemia, their biological effects being due to the release of NO. In this investigation, the effects of ISDN and GTN on NOS activity in the presence or absence of oxyhemoglobin under hypoxia and normoxia were studied. The apparent K(m) values for molecular oxygen were 21.6 +/- 1.5 and 9.4 +/- 1.3 micromol/l for nNOS and eNOS, respectively. ISDN liberated NO in a concentration- and pH-dependent manner, but no differences between hypoxia and normoxia were observed. The NO release from ISDN was also measured directly by an electron spin resonance spectral method with N-(dithiocarboxy)sarcosine-Fe complex as a NO-trapping agent. ISDN increased nNOS and eNOS activities in the presence of 30 micromol/l oxyhemoglobin under hypoxia, while it did not affect nNOS and eNOS activities under normoxia. In the absence of oxyhemoglobin, ISDN inhibited nNOS and eNOS activities under both hypoxic and normoxic experimental conditions. The rate of oxygen release from oxyhemoglobin under hypoxia was increased 3 times in the presence of 1 mmol/l ISDN. In contrast to ISDN, GTN could not release NO spontaneously, and it also did not affect nNOS and eNOS activities in the absence or presence of 30 micromol/l oxyhemoglobin under both hypoxic and normoxic conditions. These results indicated that the NO release from ISDN is different from that of GTN, and the increase of NOS activity by ISDN in the presence of oxyhemoglobin under hypoxia is ascribed to the increase in molecular oxygen concentration.     

Heroin modulates the expression of inducible nitric oxide synthase

The use of heroin (diacetylmorphine) is associated with a high incidence of infectious disease, and the immunologic alterations responsible for heroin-induced changes in resistance to infection have not been well characterized. The present study tests the hypothesis that expression of inducible nitric oxide synthase (iNOS) is modulated by the administration of heroin. The initial study using rats showed that heroin administration (0, 0.01, 0.1, or 1.0 mg/kg s.c.) results in a pronounced reduction in lipopolysaccharide (LPS)-induced expression of iNOS mRNA in spleen, lung, and liver tissue as measured by RT-PCR. Heroin also produced a reduction in the level of plasma nitrite/nitrate, the more stable end-product of nitric oxide degradation. In a subsequent study, administration of the opioid receptor antagonist, naltrexone (0.1 mg/kg) prior to the injection of heroin (1.0 mg/kg) blocked the heroin-induced reduction of iNOS expression and plasma nitrite/nitrate levels indicating that the effect is mediated via the opioid-receptor. This study provides the first evidence that heroin induces an alteration of iNOS expression, and suggests that a reduction in nitric oxide production may be involved in the increased incidence of infectious diseases amongst heroin users.     

The modulation of the action of isoforms of nitric oxide synthase

The recent patent literature covering the modulation of action of the various isoforms of nitric oxide synthase (NOS) is reviewed. Mammalian biological systems in which nitric oxide (NO) has been shown to be involved are summarised in order to give background to specific patent claims. Claims are generally for therapy of various inflammatory states and for therapy of a number of CNS indications. Emphasis is placed on inhibitors of the several isoforms, since the patent literature is most extensive in this area. Patents are covered by structural class, with amino acids and various classes of heterocycles being the most common types of claimed compounds. Patent applications covering other aspects of control of NO concentrations in mammals are very briefly reviewed, although they fall outside of the scope of NOS per se. These include agents that release NO in situ, and agents that scavenge or sequester NO.     

Biopterin analogues: novel nitric oxide synthase inhibitors with immunosuppressive action

Tetrahydrobiopterin plays an essential role in nitric oxide synthase catalysis, not only as an allosteric modulator but also as a cofactor involved in electron flow through the enzyme. In absence of tetrahydrobiopterin, all isoforms of nitric oxide synthases are virtually inactive. The present review focuses on attempts to inhibit nitric oxide synthase by biopterin analogues, and what is known about the pharmacological effects of these compounds. While several biopterin analogues are capable of inhibiting nitric oxide synthases, the 4-amino analogue of tetrahydrobiopterin (4-amino tetrahydrobiopterin) is the compound of which pharmacological actions in animals have been described. 4-Amino tetrahydrobiopterin inhibits all three isoforms of nitric oxide synthases in micromolar concentrations. In cultured cells and in aortic strips, a surprising selectivity of inhibition of the inducible isoform of nitric oxide synthases has been observed. When applied intramuscularly, 4-amino tetrahydrobiopterin spreads throughout the body within minutes, and is cleared with a half life of about an hour. A single bolus dose applied intravenously in a rat model of septic shock, saved the animals from the lethal effects of endotoxin. When applied three times a day intramuscularly in a murine model of cardiac allograft rejection, 4-amino tetrahydrobiopterin prolongs allograft survival as efficiently as high-dose cyclosporin A treatment does. Thus, 4-amino tetrahydrobiopterin is an effective immunosuppressant. The mechanism of its action is currently under investigation.     

Down-regulation of endogenous nitric oxide synthase inhibitors on endothelial SK3 expression

ObjectivesTo investigate role of endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) in down-regulation of the expression of endothelial SK3 in atherosclerosis.MethodsApolipoprotein E deficient (apo E^−/−) mice aged 11 ∼ 12 weeks were treated with ADMA (5 mg/kg per day, subcutaneous injection) for 4 weeks. Cultured human umbilical venous endothelial cells (HUVECs) were treated with different concentrations of lysophosphatidylcholine (LPC) or ADMA for 48 h. Plasma levels of ADMA were determined by high performance liquid chromatogram (HPLC); protein and mRNA levels of SK3 in the aortas of mice and cultured cells were detected by immunofluorescence, western blot and RT-PCR, respectively.ResultsConcomitantly with the elevated plasma levels of ADMA, the expressions of both SK3 protein and mRNA in aortas of apo E^−/− mice were significantly reduced in comparison to those of the wild-type mice. Moreover, 4-week treatment of ADMA made levels of SK3 expression even lower. In cultured HUVECs, either LPC or ADMA notably decreased the expressions of both SK3 protein and mRNA in a concentration dependent manner.ConclusionsEndogenous ADMA may be an important factor for down-regulation of the expression of endothelial SK3 in atherosclerotic animals.     

Effect of inhibition of nitric oxide synthase on renal cyclooxygenase in the diabetic rat

Renal cyclooxygenase (COX)-2 expression and arachidonic acid-stimulated prostaglandin release are increased in streptozotocin-diabetic rats and are reduced by tempol treatment, indicating a role for superoxide. Generation of nitric oxide (NO) and its product with superoxide, peroxynitrite, is also increased in diabetes and can induce COX-2. To investigate a role of NO, rats were treated with L-nitroarginine methyl ester (L-NAME; 100 mg/kg/day) to inhibit NO synthase (NOS) for 14-18 days. In isolated perfused kidneys from diabetic rats, prostaglandin release and vasoconstrictor responses to arachidonic acid were increased and renal cortical expression of COX-2 was 2-fold that of control rats. Treatment of diabetic rats with L-NAME reduced arachidonic acid-stimulated release of prostaglandins and the expression of COX-2. L-NAME increased vasoconstrictor responses to AA in diabetic and non-diabetic rats but abolished the differences between the two. These results, coupled with those using tempol, suggest that NO or its product with superoxide may contribute to the induction of renal COX-2 in the diabetic rat.     

Ethanol consumption increases the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and metalloproteinases in the rat kidney

Objectives The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase‐2 (MMP‐2) were studied in rat kidney. Methods Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real‐time polymerase chain reaction, respectively. MMP‐2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated. Results Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol‐treated rats showed increased activity of MMP‐2. Histopathological investigation of kidneys from ethanol‐treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol‐treated rats. Conclusions Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS‐derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol‐treated rats might contribute to progressive renal damage.     

Down-regulation of endogenous nitric oxide synthase inhibitors on endothelial SK3 expression

OBJECTIVES: To investigate role of endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) in down-regulation of the expression of endothelial SK3 in atherosclerosis. METHODS: Apolipoprotein E deficient (apo E(-/-)) mice aged 11 approximately 12 weeks were treated with ADMA (5 mg/kg per day, subcutaneous injection) for 4 weeks. Cultured human umbilical venous endothelial cells (HUVECs) were treated with different concentrations of lysophosphatidylcholine (LPC) or ADMA for 48 h. Plasma levels of ADMA were determined by high performance liquid chromatogram (HPLC); protein and mRNA levels of SK3 in the aortas of mice and cultured cells were detected by immunofluorescence, western blot and RT-PCR, respectively. RESULTS: Concomitantly with the elevated plasma levels of ADMA, the expressions of both SK3 protein and mRNA in aortas of apo E(-/-) mice were significantly reduced in comparison to those of the wild-type mice. Moreover, 4-week treatment of ADMA made levels of SK3 expression even lower. In cultured HUVECs, either LPC or ADMA notably decreased the expressions of both SK3 protein and mRNA in a concentration dependent manner. CONCLUSIONS: Endogenous ADMA may be an important factor for down-regulation of the expression of endothelial SK3 in atherosclerotic animals.     

Effect of nitric oxide synthase inhibition on antinociceptive action of different doses of acetaminophen

As demonstrated in previous studies, both cyclooxygenases (COXs) and nitric oxide synthases (NOS) localized peripherally and/or centrally participate in the antinociceptive action of acetaminophen (ACETA). We showed that opioidergic system(s) was involved in the mechanism of ACETA action, as well. In previous and recent studies, the changes in nociceptive threshold were estimated using a mechanical and chemical stimulus. In this study, the influence of nonspecific inhibitor of NOS [N(G)-nitro-L-arginine (L-NOArg)] on antinociceptive action of ACETA administered icv and it was studied in rats. ACETA increased threshold for electrical stimuli, however, its analgesic activity was not dose-dependent. Independently of the route of administration, the existence of a ceiling dose of ACETA was observed above which the activity of ACETA was self-limited. Pretreatment with L-NOArg (ip) markedly increased the action of higher doses of ACETA. It suggests that the attenuation of analgesic action of higher doses of ACETA may be due to increased activity of NOS.     

诱导型一氧化氮合酶在GK糖尿病大鼠心肌梗死后的表达

目的本研究通过制作GK糖尿病大鼠及非糖尿病大鼠心肌梗死模型,观察诱导型一氧化氮合酶(iNOS)的表达在糖尿病大鼠合并心肌梗死时与非糖尿病大鼠心肌梗死时的变化,为冠心病合并糖尿病的心脏病理改变提供理论基础与科学依据。方法应用体质量在200～250g的雄性GK糖尿病大鼠及其对照雄性Wistar大鼠通过开胸手术结扎麻醉大鼠冠状动脉左前降支制作大鼠在体心肌梗死模型。心肌梗死模型制作前测量血糖。手术成功后分组饲养大鼠3周。3周后,测量血糖,活体剪取心脏,进行iNOS的免疫组织化学染色及实时聚合酶链反应(RT-PCR)检测。结果①GK糖尿病大鼠血糖明显高于Wistar大鼠血糖(P<0.05);②Wistar心肌梗死组与GK心肌梗死组在缺血区可见到心肌细胞iNOS大量阳性染色,但GK心肌梗死组与Wistar心肌梗死组相比,iNOS阳性染色减少,二者吸光度测定值差异有统计学意义(P<0.05);③Wistar心肌梗死组与GK心肌梗死组iNOSmRNA表达在缺血区明显增加,但GK心肌梗死组iNOSmRNA表达减少,二者之间差异有统计学意义(P<0.05)。结论iNOS在糖尿病大鼠合并心肌梗死时其在缺血区的表达是降低的,表明高血糖可能抑制iNOS的表达,使体内一氧化氮(NO)生成减少,不利于梗死后血供的恢复。     

Antinociceptive properties and nitric oxide synthase inhibitory action of new ruthenium complexes

This study evaluates the actions of the new ruthenium complexes trans-[RuCl2(nic)4] (Complex I) and trans-[RuCl2(i-nic)4] (Complex II) as antinociceptives, and their interaction with nitric oxide isoenzymes and with acetylcholine-induced relaxation of rat and rabbit aorta. Complex II inhibited, in a graded manner, neuronal and inducible nitric oxide (NO) synthase, and was about two fold more effective in inhibiting the neuronal NO synthase than the inducible form of the enzyme. Complex I was inactive. Both complexes failed to interfere with constitutive endothelial nitric oxide synthase because they did not change the mean arterial blood pressure of rats. The vasorelaxant effect of acetylcholine was markedly antagonised by the Complexes I and II in rings of both rat and rabbit aorta. Complexes I and II, given intraperitoneally, like N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(G)-nitro-L-arginine (L-NOARG), inhibited, in a graded manner, both phases of the pain response induced by formalin. The actions of L-NAME, L-NOARG and Complex II, but not that of Complex I, were largely reversed by L-arginine. Both complexes failed to affect the motor response of animals in the rota-rod test and had no effect in the hot-plate assay. Together, these findings provide indications that the new ruthenium complexes, especially Complex II and its derivatives, might be of potential therapeutic benefit in the management of pain disorders.     

Elevated neuronal nitric oxide synthase expression in chronic haloperidol-treated rats

Long-term administration of neuroleptic drugs, such as haloperidol, in the management of psychiatric disorders may adversely cause an irreversible neurological syndrome of tardive dyskinesia, which is associated with dopamine (DA(2)) receptor supersensitivity in the basal ganglia. Recent studies also indirectly suggest an involvement of nitric oxide synthase (NOS) in dopaminergic supersensitivity; however, chronic neuroleptic effects on neuronal NOS (nNOS) expression in the basal ganglia have not been reported. In this investigation, we treated rats with saline or haloperidol (1 mg/kg, s.c.) daily for 21 days. Five days later, we detected a significant increase of NOS activity in the striatum of haloperidol-treated rats when compared to saline controls. This effect was associated with elevated levels of nNOS mRNA and protein expression in the striatum, but not in the nucleus accumbens, as evidenced by the use of in situ hybridization, Western blot and immunohistochemical techniques. The involvement of the nNOS system after chronic haloperidol treatment coincides with increases of striatal DA(2) receptor sites, calmodulin kinase II activity, animal locomotor and stereotypy behaviors. This study suggests an integral role between nNOS, DA(2) receptor and calmodulin system in the development of dopaminergic behavioral supersensitivity resulting from chronic neuroleptic drug treatment. Furthermore, the toxic effect of chronic haloperidol on NOS system selectivity takes place in the neostriatum.