NOS （IC50 = 70 μM）

Nitric oxide synthase (NOS) activity is commonly inhibited by L-arginine analogs, with L-NAME (Nw-nitro-L-arginine methyl ester) being the most effective [2]. L-NAME, when dissolved fresh, is a pure inhibitor of brain NOS with an average IC50 of 70 μM, which is 50 times less than that of L-NOARG (IC50 = 1.4 μM). Despite this, L-NAME's apparent inhibitory potency is comparable to that of L-NOARG. For extended periods, incubate at pH values that are neutral or alkaline. According to HPLC studies, the drug's hydrolysis into L-NOARG is intimately linked to L-NAME's suppression of NOS [1].

L-NAME hydrochloride may be used to induce hypertension in animal models [6].
Pathogenic principle: L-NAME hydrochloride causes hypertension by reducing the release of nitric oxide (NO) in animals and inhibiting the activity of endothelial nitric oxide synthase (eNOS).
The mouse is the most used animal for studying the genetic basis of cardiovascular diseases. However, the mechanisms of regulation of cardiovascular function in this animal are not yet well understood. The goal of this study was to evaluate the baroreflex, the Bezold-Jarisch cardiopulmonary reflex (BJR), and the chemoreflex in mice with hypertension induced by inhibition of NO using Nomega-nitro-L-arginine-methyl ester (L-NAME). Basal mean arterial pressure (MAP) measured under anesthesia (urethane, 1 mg/g IP) was significantly higher in L-NAME (400 microgram/g IP for 7 days)-treated (HT) mice (n=7) compared with vehicle-treated (NT; n=10) animals (126+/-9 versus 79+/-2 mm Hg) without differences in heart rate (HR). Baroreflex sensitivity, evaluated using phenylephrine (1 microgram/g IV) was enhanced in HT mice compared with NT mice (-9.8+/-1.4 versus -4.9+/-0.5 bpm/mm Hg). The BJR, induced by phenylbiguanide (40 ng/g IV), was significantly attenuated in HT animals (MAP, -13+/-5%; HR, -39+/-6%) compared with NT animals (MAP, -38+/-5%; HR, -66+/-2%). The chemoreflex, induced by potassium cyanide (0.26 microgram/g IV), was significantly attenuated in HT animals (MAP, +14+/-4%; HR, -8+/-2%) compared with NT animals (MAP, +29+/-4%; HR, -15+/-4%). As has been observed in rats, chronic inhibition of NO synthase in mice results in arterial hypertension. Enhancement of baroreflex sensitivity and attenuation of BJR and chemoreflex seem to be mainly caused by inhibition of NO synthesis because individual analyses did not show positive correlation between changes in these reflexes and MAP levels in the HT group[6].

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The combined treatment of parenteral arginine and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) have been shown to improve liver function and systemic inflammation in subacute peritonitic rats. Here, we investigated the effects of single and combined parenteral arginine and L-NAME treatments on leukocyte and splenocyte immunity. Male Wistar rats were subjected to cecal punctures and were intravenously given total parenteral nutrition solutions with or without arginine and/or L-NAME supplementations for 7 days. Non-surgical and sham-operated rats with no cecal puncture were given a chow diet and parenteral nutrition, respectively. Parenteral feeding elevated the white blood cell numbers and subacute peritonitis augmented the parenteral nutrition-induced alterations in the loss of body weight gain, splenomegaly, and splenocyte decreases. Parenteral arginine significantly increased the B-leukocyte level, decreased the natural killer T (NKT)-leukocyte and splenocyte levels, alleviated the loss in body weight gain and total and cytotoxic T-splenocyte levels, and attenuated the increases in plasma nitrate/nitrite and interferon-gamma production by T-splenocytes. L-NAME infusion significantly decreased NKT-leukocyte level, tumor-necrosis factor (TNF)-alpha production by T-splenocytes and macrophages, and interferon-gamma production by T-leukocytes, monocytes, and T-splenocytes, as well as increased interleukin-6 production by T-leukocytes and monocytes and nitrate/nitrite production by T-leukocytes. Combined treatment significantly decreased plasma nitrate/nitrite, the NKT-leukocyte level, and TNF-alpha production by T-splenocytes. Parenteral arginine may attenuate immune impairment and L-NAME infusion may augment leukocyte proinflammatory response, eliminate splenocyte proinflammatory and T-helper 1 responses, and diminish arginine-induced immunomodulation in combined treatment in subacute peritonitic rats[3].

1. The L-arginine derivatives NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work was carried out to investigate whether L-NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L-NOARG for inhibition of the enzyme. 2. A bolus of L-NAME and L-NOARG (0.25 micromol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 +/- 0.8 mmHg; n = 5), but the effect developed more rapidly following addition of L-NOARG than L-NAME (mean half-time: 0.7 vs 4.2 min). The time-dependent onset of the inhibitory effect of L-NAME was paralleled by the appearance of L-NOARG in the coronary effluent. 3. Freshly dissolved L-NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 microM) than L-NOARG (IC50 = 1.4 microM), but the apparent inhibitory potency of L-NAME approached that of L-NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L-NAME closely correlated with hydrolysis of the drug to L-NOARG. 4. Freshly dissolved L-NAME contained 2% of L-NOARG and was hydrolyzed with a half-life of 365 +/- 11.2 min in buffer (pH 7.4), 207 +/- 1.7 min in human plasma, and 29 +/- 2.2 min in whole blood (n = 3 in each case). When L-NAME was preincubated in plasma or buffer, inhibition of NOS was proportional to formation of L-NOARG, but in blood the inhibition was much less than expected from the rates of L-NAME hydrolysis. This was explained by accumulation of L-NOARG in blood cells. 5. These results suggest that L-NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L-NOARG. Bioactivation of L-NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium[1].

L-arginine analogues are widely used inhibitors of nitric oxide synthase (NOS) activity both in vitro and in vivo, with N(ω)-nitro-L-arginine methyl ester (L-NAME) being at the head. On the one hand, acute and chronic L-NAME treatment leads to changes in blood pressure and vascular reactivity due to decreased nitric oxide (NO) bioavailability. However, lower doses of L-NAME may also activate NO production via feedback regulatory mechanisms if administered for longer time. Such L-NAME-induced activation has been observed in both NOS expression and activity and revealed considerable differences in regulatory mechanisms of NO production between particular tissues depending on the amount of L-NAME. Moreover, feedback activation of NO production by L-NAME seems to be regulated diversely under conditions of hypertension. This review summarizes the mechanisms of NOS regulation in order to better understand the apparent discrepancies found in the current literature[2].

There is increasing evidence that nitric oxide may be involved in learning and memory. However, there remain comparatively few studies that have explored the relationship between nitric oxide signaling and fear extinction, an inhibitory learning model. In the present study, we tested the effects of nitric oxide synthase inhibitor l-NAME on three tone fear extinction tasks in rats. In task 1, rats received fear conditioning, extinction training and extinction test in the same context (AAA design). In task 2, rats received fear conditioning in context A, extinction training in context B and extinction test in context A (ABA design). In task 3, rats received fear conditioning in context A, extinction training and extinction test in context B (ABB design). l-NAME (10, 20 and 40 mg/kg) was injected intraperitoneally 30 min prior to extinction training in each task. Percent of time spent freezing was used to measure conditioned fear response. We found that l-NAME administrations had no effect on freezing in task 1 and 2 but produced a dose-dependent increase in task 3. Further results indicated that the increased freezing in task 3 was not attributed to state-dependency effects or nonspecific changes of locomotor activity that followed l-NAME injection. These results showed that l-NAME produced a task-dependent impairment of fear extinction, and implied that nitric oxide signaling was involved in memory process of certain extinction tasks.[4]

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Chronic NG-nitro-l-arginine methyl ester (L-NAME) administration induces cardiac hypertrophy in rodent models. Our aims is to determine the role of c-kit expression in L-NAME induced cardiac hypertrophy. 12-20 week old C57BL/6J mice (5 per group) were administered L-NAME (0.325mg/ml) in the drinking water. Hearts were excised at 1-day, 2-days, 5-days, 2-weeks or 6-weeks; or controls which received no L-NAME. Ventricular cross-sectional wall thickness and individual cardiac myocytes cross-sectional area and cardiomyocyte/nuclear ratio to determine cardiac hypertrophy. Immuno-histochemical staining for c-kit, sca-1 and BCRP undertaken. Six weeks L-NAME administration induced significant cardiac hypertrophy compared to control hearts, evidenced by an increase in the thickness of the cross-sectional free ventricular wall (p<0.05) and an increase in mean individual cross-sectional area of cardiac myocytes in the LV wall (p<0.007). We observed c-kit(+) cells (predominately non-mast cell sub-types) in both healthy mice and in the L-NAME treated mice. C-kit staining in the left ventricular cross sections following L-NAME remained stable at 1 and 2 days compared to controls (p=NS). After 5 days of L-NAME we observed c-kit expression to decrease below control levels (p<0.05) and these lower levels were sustained at 2 and 6 weeks. C-kit expression does not decrease during two days of L-NAME administration, suggesting, firstly, that the later decrease in c-kit is not due to NOS inhibition directly and, secondly, there is the possibility for c-kit(+) cell differentiation into other cell types, possibly inducing myocardial cellular hyperplasia, without significant replacement of the original pool of c-kit(+) cells.[5]

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Despite major scientific advances in its prevention, treatment and care, hypertension remains a serious condition that might lead to long-term complications such as heart disease and stroke. The great majority of forms of hypertension eventually result from an increased vasomotor tone activity that is regulated by endothelial NOS (eNOS) in vascular endothelium. Here, we examined the effect of fucoidan on eNOS activation in human umbilical vein endothelial cells (HUVECs). We also examined the effects of functional components of Undaria pinnatifida fucoidan on blood pressure and vascular function in eNOS inhibition-induced hypertensive rats in vivo. Our results suggest that fucoidan increased nitric oxide production by activating eNOS and Akt phosphorylation, which could be impaired by Akt or eNOS inhibitors. In the hypertensive rat model, treatment of fucoidan resulted in potent and persistent reduction of high blood pressure (BP) even after drug withdrawal. Our results showed that the mechanisms might involve protection against vascular structure damage, enhanced endothelium-independent vascular function and inhibition of abnormal proliferation of smooth muscle cells, which are mediated by the Akt-eNOS signaling pathway. Moreover, fucoidan treatment reduced the vascular inflammation and oxidative stress control caused by iNOS expression. Together, these results support a putative role of fucoidan in hypertension prevention and treatment.[7]

[1]. Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement forbioactivation to the free acid, NG-nitro-L-arginine. Br J Pharmacol. 1996 Jul;118(6):1433-40.

[2]. L-NAME in the cardiovascular system - nitric oxide synthase activator? Pharmacol Rep. 2012;64(3):511-20.

[3]. The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Methyl Ester Diminishes the Immunomodulatory Effects of Parental Arginine in Rats with Subacute Peritonitis. PLoSOne. 2016 Mar 23;11(3):e0151973.

[4]. Effect of nitric oxide synthase inhibitor L-NAME on fear extinction in rats: a task-dependent effect. Neurosci Lett. 2014 Jun 20;572:13-8.

[5]. Chronic NG-nitro-l-arginine methyl ester (L-NAME) administration in C57BL/6J mice induces a sustained decrease in c-kit positive cells during development of cardiac hypertrophy. J Physiol Pharmacol. 2013 Dec;64(6):727-36.

[6]. Cardiovascular neural reflexes in L-NAME-induced hypertension in mice. Hypertension. 2001, 38, 3.

[7]. Fucoidan from Undaria pinnatifida prevents vascular dysfunction through PI3K/Akt/eNOS-dependent mechanisms in the l-NAME-induced hypertensive rat model. Food Funct. 2016, 7, 5.

N(gamma)-nitro-L-arginine methyl ester is an alpha-amino acid ester that is the methyl ester of N(gamma)-nitro-L-arginine. It has a role as an EC 1.14.13.39 (nitric oxide synthase) inhibitor. It is an alpha-amino acid ester, a L-arginine derivative, a N-nitro compound and a methyl ester. It is a conjugate base of a N(gamma)-nitro-L-arginine methyl ester(1+).
L-NAME has been investigated for the treatment of Hypotension and Spinal Cord Injury.
A non-selective inhibitor of nitric oxide synthase. It has been used experimentally to induce hypertension.

C7H15N5O4

233.2251

233.112

C, 36.05; H, 6.48; N, 30.03; O, 27.44

50903-99-6

51298-62-5 (HCl); 50903-99-6; 50912-92-0 (D-NAME)

39836

Typically exists as solid at room temperature

1.48g/cm3

383.5ºC at 760mmHg

185.8ºC

0.677

3

6

7

16

274

1

COC(=O)C(CCCN=C(N)N[N+](=O)[O-])N

KCWZGJVSDFYRIX-YFKPBYRVSA-N

InChI=1S/C7H15N5O4/c1-16-6(13)5(8)3-2-4-10-7(9)11-12(14)15/h5H,2-4,8H2,1H3,(H3,9,10,11)/t5-/m0/s1

methyl (2S)-2-amino-5-[[amino(nitramido)methylidene]amino]pentanoate

L-NAME; NG-NITROARGININE METHYL ESTER; 50903-99-6; N(G)-Nitro-L-arginine methyl ester; N-Nitroarginine methyl ester; N(G)-Nitroarginine methyl ester; Ng-nitro-L-arginine methyl ester; N-Nitro-L-arginine methylester;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)