NOS/nitric oxide synthaseNOS

In the absence of Aβ1-42, S-MTC (10 or 100 μM) decreased cellular NO emission. S-MTC reduced cell viability at 100 μM. Comparing S-MTC (100 μM) to the control (no exposure to NOS inhibitor; 19.6±1.2 μM), nitrite generation was dramatically reduced (11.2±1.1 μM). After treatment with Aβ1-42 and L-NOARG (100 μM) or Aβ1-42 and S-MTC (100 μM), the amount of nitrite produced was notably less than when Aβ1-42 was used alone (33.5±2.0 and 34.5±1.6 μM, respectively). S-MTC (100 μM) was able to considerably lower nitrite generation (25.2±1.1 μM) when provided one hour after Aβ1-42 as opposed to Aβ1-42 therapy alone (38.3±2.7 μM). MTT (87±1% of control) and NR (80±1% of control) levels were lowered by S-MTC (100 μM) concentration. The effects of Aβ1-42 alone were dramatically negated (72±2% vs. 61±2% of control) when S-MTC (100 μM) and Aβ1-42 were administered together [1].

S-methyl-L-thiocitrulline, or S-MTC, is a neuronal NOS inhibitor that is selective. S-MTC pretreatment significantly inhibited HBO2-induced analgesia (icv). In the second experiment, several mouse groups received treatments 15–30 minutes prior to HBO2 treatment, including naltrexone hydrochloride (NTX) (3.0 mg/kg, intraperitoneal injection), L-NAME (1.0 μg/mouse, icv), S-MTC (1.0 μg/mice, icv), or N5-(1-iminoethyl)-L-ornithine (L-NIO) (3.0 mg/kg, sc). Ninety minutes after HBO2 treatment, the analgesic impact was evaluated. It was found that NTX and L-NAME entirely eliminated the effect, S-MTC antagonized two-thirds of it, and L-NIO had no effect at all (F=25.57, p<0.0001) [2]. At a dosage of 0.3 mg/kg, S-MTC (SMTC) raises mean blood pressure (BP). S-MTC induced vasoconstriction, an increase in blood pressure, and a decrease in heart rate in all three vascular beds at dosages of 1.0, 3.0, and 10 mg/kg [3].

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1. The regional haemodynamic effects of the putative nNOS inhibitor, S-methyl-L-thiocitrulline (SMTC), were compared with those of the nonselective NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), in conscious, male Sprague-Dawley rats. 2. SMTC (0.3 mg kg(-1) bolus) produced a significant, short-lived, pressor effect associated with renal, mesenteric and hindquarters vasoconstriction; the same dose of L-NAME did not affect mean blood pressure (BP), although it caused bradycardia and mesenteric vasoconstriction. 3. At the highest dose tested (10 mg kg(-1)), L-NAME produced a significantly greater bradycardia and fall in mesenteric vascular conductance than SMTC, although the initial pressor response to SMTC was greater, but less sustained, than that to L-NAME. 4. Infusion of SMTC or L-NAME (3 mg kg(-1) h(-1)) induced rises in BP and falls in renal, mesenteric and hindquarters vascular conductances, but the effects of L-NAME were greater than those of SMTC, and L-NAME also caused bradycardia. 5. The renal vasodilator response to acetylcholine was markedly attenuated by infusion of L-NAME, but unaffected by SMTC. The hindquarters vasodilatation induced by salbutamol was attenuated by L-NAME, but not by SMTC. The mesenteric vasodilator response to bradykinin was modestly enhanced by SMTC, but not by L-NAME. The depressor and renal, mesenteric and hindquarters vasodilator responses to sodium nitroprusside were enhanced by L-NAME, whereas SMTC modestly enhanced the hypotensive and renal vasodilator effects of sodium nitroprusside, but attenuated the accompanying tachycardia. 6. The results are consistent with the cardiovascular effects of low doses of SMTC being attributable to nNOS inhibition[3].

Assessment of NO release[1]
NO is rapidly converted to nitrate and nitrite in aqueous solutions. NO released by cultured cells after Aβ1–42 and specific NOS inhibitor (e.g. SMTC) treatments were inferred by converting the nitrate produced into nitrite by nitrate reductase, followed by the addition of the Griess reagent (NO colorimetric assay kit), which measured total nitrite production (Nims et al., 1996).

On day 7 after plating, the culture medium was removed and replaced with freshly prepared culture medium in the presence of either Aβ1–42 (1, 5, 10, or 20 μM), Aβ42–1, or peroxynitrite (100 or 200 μM) with or without either NG-nitro-L-arginine (L-NOARG, a type I (and III) NOS inhibitor (Furfine et al., 1993); 10 or 100 μM), S-methyl-L-thiocitrulline (SMTC; a type I NOS inhibitor (Furfine et al., 1994); 10 or 100 μM), N-iminoethyl-L-lysine (L-NIL, a type II NOS inhibitor (Moore et al., 1994); 10 or 100 μM), N-(3-(aminomethyl)benzyl)acetamidine (1400W, a type II NOS inhibitor (Garvey et al., 1997); 1 or 5 μM), 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, a NO scavenger (Hogg et al., 1995); 10 or 100 μM), or 6-hydorxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a vitamin E analogue with antioxidative properties against peroxynitrite-mediated oxidative stress (Salgo & Pryor, 1996); 10 or 100 μM) alone or in combination. The cultured cells were then incubated for 20 h at the conditions described above. For the time-course studies, the cultured cells were pre-treated with the described culture medium containing Aβ1–42 (10 μM). Either L-NIL (100 μM), L-NOARG (100 μM), 1400W (5 μM), SMTC (100 μM), carboxy-PTIO (100 μM) or Trolox (100 μM) were administered at 1, 4, and 8 h later. Assessments were carried out 20 h after Aβ1–42 administration. To examine the combining effects of these drugs, they were paired with each other at half of the maximum concentrations used, except for 1400W, where 3 μM was used[1].

NTX, L-NAME, SMTC and L-NIO were freshly prepared in 0.9% physiological saline solution. NTX and L-NIO were administered systemically (30-min pretreatment time) and L-NAME and SMTC were administered i.c.v. (15-min pretreatment time). In one set of experiments (#1, #2, and #3), opioid antagonists and NOS-inhibitors were administered 15–30 min prior to the 60-min HBO2 treatment (180 min prior to antinociceptive testing). In another experiment (#4), opioid antagonist and NOS-inhibitor pretreatment was administered 60 min following cessation of the 60-min HBO2 treatment (15–30 min prior to antinociceptive testing). For i.p. or s.c. pretreatments, the volume of injection was 0.1 ml/10 g body weight with control animals receiving an i.p. or s.c. injection of vehicle (sterile saline) only. For i.c.v. pretreatments, the volume of microinjection was 5.0 μl per mouse with control animals receiving an i.c.v. microinjection of vehicle (sterile saline) only[2].

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Cardiovascular responses to SMTC or L-NAME[3]
On the day after catheterisation (day 1), animals (n=7) received bolus i.v. injections (0.1 ml) of either saline (vehicle), and 0.3 and 3 mg kg−1 SMTC (n=4), or 0.1, 1 and 10 mg kg−1 SMTC (n=3). On day 3, the dose regimen was switched to ensure that each animal had received all the doses of SMTC. On each day, drugs were given in ascending dose-order, and at least 60 min was allowed between doses. The intervening day (day 2) was allowed for wash-out of any drug effects. This protocol was repeated with L-NAME in a different group of rats (n=8).

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Effects of infusion of SMTC or L-NAME on resting cardiovascular variables and on responses to acetylcholine, salbutamol, sodium nitroprusside and bradykinin[3]
On day 1, animals in group 1 (n=8) and group 2 (n=9) received an i.v. infusion (0.4 ml h−1) of saline (vehicle). After 90 min, during continued infusion of the vehicle, animals were given 3 min infusions (0.15 ml min−1) of acetylcholine (10 μg kg−1 min−1), salbutamol (0.6 μg kg−1 min−1), sodium nitroprusside (20 μg kg−1 min−1) and bradykinin (38 μg kg−1 min−1). The order of administration was randomised between animals within the groups, with at least 10 min between each substance to allow return to baseline values. The doses of vasodilators were chosen on the basis of previous experiments (Gardiner et al., 1991b; Randall et al., 1996; Gardiner et al., 1998), which showed that they produced robust, steady-state responses.[3]
On day 3, group 1 received SMTC and group 2 received L-NAME (both at 3 mg kg−1 h−1 i.v.). Starting 90 min later, while the infusions of SMTC or L-NAME were continued, animals received 3 min infusions of acetylcholine, salbutamol, sodium nitroprusside and bradykinin using the same doses and protocol as for day 1.[3]

[1]. Law A, et al. Neuroprotective and neurorescuing effects of isoform-specific nitric oxide synthase inhibitors, nitric oxide scavenger, and antioxidant against beta-amyloid toxicity. Br J Pharmacol. 2001 Aug;133(7):1114-24.
[2]. Zelinski LM, et al. A prolonged nitric oxide-dependent, opioid-mediated antinociceptive effect of hyperbaric oxygenin mice. J Pain. 2009 Feb;10(2):167-72.
[3]. Wakefield ID, et al. Comparative regional haemodynamic effects of the nitric oxide synthase inhibitors, S-methyl-L-thiocitrulline and L-NAME, in conscious rats. Br J Pharmacol. 2003 Jul;139(6):1235-43

S-methyl-L-thiocitrulline is an L-arginine derivative in which the guanidino NH2 group of L-arginine is replaced by a methylsufanyl group. It has a role as an EC 1.14.13.39 (nitric oxide synthase) inhibitor and a neuroprotective agent. It is a L-arginine derivative, a L-ornithine derivative, a non-proteinogenic L-alpha-amino acid and an imidothiocarbamic ester.

C7H15N3O2S

205.2779

205.088

156719-41-4

107968

Typically exists as solid at room temperature

1.35 g/cm3

405ºC at 760 mmHg

60ºC

198.7ºC

1.572

1.347

3

5

6

13

196

1

N[C@@H](CCCNC(SC)=N)C(O)=O

NGVMVBQRKZPFLB-YFKPBYRVSA-N

InChI=1S/C7H15N3O2S/c1-13-7(9)10-4-2-3-5(8)6(11)12/h5H,2-4,8H2,1H3,(H2,9,10)(H,11,12)/t5-/m0/s1

(2S)-2-amino-5-[[amino(methylsulfanyl)methylidene]amino]pentanoic acid

S-Methyl-L-thiocitrulline; S-Methylthiocitrulline; 156719-41-4; L-S-Methylthiocitrulline; S-MTC; N(delta)-(S-Methylisothioureido)norvaline; L-Ornithine, N5-[imino(methylthio)methyl]-; N5-(Imino(methylthio)methyl)-L-ornithine;

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.)