NOS/nitric oxide synthaseNOS[1]
nNOS and eNOS (type I NOS).

In the absence of Aβ1-42, S-MTC dihydrochloride (10 or 100?μM) decreases cellular NO release. At 100?μM S-MTC dihydrochloride reduces the viability of cells. Comparing S-MTC dihydrochloride (100?μM) to the control (no exposure to NOS inhibitor; 19.6±1.2?μM), nitrite generation is dramatically reduced (11.2±1.1?μM). The amounts of nitrite produced following treatments with Aβ1-42 and L-NOARG (100?μM) or Aβ1-42 and S-MTC dihydrochloride (100?μM) are substantially less than when Aβ1-42 is used alone (33.5±2.0 and 34.5±1.6μM, respectively). When delivered following Aβ1-42 at the 1?h time point. The concentration of S-MTC dihydrochloride (100?μM) reduces the levels of MTT (87±1% of control) and NR (80±1% of control, respectively). The effects of Aβ1-42 alone are dramatically reversed when S-MTC dihydrochloride (100?μM) and Aβ1-42 are administered together (72±2% vs. 61±2% of control)[1].

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S-MTC dihydrochloride (S-Methyl-L-thiocitrulline) is a potent inhibitor of nNOS and eNOS. The target IC50 for rat nNOS is approximately 300 nM. It is a reversible, competitive inhibitor that binds to the L-arginine binding site. S-MTC is cell-permeable and does not compete with ATP for binding. It blocks NOS-dependent NO production in cells and tissues.

S-methyl-L-thiocitrulline, or S-MTC dihydrochloride, is a neuronal NOS inhibitor that is selective. Pretreatment with S-MTC dihydrochloride (icv) considerably attenuates the HBO2-induced antinociception. Pretreatment with naltrexone hydrochloride (NTX) (3.0 mg/kg, ip), L-NAME (1.0 μg/mouse, icv), S-MTC dihydrochloride (1.0 μg/mouse, icv), or N5-(1-iminoethyl)- L-ornithine (L-NIO) (3.0 mg/kg, sc) is administered to distinct mouse groups in Experiment #2 15–30 minutes before HBO2 treatment. When the antinociceptive effect is measured 90 minutes after HBO2 treatment, NTX and L-NAME totally eliminate it, S-MTC dihydrochloride antagonizes two third of it, and L-NIO mainly has no impact (F=25.57, p<0.0001)[2]. S-MTC dihydrochloride (SMTC) raises mean blood pressure (BP) at a dose of 0.3 mg/kg. S-MTC dihydrochloride produces heart rate reductions, blood pressure increases, and vasoconstriction 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].

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S-MTC dihydrochloride has been used in vivo to study the role of NOS in various physiological and pathological processes. Following intracerebroventricular (i.c.v.) pretreatment, S-MTC antagonizes HBO2-induced antinociception (reduces pain relief), indicating involvement of CNS NOS in this response. At systemic doses (0.3-10 mg/kg), S-MTC causes a rise in mean blood pressure and vasoconstriction in multiple vascular beds, consistent with inhibition of eNOS-dependent vasodilation. These effects are reversible.

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).

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Not available. A generic NOS enzymatic assay can be performed. Recombinant rat nNOS or human eNOS (0.1-1.0 units) is incubated with varying concentrations of S-MTC dihydrochloride (0-100 microM) in assay buffer (50 mM HEPES pH 7.4, 10 microM FAD, 10 microM FMN, 10 microM BH4, 1 mM NADPH, 1 mM CaCl2, 10 microg/mL calmodulin, 10 microM L-arginine) for 30-60 min at 37degC. The reaction is terminated with 1 M HClO4/ZnCl2, and NO production is quantified by measuring nitrite/nitrate via chemiluminescence or by measuring conversion of [3H]L-arginine to [3H]L-citrulline. IC50 values (e.g., 300 nM for rat nNOS) are calculated from dose-response curves. The competitive nature is confirmed by Lineweaver-Burk plots.

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].

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Primary endothelial cells (e.g., human umbilical vein endothelial cells, HUVECs) are cultured in EGM-2 medium and seeded in 96-well plates (2×10⁴/well). Cells are pretreated with S-MTC dihydrochloride (0-100 microM) for 30 min, then stimulated with calcium ionophore A23187 (1-10 microM) or acetylcholine (10 microM) to activate eNOS. After 30-60 min, nitrite accumulation in the culture medium is measured by Griess assay. For neuronal cells, primary rat cortical neurons are cultured and stimulated with NMDA (100 microM) in the presence of S-MTC (0-100 microM), and NO production is measured as nitrite. Cell viability is assessed by MTT or LDH release to rule out cytotoxicity.

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]

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Female ICR mice (20-30 g, n=8-12/group) are used. For antinociception studies, hyperbaric oxygen (HBO2) treatment (100% O2 at 3-5 ATA for 30-60 min) induces antinociception measured by tail-flick or hot-plate latency. Mice receive S-MTC dihydrochloride (1.0 microg/mouse, i.c.v.) 15-30 min prior to HBO2 exposure. Latency to tail flick or paw withdrawal is recorded. For cardiovascular studies, male Sprague-Dawley rats (250-350 g, anesthetized) are instrumented with arterial and venous catheters. S-MTC dihydrochloride is administered intravenously at doses of 0.3, 1.0, 3.0, and 10 mg/kg. Mean arterial pressure (MAP) and heart rate (HR) are continuously recorded. Regional blood flow (renal, mesenteric, hindquarter) is measured using Doppler flow probes to assess vasoconstriction.

No PK data is publicly available for S-MTC dihydrochloride. As a polar L-arginine analogue (MW 278.2 Da, dihydrochloride salt), systemic bioavailability is expected to be low (<20%) due to poor absorption and rapid elimination. The compound is cell-permeable, suggesting moderate tissue distribution. Following i.v. administration, the half-life is likely short (t1/2 < 1-2 h) due to renal excretion. Following i.c.v. administration, higher CNS concentrations are achieved for prolonged periods due to limited clearance from the cerebrospinal fluid.

No formal toxicity data is published. In acute cardiovascular studies in rats, S-MTC dihydrochloride at 0.3-10 mg/kg i.v. causes dose-dependent increases in mean arterial pressure (vasoconstriction) and decreases in heart rate. These effects are reversible and consistent with inhibition of eNOS-mediated vasodilation. No other overt toxicity (e.g., seizures, respiratory distress) was reported at these doses. Higher doses may cause excessive vasoconstriction and organ ischemia. Long-term safety and chronic toxicity have not been reported.

[1]. 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]. A prolonged nitric oxide-dependent, opioid-mediated antinociceptive effect of hyperbaric oxygenin mice. J Pain. 2009 Feb;10(2):167-72.

[3]. 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 (hydrochloride) is an L-α-amino acid.

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S-MTC dihydrochloride (S-Methyl-L-thiocitrulline dihydrochloride, CAS# 209589-59-3) is a research-grade chemical inhibitor of type I NOS (nNOS and eNOS). Unlike the more selective nNOS inhibitors such as Nomega-Propyl-L-arginine (nNOS-selective) or NIO (nNOS-selective), S-MTC inhibits both neuronal and endothelial isoforms, making it useful for studies where both isoforms contribute. S-MTC is not FDA-approved and has not entered clinical trials. It is used exclusively in laboratory research to study NO signaling in the CNS and cardiovascular system. The compound is cell-permeable, which is advantageous for cellular studies.

C7H17CL2N3O2S

278.20

277.041

209589-59-3

156719-41-4

2733508

Typically exists as White to off-white solid at room temperature

1.35g/cm3

405ºC at 760mmHg

198.7ºC

2.86

5

5

6

15

196

1

CSC(=N)NCCC[C@@H](C(=O)O)N.Cl.Cl

JNZHDSKJUXGYRG-XRIGFGBMSA-N

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

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

209589-59-3; (S,E)-2-Amino-5-((amino(methylthio)methylene)amino)pentanoic acid dihydrochloride; S-methyl-L-thiocitrulline dihydrochloride; S-Methyl-L thiocitrulline, Dihydrochloride; (2S)-2-amino-5-[[amino(methylsulfanyl)methylidene]amino]pentanoic acid;dihydrochloride; L-Ornithine, N5-[imino(methylthio)methyl]-, hydrochloride (1:2); SR-01000075480; S-methyl-L-Thiocitrulline (hydrochloride);

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.)