Platelet aggregation

Under normoxic conditions, S-Nitroso-N-acetyl-DL-penicillamine (10 mM; 8 hours) causes about 80% toxicity via releasing nitric oxide (NO) after 6 hours [1]. In ventricular myocytes ex vivo, S-Nitroso-N-acetyl-DL-penicillamine (100 μM; 30 min) produced a half-life of almost 6 hours [3].

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1. The effects of two new analogues of S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-formyl-DL-penicillamine (SNFP) and S-nitroso-DL-penicillamine (SNPL), on platelet function were examined in vitro. 2. SNAP and its analogues were potent inhibitors of platelet aggregation and inducers of disaggregation. 3. All compounds inhibited fibrinogen binding to platelets. 4. They also decreased the release of P-selectin from platelets. 5. Both inhibition of fibrinogen binding and release of P-selectin correlated with an increase in intraplatelet cyclic GMP concentrations. 6. At concentrations sufficient to inhibit platelet function and induce cyclic GMP formation (0.01-3 microM), the release of NO could be detected from SNPL but not from SNAP and SNFP. 7. Release of NO from all compounds was detected at concentrations > or = 10 microM. 8. Thus, the spontaneous release of NO from SNPL explains the actions of this compound on platelet function; however, platelet-mediated mechanisms may be involved in the release of NO from SNAP and SNFP.[1]

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S-Nitroso-N-acetyl-DL-penicillamine (SNAP) and sodium nitroprusside (SNP), both of which are known to release nitric oxide (.NO), exhibited cytotoxicity against cultivated endothelial cells. Under hypoxic conditions 5 mM SNAP and 20 mM SNP induced a loss in cell viability of about 90% and 80% respectively, after an 8 h incubation. Under normoxic conditions, cell death was only 45% and 42% respectively within the same time period. Concentrations of .NO liberated from SNAP and SNP were measured by the oxyhaemoglobin method and by two of the recently developed nitric oxide cheletropic traps (NOCTs). The .NO concentrations from SNAP and SNP increased from 74 microM and 28 microM to 136 microM and 66 microM respectively within 15 min of hypoxic incubation, and then decreased to 36 microM and 28 microM. In the respective normoxic incubations the .NO levels from SNAP and SNP remained in the region of about 30 microM and 20 microM respectively. In contrast, spermine/NO adduct (spermineNONOate) was shown to be more toxic under normoxic than under hypoxic conditions. Under either of these conditions, the concentration of .NO liberated from 2 mM spermineNONOate was about 20 microM. The results demonstrate that the cytotoxicity of SNAP and SNP, but not of spermineNONOate, is significantly enhanced under hypoxic compared with normoxic incubations. Studies on the .NO-releasing behaviour of these compounds indicate that the increased toxicity of SNAP and SNP under hypoxic conditions is related to the influence of O2 on the chemical processes by which .NO is produced from the precursors, rather than to an increased sensitivity of the hypoxic cells towards .NO. [2]

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1. Previous studies suggest that exogenous nitric oxide (NO) and NO-dependent signalling pathways modulate intracellular pH (pH(i)) in different cell types, but the role of NO in pH(i) regulation in the heart is poorly understood. Therefore, in the present study we investigated the effect of the NO donors S-nitroso-N-acetyl-DL-penicillamine, spermine NONOate and propylamine propylamine NONOate on pH(i) in rat isolated ventricular myocytes. 2. Cells were isolated from the hearts of adult Wistar rats and pH(i) was monitored using the pH-sensitive fluorescent indicator 5-(and-6)-carboxy seminaphtharhodafluor (SNARF)-1 (10 μmol/L) and a confocal microscope. To test the effect of NO donors on the Na⁺/H⁺ exchanger (NHE), basal pH(i) in Na⁺-free buffer and pH(i) recovery from intracellular acidosis after an ammonium chloride (10 mmol/L) prepulse were monitored. The role of carbonic anhydrase was tested using acetazolamide (50 μmol/L). 4,4-Diisothiocyanatostilbene-2,2'-disulphonic acid (0.5 mmol/L; DIDS) was used to inhibit the Cl⁻/OH⁻ and Cl⁻/HCO₃-exchangers. Acetazolamide and DIDS were applied via the superfusion system 1 and 5 min before the NO donors. 3. All three NO donors acutely decreased pH(i) and this effect persisted until the NO donor was removed. In Na⁺-free buffer, the decrease in basal pH(i) was increased, whereas inhibition of carbonic anhydrase and Cl⁻/OH⁻ and Cl⁻/HCO₃⁻ exchangers did not alter the effects of the NO donors on pH(i). After an ammonium preload, pH(i) recovery was accelerated in the presence of the NO donors. 4. In conclusion, exogenous NO decreases basal pH(i), leading to increased NHE activity. Carbonic anhydrase and chloride-dependent sarcolemmal HCO₃⁻ and OH⁻ transporters are not involved in the NO-induced decrease in pH(i) in rat isolated ventricular myocytes. [3]

In the trachea of guinea pigs, SNAP (100 μM, 300 μM) causes a slight but noteworthy increase in electrically induced [3H]-miccholine release [4].

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The effects of the nitric oxide (NO) donor S-nitroso-N-acetyl-DL-penicillamine (SNAP) and the NO synthase inhibitor L-N(G)-nitroarginine (L-NOARG) on the electrically evoked [(3)H]-acetylcholine release were studied in an epithelium-free preparation of guinea-pig trachea that had been preincubated with [(3)H]-choline. SNAP (100 and 300 microM) caused small but significant increases of the electrically evoked [(3)H]-acetylcholine release (121+/-4% and 124+/-10% of control). Resting outflow of [(3)H]-ACh was not affected by SNAP. The increase by SNAP was abolished by the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 1 microM). The facilitatory effect of SNAP (100 and 300 microM) was reversed into inhibition of release (to 74+/-4% and to 78+/-2%) after pretreatment of the trachea with capsaicin (3 microM). ODQ prevented the inhibition. Capsaicin pretreatment alone did not significantly alter the release of [(3)H]-acetylcholine. A significant inhibition by SNAP (100 microM) of [(3)H]-acetylcholine release (78+/-3%) was also seen in the presence of the NK(2) receptor antagonist SR 48968 (30 nM). L-NOARG (10 and 100 microM) significantly enhanced the electrically-evoked smooth muscle contractions, but caused no significant increases of the evoked release from capsaicin pretreated trachea strips. This might indicate that the inhibitory effect of endogenous NO on acetylcholine release is too small to be detected by overflow studies. It is concluded that NO has dual effects on the evoked acetylcholine release. NO enhances release in the absence of modifying drugs, but NO inhibits acetylcholine release after blockade of the NK(2) receptor or after sensory nerve depletion with capsaicin. This suggests that NO and endogenous tachykinins act in series to produce an increase in acetylcholine release. [4]

Measurement of pHi [3]
pHi in intact myocytes was determined as previously described.20 Cells were loaded with 5-(and-6)-carboxy SNARF-1 and acetoxymethyl ester (SNARF-1; 10 µM) in bicarbonate-free Tyrode solution at room temperature for 15 min.20, 22 After dye loading, the cells were superperfused with the same solution (without dye) for additional 20 min before recordings started. SNARF-1 was excited at 543 nm with a green HeNe laser, and emitted fluorescence was simultaneously collected at 590 and 650 nm. The 650 nm/ 590 nm emission ratio from each cell was calculated and converted to pHi values using nigericin calibration technique.23, 24 Calibration curve was made by exposing the cells to different external pH (6.0, 7.5, and 9.0) buffers in a depolarizing high K+ buffer (140 mM KCl, 1.0 mM MgCl2, 5 mM D-glucose, 10 mM HEPES, and in the presence of 10 µM nigericin).25 Nigericin, a H+/K+ exchanger ionophore sets [K+]o = [K+]I, and, therefore, in the presence of nigericin the pHo = pHi. The pH calibration data were obtained separately from individual cells and were averaged for more than 15 cells. NHE activity was assessed after myocytes were exposed to an acid load consisting of 10 mM ammonium-chloride (NH4Cl) for 3 min.3 The experiments were performed in the absence of bicarbonate to assure that the pHi recovery after NH4Cl readdition was because of NHE activation. In addition, experiments were conducted in sodium-free solution by replacing sodium with 132 mM N-methyl-D-glucamine to block NHE activity.3 Intracellular carbon dioxide hydration by CA results in the generation of HCO3− and H+-ions, leading to a fall of pHi. To examine the effect of NO donors on CA, a CA inhibitor acetazolamide (50 µM) was dissolved in DMSO and added to superfusion solution shortly before NO donor was applied. 4 To determine whether NO induces pHi changes due to increased base efflux through Cl−-OH− and Cl−-HCO3− exchangers, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 0.5 mM), a nonselective anion transport inhibitor, was administered 5 min before superfusion with NO donor.

Cell Viability Assay[1]
Cell Types: Rat Liver Sinusoidal Endothelial Cells
Tested Concentrations: 2 mM, 5 mM, 10 mM
Incubation Duration: 2 hrs (hours), 4 hrs (hours), 6 hrs (hours), 8 hrs (hours)
Experimental Results: Demonstrated sustained decrease in basal pHi[3 ]. Cytotoxicity against cultured endothelial cells.

Release of [3H]-acetylcholine [4]
After 30 min equilibration superfusion was stopped and the tracheal strips were incubated with [3H]-choline (185 KBq ml−1) for 1 h during which the tissue was stimulated electrically with square wave pulses of 20 Hz and 1 ms duration for 5 s every 30 s (Grass S6 stimulator), via two platinum electrodes that were positioned parallel to the strips (distance 0.6 cm; voltage drop 10 V cm−1). The strips were then again superfused (2 ml min−1) with the physiological salt solution which contained in addition 10 μM hemicholinium-3. After a washout period of 90 min the superfusate was collected in 3 min fractions and the tritium content of the samples measured by liquid scintillation spectrometry. The strips were stimulated twice 30 min apart (S1, S2). Each stimulation period consisted of 600 pulses applied in trains of 100 pulses at a frequency of 20 Hz in intervals of 30 s. The stimulation-evoked outflow of [3H]-radioactivity was calculated from the difference between the total outflow during and after stimulation, and the basal outflow calculated by interpolation from two samples before and after stimulation. Previous experiments have shown that the electrically evoked outflow of [3H]-radioactivity from this preparation consists only of [3H]-acetylcholine (Kilbinger et al., 1991). SNAP and L-NOARG were added to the superfusion solution 20 min before S2. The effects of SNAP and L-NOARG on the electrically-evoked outflow was calculated by expressing the ratio S2/S1 as a percentage of the equivalent ratio obtained in corresponding control experiments. In interaction experiments, the tachykinin receptor antagonists, SR 48968 (30 nM) and CP 99994 (100 nM) were added 90 min before SNAP and remained in the medium up to the end of the experiment.

[1]. Comparative pharmacology of analogues of S-nitroso-N-acetyl-DL-penicillamine on human platelets. Br J Pharmacol. 1994 Aug;112(4):1071-6.

[2]. Enhanced release of nitric oxide causes increased cytotoxicity of S-nitroso-N-acetyl-DL-penicillamine and sodium nitroprusside under hypoxic conditions. Biochem J. 1996 Sep 15;318 ( Pt 3):789-95.

[3]. Effect of nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine, spermine NONOate and propylamine propylamine NONOate on intracellular pH in cardiomyocytes. Clin Exp Pharmacol Physiol. 2012 Sep;39(9):772-8.

[4]. Modulation of acetylcholine release in the guinea-pig trachea by the nitric oxide donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP). Br J Pharmacol. 2000 Sep;131(1):94-8.

A sulfur-containing alkyl thionitrite that is one of the NITRIC OXIDE DONORS.
See also: S-Nitroso-N-Acetylpenicillamine (annotation moved to).

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SNAP inhibited the electrically-evoked acetylcholine release when the NK2 receptor could not be stimulated by an endogenous tachykinin, i.e. in the presence of SR 48968 or after depletion of sensory nerves by capsaicin. The mechanisms by which SNAP diminished acetylcholine release are unknown, but may be similar to those described for guinea-pig myenteric neurones where the soluble guanylyl cyclase inhibitor, ODQ, abolished the inhibitory effect of SNAP as well (Hebeiss & Kilbinger, 1998). In the presence of the NK1 receptor antagonist CP 99994, SNAP caused neither facilitation nor inhibition of acetylcholine release. This suggests that NK1 receptors are also involved in the excitatory effects of tachykinins on cholinergic neurones as was proposed by Watson et al. (1993). However, the predominant pathway seems to involve NK2 receptors, since only blockade of NK2 receptors unmasked an inhibitory action of SNAP. NK3 receptor antagonist were not studied since there is evidence that NK3 receptors are not involved in the facilitation by tachykinins of cholinergic neurotransmission (Watson et al., 1993). [4]

C7H12N2O4S

220.2462

220.051

67776-06-1

5231

Light green to green solid powder

1.4±0.1 g/cm3

151ºC

1.560

1.13

2

6

4

14

254

0

ZIIQCSMRQKCOCT-UHFFFAOYSA-N

InChI=1S/C7H12N2O4S/c1-4(10)8-5(6(11)12)7(2,3)14-9-13/h5H,1-3H3,(H,8,10)(H,11,12)

2-acetamido-3-methyl-3-nitrososulfanylbutanoic acid

67776-06-1; snap; S-Nitroso-N-acetyl-DL-penicillamine; Valine,N-acetyl-3-(nitrosothio)-; S-Nitroso-N-acetyl-D,L-penicillamine; 2-acetamido-3-methyl-3-(nitrososulfanyl)butanoic acid; 152971-80-7; DL-Valine, N-acetyl-3-(nitrosothio)-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO : ~250 mg/mL (~1135.07 mM)
H2O : ~11.11 mg/mL (~50.44 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (9.44 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (9.44 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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