phospholipase C (PLC); 5-LO (5-lipoxygenase)

In membranes separated from PMNs, U-73122 TFA efficiently suppresses receptor-coupled activation of PLC [1]. Human polymorphonuclear neutrophil (PMN) aggregation induced by N-formyl-methionyl-leucyl-phenylalanine and the corresponding synthesis of diacylglycerol and IP3 are inhibited by U-73122 TFA [2]. In digitonin-permeabilized cells, U-73122 TFA dramatically inhibits phosphoinositide release caused by oxotremorine-M or guanosine-5'-O- (3-thiotriphosphate), but not by the addition of Ca2+ [3].

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Aggregation of human platelets induced by a variety of agonists was inhibited by 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dionel (U-73122) (IC50 values 1-5 microM), but not by the close analog 1-[6-[[17 beta-3-methoxyestra- 1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343) in which pyrrolidinedione was substituted for pyrroledione. Inhibition by U-73122 was not mediated by an increase in intracellular cyclic AMP. In contrast, the production of inositol 1,4,5-trisphosphate (IP3) and the subsequent rapid increase in cytosolic Ca++ induced by either thrombin or the thromboxane-mimetic, (5Z,9 alpha, 11 alpha, 13E, 15S) 15-hydroxy-11,9-(epoxymethano)prosta- 5,13,-dien-1-oic acid (U-46619), was inhibited by U-73122 but not by U-73343. Reduction of IP3 levels appeared to reflect an inhibition of IP3 production because the hydrolysis of phosphatidyl[3H]inositol and phosphatidyl[3H]inositol 4,5-bisphosphate catalyzed by a soluble fraction from platelets was inhibited by U-73122 (Ki = 9 and 40 microM, respectively). In addition, U-73122 inhibited thromboxane B2 production induced by collagen but not that supported by exogenously added arachidonic acid, suggesting that U-73122 also inhibited receptor-coupled mobilization of arachidonic acid. After preincubation of platelets with [3H]arachidonic acid, the loss of [3H]phosphatidylinositol and accumulation of [3H]phosphatidic acid induced by thrombin was attenuated by U-73122. U-73122 did not inhibit the activities of phospholipases A2 purified either from porcine pancreas or from the venoms of Crotalus adamanteus and Naja naja. Although U-73122 inhibited neither the conversion of exogenous arachidonic acid to thromboxane B2 nor the binding of the thromboxane receptor antagonist [1S-[1 alpha, 2 beta (5Z), 3 beta, 4 alpha]]-7-[3-[[2- [2-[(phenylamino)-carbonyl]- hydrazino]methyl]-7-oxabicyclo [2.2.1]-hept-2-yl-5-heptenoic acid to platelet membranes, it was an effective inhibitor of arachidonic acid-induced aggregation of platelets. These data are consistent with the observed inhibition by U-73122 of platelet activation by the thromboxane receptor agonist, U-46619, via a mechanism that involves inhibition of a phospholipase C-dependent component(s) of signal transduction. U-73122, but not U-73343, inhibited also N-formyl-methionyl-leucyl-phenylalanine-induced aggregation of human polymorphonuclear neutrophils (PMN) and the associated production of IP3 and diacyglycerol. Diradylglycerol produced in PMN stimulated with N-formyl- methionyl-leucyl-phenylalanine was 74 +/- 7% saponifiable and inhibited by U-73122 (Ki = 2 microM). [2]

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The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules. [3]

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U-73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U-73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors [6].

In endotoxemic mice, U73122 TFA markedly enhanced cardiac work, contraction and relaxation rates, without changing heart rate, while it had no effect on sham animals and greatly decreased TNF-α mRNA expression [4]. When compared to vehicle infusion into the VTA, U73122 TFA (400 nM/μL) significantly decreased the overall length of lordosis in hamsters that were induced with estrogen and progesterone. The locomotor behavior of the hamsters in the activity monitor was not affected by the VTA infusion of U73122 TFA; however, muscimol significantly decreased the overall number of beam interruptions when compared to hamsters given SKF38393 [5].

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In Experiment 1, ovariectomised hamsters, primed with oestradiol (10 microg; h 0) + progesterone (100 microg; h 45), were pretested for lordosis and motor behaviour (h 48) and then infused with the PLC inhibitor, U-73122 (400 nM/side), or vehicle. Thirty minutes later, hamsters were retested and then received infusions of SKF38393 (100 ng/side), muscimol (100 ng/side), or vehicle to the VTA. Hamsters were post-tested for lordosis and motor behaviour 30 min later. In Experiment 2, a similar protocol was utilised except that instead of the PLC inhibitor hamsters were infused with the PKC inhibitor, bisindolylmaleimide (75 nM/side). Systemic progesterone, SKF38393-, and muscimol-facilitated lordosis was attenuated by infusion of the PLC inhibitor, U73122, or the PKC inhibitor, bisindolylmaleimide, compared to vehicle to the VTA. Thus, the actions of progestins in the VTA to enhance lordosis through D(1) and/or GABA(A) may include downstream activity of PLC and PKC. [5]

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Experiment 1: effects of U-73122 pretreatment on SKF38393- or muscimol-mediated increases in progesterone-facilitated lordosis of hamsters [5]
Site analyses revealed that 39 hamsters received bilateral infusions to the VTA and three hamsters received bilateral infusions to the substantia nigra. Data from the three hamsters that received bilateral infusions to the substantia nigra were excluded from statistical analyses (data not shown). Hamsters that received infusions to the substantia nigra did not demonstrate similar behavioural patterns as did hamsters that received infusions to the VTA.

Infusions of the PLC inhibitor, U-73122, to the VTA significantly reduced total lordosis durations, compared to vehicle infusions to the VTA, of oestradiol and progesterone-primed hamsters [F(1,111) = 146.44, P < 0.01]. Infusions of the D1 agonist, SKF38393, or the GABAA agonist, muscimol, to the VTA significantly increased total lordosis durations, compared to vehicle infusions to the VTA [F(2,111) = 25.53, P < 0.01]. There was a significant interaction between these variables [F(2,111) = 25.40, P < 0.01] due to U73122, but not vehicle, infusions attenuating progesterone-facilitated lordosis and blocking the enhancing effects of SKF38393 or muscimol on progesterone-mediated lordosis of oestradiol-primed hamsters (Fig. 2).

VTA infusions of U-73122 did not alter motor behaviour of hamsters in the activity monitor, but there was a significant effect of muscimol to decrease total number of beam breaks compared to hamsters administered SKF38393 [F(2,111) = 4.13, P < 0.02](Table 1).

Table 1. Experiment 1: Total Number of Bream Breaks Made in the Horizontal Crossing Chamber of Oestradiol and Progesterone-Primed Hamsters That Received Infusions of U-73122 or Vehicle and SKF38393, Muscimol or Vehicle to the Ventral Tegmental Area.

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To investigate the role of PLCgamma1 in endotoxemia in vivo, wild-type and heterozygous PLCgamma1 knockout (PLCgamma1(+/-)) mice were pre-treated with either U-73122, or its inactive analog U73343, or vehicle for 15 min, followed by LPS for 4 h. Inhibition of PLCgamma1 by U73122 or by heterozygous deletion of the PLCgamma1 gene decreased cardiac TNF-alpha expression. More importantly, LPS-induced myocardial dysfunction was also attenuated in PLCgamma1(+/-) mice or by U73122 treatment. [4]
The role of PLCγ1 in myocardial depression induced by endotoxemia was also examined by using a pharmacological inhibitor, U-73122. Wild-type mice were pre-treated with U73122 or its inactive analogue U73343 (9 mg/kg, i.p.) for 15 min, followed by vehicle or LPS (4 mg/kg, i.p.) for 4 h. Cardiac function was assessed as above. U73122 had no effect on sham animals, but significantly increased heart work and rate of contraction and relaxation without affecting heart rate in endotoxemic mice, compared with U73343 (Figure 6). These data further support the contribution of PLCγ1 to myocardial dysfunction in endotoxemia[4].

Aggregation of human platelets induced by a variety of agonists was inhibited by 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dionel (U-73122 TFA) (IC50 values 1-5 microM), but not by the close analog 1-[6-[[17 beta-3-methoxyestra- 1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343) in which pyrrolidinedione was substituted for pyrroledione. Inhibition by U-73122 TFA was not mediated by an increase in intracellular cyclic AMP. In contrast, the production of inositol 1,4,5-trisphosphate (IP3) and the subsequent rapid increase in cytosolic Ca++ induced by either thrombin or the thromboxane-mimetic, (5Z,9 alpha, 11 alpha, 13E, 15S) 15-hydroxy-11,9-(epoxymethano)prosta- 5,13,-dien-1-oic acid (U-46619), was inhibited by U-73122 TFA but not by U-73343. Reduction of IP3 levels appeared to reflect an inhibition of IP3 production because the hydrolysis of phosphatidyl[3H]inositol and phosphatidyl[3H]inositol 4,5-bisphosphate catalyzed by a soluble fraction from platelets was inhibited by U-73122 TFA (Ki = 9 and 40 microM, respectively). In addition, U-73122 TFA inhibited thromboxane B2 production induced by collagen but not that supported by exogenously added arachidonic acid, suggesting that U-73122 TFA also inhibited receptor-coupled mobilization of arachidonic acid. After preincubation of platelets with [3H]arachidonic acid, the loss of [3H]phosphatidylinositol and accumulation of [3H]phosphatidic acid induced by thrombin was attenuated by U-73122 TFA. U-73122 TFA did not inhibit the activities of phospholipases A2 purified either from porcine pancreas or from the venoms of Crotalus adamanteus and Naja naja. Although U-73122 TFA inhibited neither the conversion of exogenous arachidonic acid to thromboxane B2 nor the binding of the thromboxane receptor antagonist [1S-[1 alpha, 2 beta (5Z), 3 beta, 4 alpha]]-7-[3-[[2- [2-[(phenylamino)-carbonyl]- hydrazino]methyl]-7-oxabicyclo [2.2.1]-hept-2-yl-5-heptenoic acid to platelet membranes, it was an effective inhibitor of arachidonic acid-induced aggregation of platelets. These data are consistent with the observed inhibition by U-73122 TFA of platelet activation by the thromboxane receptor agonist, U-46619, via a mechanism that involves inhibition of a phospholipase C-dependent component(s) of signal transduction. U-73122 TFA, but not U-73343, inhibited also N-formyl-methionyl-leucyl-phenylalanine-induced aggregation of human polymorphonuclear neutrophils (PMN) and the associated production of IP3 and diacyglycerol. Diradylglycerol produced in PMN stimulated with N-formyl- methionyl-leucyl-phenylalanine was 74 +/- 7% saponifiable and inhibited by U-73122 TFA (Ki = 2 microM) [2].

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Sample preparation for mass spectrometry [6]
Purified 5-LO was concentrated and desalted using centrifugal concentrators. The protein was diluted to a final concentration of 150 μg·mL− 1 in PBS pH 7.4 and a solution of 1 mM U-73122 (in ethanol) was added to obtain a final concentration of 30 μM. After incubation for 30 min at 37 °C the protein solution was separated from unbound U-73122 by centrifugal concentrators with 50 mM NH4HCO3 pH 7.8 as washing buffer. Disulfide bonds within the protein were reduced for 1 h at 57 °C in the presence of 5 mM dithiothreitol. Alkylation of free cysteines was achieved by incubation for 1 h with 35 mM iodoacetamide at room temperature in the dark. Excessive iodoacetamide was quenched by addition of dithiothreitol at a final concentration of 35 mM. Digestion of 5-LO was carried out with trypsin in an trypsin/5-LO ratio of 1:50 (w:w) at 37 °C for 12 h. For the wild type 5-LO, a second step of reduction and alkylation was necessary after the digestion in order to enhance the signal of peptides which contain two cysteines in close proximity.

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MALDI-MS [6]
1 μL of the digested sample (~ 3 pmol 5-LO) was mixed on a stainless steel MALDI target with 1 μL of 2.5 mg·mL− 1 CHCA solution in 70% acetonitrile and 0.1% trifluoroacetic acid. After crystallization, MS spectra were acquired using an Ultraflex I MALDI-TOF/TOF instrument). The spectra were internally calibrated. The minimum signal to noise ratio was set to 3 for peak detection and error tolerance up to 30 ppm for peak assignment. Methionine oxidation, pyro-Glu formation from glutamine as well as alkylation of cysteine by U-73122, by carbamidomethylation and pyro-carbamidomethylation were included as optional modifications for peak assignment.

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Radioligand Binding Assays[2]
The binding of either [3H]NMS or [3H]scopolamine to intact cells (resuspended in buffer A) or digitoninpermeabilized cells (resuspended in KGEH buffer) was measured as previously described.

Agonist-induced production of IP3 in PMN is measured by use of the competitive radiobinding assy. PMN (2 x 106-107) in 0.2 mL of phosphate-buffered saline, pH 7.4 [NaC1 (138 mM), Na2HPO4 (8.1 mM), KH2PO4 (1.5 mM), KCI (2.7 mM), CaCl2 (1.0 mM), MgC12 (1.0 mM) and glucose (0.1%, w/v)] are incubated in conical polypropylene tubes at 37°C in a shaking water bath. U-73122 TFA or U-73343 is added (in 1 μL of DMSO) 3 min before the addition of agonist, FMLP (0.1 μM) plus cytochalasin B (5 μg/mL). FMLP and cytochalasin B are added in 1 μL each of DMSO and ethanol, respectively. Appropriate vehicle controls are included in each experiment. PMN incubation mixtures are quenched with the addition of 0.07 mL of ice-cold TCA (20%, w/v) and a portion (0.2 mL) of the TCA extract is processed for the measurement of IP3 by competitive radiobinding as described above for platelets [2].

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Cell Culture Conditions [2]
Human SK-N-SH neuroblastoma cells (passage number unknown) were cultured under conditions that have been previously described. Cells that were 10-20 days postpassage were used for all experiments. After aspiration of culture medium, cells were detached from the tissue culture flasks by the addition of Puck's Dl solution, collected by centrifugation (300 X g for 1 min), and unless stated otherwise, resuspended in buffer A (142 mM NaC1, 5.6 mM KCl, 2.2 mM CaC12, 3.6 mM NaHC03, 1 mM MgCl,, 5.6 mM D-glUCOSe, and 30 mM Na+-HEPES buffer, pH 7.4).

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Measurement of PPI [2]
Hydrolysis-SK-N-SH cells were prelabeled for 2 or 3 days at 37 "C in Dulbecco's modified Eagle's medium/fetal calf serum containing 10 pCi/ml of [3H]inositol in an atmosphere of 90% air, 10% COZ. Under these conditions, the labeling of the inositol lipids achieves isotopic equilibrium. For intact cells, the accumulation of a total [3H]inositol phosphate fraction was monitored in the presence of Li+, as previously described. Identification of the individual inositol phosphate isomers present in such fractions has been reported previously. For measurement of PPI hydrolysis in permeabilized cells, the prelabeled cells were washed once with Puck's Dl solution and then resuspended in KGEH buffer (139 mM K+- glutamate, 2 mM ATP,4 mM MgCl,, 10 mM LiCl, 10 mM EGTA, and 30 mM Na+-HEPES buffer, pH 7.4) containing 20 p~ digitonin. Cells were allowed to permeabilize for 5 min at 37 "C at a protein concentration of approximately 3-4 mg/ml. Permeabilized cells were then centrifuged and washed with an equal volume of KGEH buffer (minus digitonin) and resuspended in the same buffer. Incubations (free [Ca2+] = 60 nM) were routinely allowed to proceed for 30 min, after whlch tlme reactions were terminated and inositol phosphate release quantitated as previously described).

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Measurement of Cytoplasmic Ca2+ Concentrations[2]
Cytoplasmic Caz+ concentrations ([Ca2+Ii) in SK-N-SH cells were determined by monitoring fura-2 fluorescence in a Shimadzu RF-5000 spectrofluorophotometer as previously described, with the exception that the dual wavelength method of Grynkiewicz et al. was routinely employed. Under these conditions, [Caz+]i = (R -R,i,/Rm.. - R) B. Kd, where R, Rmi,, and Rmax are the ratios of the fluorescence obtained at excitation wavelengths of 340 and 380 nm (Xernisaion = 505 nm). B is the ratio of the fluorescence of Ca2+-free/Ca'+-saturated signals at 380 nm, and Kd is the affinity constant of fura-2 for Ca2+ (224 nM). Tissue autofluorescence at either excitation wavelength was negligible (<6%).

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Measurement of Intracellular CAMP[2]
Intact cells (0.3 mg of protein/ml) were incubated with the various agents for 30 min at 37 "C in the presence of 1 mM 3-isobutyl-1-methylxanthine. Reactions were terminated and CAMP extracted and quantitated by means of a radioimmunoassay as previously described.

Hamsters are hormone-primed with 17β-oestradiol at h 0 and progesterone at h 45. At h 48, hamsters are pretested for motor behaviour, followed by sexual behaviour testing, and bilateral infusions of U73122 TFA (400 nM/μL) or saline vehicle. Thirty minutes after infusions, hamsters are re-tested for sexual behaviour (post inhibitor infusion test) and, immediately after testing, infused bilaterally with SKF38393 (100 ng/μL), muscimol (100 ng/μL), or saline vehicle. Thirty minutes after the agonist or vehicle infusions, lordosis and motor behaviour of hamsters is reassessed (post agonist infusion test). All hamsters are assigned to one pretreatment condition, U73122 TFA or vehicle, and are tested once a week for 3 weeks until all infusion conditions (SKF38393, muscimol or vehicle), are received. The order in which hamsters receive SKF38393, muscimol or vehicle infusions is counterbalanced across the group [5]. he PLC inhibitor, U73122, was dissolved in saline at a concentration of 400 nM/µl. The concentration of PLC utilised was based upon published reports and our pilot data [5].

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Effect of U-73122 on myocardial function in endotoxemia. Wild-type mice were pre-treated with either U-73122 or U73343 (9 mg/kg, i.p.) for 15 min, followed by vehicle or lipopolysaccharide (4 mg/kg, i.p.) for 4 h. Changes in heart rate (A), heart work (B), rate of contraction (+dF/dtmax, C) and relaxation (−dF/dtmin, D) are presented. Heart work and rate of contraction and relaxation were significantly improved in U73122 + lipopolysaccharide compared with U73343 + lipopolysaccharide. Data are mean ± SEM, n = 7–9 per group, *P < 0.05 vs. Sham; †P < 0.05 vs. lipopolysaccharide + U73122. [4]

[1]. Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther. 1990 May;253(2):688-97.

[2]. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther. 1990 Nov;255(2):756-68.

[3]. The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation. J Biol Chem. 1991 Dec 15;266(35):23856-62.

[4]. Disruption of phospholipase Cgamma1 signalling attenuates cardiac tumor necrosis factor-alpha expression and improves myocardial function during endotoxemia. Cardiovasc Res. 2008 Apr 1;78(1):90-7.

[5]. In the ventral tegmental area, the membrane-mediated actions of progestins for lordosis of hormone-primed hamsters involve phospholipase C and protein kinase C. J Neuroendocrinol. 2007 Sep;19(9):717-24.

[6]. Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416. Biochim Biophys Acta. 2012 Feb;1821(2):279-86.

[7]. 3Beta-hydroxy-6-aza-cholestane and related analogues as phosphatidylinositol specific phospholipase C (PI-PLC) inhibitors with antitumor activity. Bioorg Med Chem. 2000 Apr;8(4):699-706.

U-73122 is an aza-steroid that is 3-O-methyl-17beta-estradiol in which the 17beta-hydroxy group is replaced by a 6-(maleimid-1-yl)hexylamino group. An inibitor of phospholipase C. It has a role as an EC 3.1.4.11 (phosphoinositide phospholipase C) inhibitor. It is an aza-steroid, a member of maleimides and an aromatic ether. It is functionally related to a 17beta-estradiol.

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1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]- 1H-pyrrole-2,5-dione (U-73122), an inhibitor of phospholipase C (PLC)-dependent processes in human platelets, was found to be a potent inhibitor of human polymorphonuclear neutrophil (PMN) activation by structurally unrelated receptor-specific agonists. U-73122 caused a time- and concentration-dependent (0.1-1 microM) inhibition of myeloperoxidase and vitamin B12-binding protein release from PMNs exposed to N-formyl-methionyl-leucyl-phenylalanine, recombinant human C5a, leukotriene B4 and platelet-activating factor. Activation of the respiratory burst, as measured by superoxide anion production, in PMNs stimulated with these agonists was also suppressed by U-73122. These data suggested that U-73122 inhibited a component of signal transduction that was common to the mechanisms of action of these stimuli. Production of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol and the rise in the cytosolic free calcium concentration, which are early postreceptor events in PMN activation, were all suppressed in U-73122-treated PMNs stimulated with the agonists. These signal transduction events require activation of PLC. Receptor-coupled activation of PLC in membranes isolated from PMNs was potently inhibited by U-73122. U-73122, however, had no direct effect on PMN protein kinase C activity. 1-[6-[[17 beta-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] -2,5- pyrrolidine-dione (U-73343), a close analog of U-73122 that does not suppress PLC activity, did not inhibit receptor-specific agonist-induced PMN responsiveness. U-73122, therefore, is a novel reagent that is useful in investigating PLC function in receptor-mediated PMN activation.[1]

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In conclusion, the results obtained demonstrate that inclusion of the aminosteroid U-73122 leads to an inhibition of both mAChR-stimulated PPI hydrolysis and receptor sequestration in SK-N-SH cells. Because the primary site of action of U-73122 appears to be an inhibition of G, regulation of phospholipase C, we conclude that activation of this G-protein either directly or indirectly is required for mAChR sequestration. [2]

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The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.[3]

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U-73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.[6]

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We could show that U-73122 is a 5-LO inhibitor that covalently and irreversibly binds to Cys416 and that this interaction is essential for inhibition of the human 5-LO enzyme by this compound. The data suggest that the surface area close to Cys416 could be an interesting target for the development of novel 5-LO inhibitors. [6]

C29H40N2O3.CF3COOH

578.66

464.303

C, 74.96; H, 8.68; N, 6.03; O, 10.33

112648-68-7

104794

Off-white to yellow solid

1.2±0.1 g/cm3

617.1±55.0 °C at 760 mmHg

327.0±31.5 °C

0.0±1.8 mmHg at 25°C

1.589

6.59

1

4

9

34

763

5

O(C([H])([H])[H])C1C([H])=C([H])C2=C(C=1[H])C([H])([H])C([H])([H])[C@]1([H])[C@]2([H])C([H])([H])C([H])([H])[C@]2(C([H])([H])[H])[C@]([H])(C([H])([H])C([H])([H])[C@]21[H])N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])N1C(C([H])=C([H])C1=O)=O

LUFAORPFSVMJIW-ZRJUGLEFSA-N

InChI=1S/C29H40N2O3/c1-29-16-15-23-22-10-8-21(34-2)19-20(22)7-9-24(23)25(29)11-12-26(29)30-17-5-3-4-6-18-31-27(32)13-14-28(31)33/h8,10,13-14,19,23-26,30H,3-7,9,11-12,15-18H2,1-2H3/t23-,24-,25+,26+,29+/m1/s1

1-(6-(((8R,9S,13S,14S,17S)-3-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione

U 73122; U73122 TFA; 112648-68-7; U-73122 TFA; U73122 TFA; U 73122; 1-(6-((3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U-73,122; U-73122 TFA hydrate; 1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione; U73122 TFA.

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)

Solubility in Formulation 1: ≥ 0.62 mg/mL (1.33 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 6.2 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: ≥ 0.62 mg/mL (1.33 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 6.2 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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Solubility in Formulation 3: 2% Tween 80+saline: 5mg/mL

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Solubility in Formulation 4: 10 mg/mL (21.52 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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