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This experiment was designed to explore the protection of sphingosine1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation acting via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal pathway. The data showed that S1P could significantly increase cell viability, lower the rate of apoptosis, decrease the content of lactate dehydrogenase (LDH) and caspase3 activity in the culture medium, increase the activity of total superoxide dismutase (T-SOD) and manganese superoxide dismutase (Mn-SOD), reduce the loss of mitochondrial membrane potential and the fluorescence intensity of intracellular calcium, as well as increase the phosphorylation of JAK2 and STAT3 in comparison with the H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Our date shows that S1P protects H9c2 cells from hypoxia/reoxygenation injury and that the protection by S1P was inhibited by AG490 and stattic. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via the JAK-STAT pathway., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Signal transducers and activators of transcription (STATs) comprise a family of transcription factors that are structurally related and which participate in signaling pathways activated by cytokines, growth factors and pathogens. Activation of STAT proteins is mediated by the highly conserved Src homology 2 (SH2) domain, which interacts with phosphotyrosine motifs for specific contacts between STATs and receptors and for STAT dimerization. By generating new models for human (h)STAT1, hSTAT2 and hSTAT3 we applied comparative in silico docking to determine SH2-binding specificity of the STAT3 inhibitor stattic, and of fludarabine (STAT1 inhibitor). Thus, we provide evidence that by primarily targeting the highly conserved phosphotyrosine (pY+0) SH2 binding pocket stattic is not a specific hSTAT3 inhibitor, but is equally effective towards hSTAT1 and hSTAT2. This was confirmed in Human Micro-vascular Endothelial Cells (HMECs) in vitro, in which stattic inhibited interferon-α-induced phosphorylation of all three STATs. Likewise, fludarabine inhibits both hSTAT1 and hSTAT3 phosphorylation, but not hSTAT2, by competing with the highly conserved pY+0 and pY-X binding sites, which are less well-preserved in hSTAT2. Moreover we observed that in HMECs in vitro fludarabine inhibits cytokine and lipopolysaccharide-induced phosphorylation of hSTAT1 and hSTAT3 but does not affect hSTAT2. Finally, multiple sequence alignment of STAT-SH2 domain sequences confirmed high conservation between hSTAT1 and hSTAT3, but not hSTAT2, with respect to stattic and fludarabine binding sites. Together our data offer a molecular basis that explains STAT cross-binding specificity of stattic and fludarabine, thereby questioning the present selection strategies of SH2 domain-based competitive small inhibitors., (© 2013 Elsevier B.V. All rights reserved.)

Neurokinins are known to induce neurogenic inflammation related to respiratory diseases. The effects of CS-003 ([1-{2-[(2R)-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl}spiro[benzo[c]thiophene-1(3H),4'-piperidine]-(2S)-oxide hydrochloride]), a novel triple neurokinin receptor antagonist, on several respiratory disease models were evaluated in guinea pigs. As we have already shown that CS-003 is intravenously effective, we first determined if CS-003 was orally effective. CS-003 dose-dependently inhibited substance P-induced tracheal vascular hyperpermeability, neurokinin A- and neurokinin B-induced bronchoconstriction with ID(50) values of 3.6, 1.3 and 0.89 mg/kg (p.o.), respectively. CS-003 (10 mg/kg, p.o.) inhibited the number of coughs induced by capsaicin aerosol (P<0.01) and the antitussive effect was comparable to that of codeine. CS-003 (10 mg/kg, p.o.) also inhibited airway hyperresponsiveness to methacholine chloride in ovalbumin-induced asthma models (P<0.01), a milder one and a severer one. On the other hand, montelukast (10 mg/kg, p.o.), a leukotriene receptor antagonist, significantly inhibited the hyperresponsiveness only in the milder model (P<0.05). In an ovalbumin-induced rhinitis model, oral administration of CS-003 inhibited nasal blockade in a dose-dependent manner and the inhibitory effect was comparable to that of dexamethasone (10 mg/kg, p.o.). CS-003 (i.v.) also dose-dependently inhibited cigarette smoke-induced bronchoconstriction, tracheal vascular hyperpermeability and mucus secretion. These data show that CS-003, a potent orally active triple neurokinin receptor antagonist, may be useful for the treatment of respiratory diseases associated with neurokinins, such as allergic asthma, allergic rhinitis, chronic obstructive pulmonary disease and cough.

Neurokinins are known to induce neurogenic inflammation related to respiratory diseases, though there is little information on triple neurokinin receptor antagonists. The pharmacological properties of the novel triple neurokinin 1, 2 and 3 receptor antagonist [1-(2-[(2R)-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl)spiro[benzo[c]thiophene-1(3H),4'-piperidine]-(2S)-oxide hydrochloride] (CS-003) were evaluated in this study. The binding affinities of CS-003 were evaluated with human and guinea pig neurokinin receptors. As well, the in vivo antagonism of CS-003 against exogenous neurokinins and effects on capsaicin-induced and citric acid-induced responses were investigated in guinea pigs. CS-003 exhibited high affinities for human neurokinin 1, neurokinin 2 and neurokinin 3 receptors with Ki values (mean+/-S.E.M.) of 2.3+/-0.52, 0.54+/-0.11 and 0.74+/-0.17 nM, respectively, and for the guinea pig receptors with Ki values of 5.2+/-1.4, 0.47+/-0.075 and 0.71+/-0.27 nM, respectively. Competitive antagonism was indicated in a Schild analysis of substance P-, neurokinin A- and neurokinin B-induced inositol phosphate formation with pA2 values of 8.7, 9.4 and 9.5, respectively. CS-003 inhibited substance P-induced tracheal vascular hyperpermeability, neurokinin A- and neurokinin B-induced bronchoconstriction with ID50 values of 0.13, 0.040 and 0.063 mg/kg (i.v.), respectively. CS-003 also inhibited capsaicin-induced bronchoconstriction (ID50: 0.27 mg/kg, i.v.), which is caused by endogenous neurokinins. CS-003 significantly inhibited citric acid-induced coughs and the effect was greater than those of other selective neurokinin receptor antagonists. CS-003 is a potent antagonist of triple neurokinin receptors and may achieve the best therapeutic efficacy on respiratory diseases associated with neurokinins compared to selective neurokinin receptor antagonists.

Partial bladder outlet obstruction of the pig is considered as a valuable preclinical model for evaluating the profile of compounds for the treatment of bladder overactivity. In this study, we characterized the pharmacological properties of isolated bladder smooth muscle from pigs following partial outlet obstruction and its sensitivity to potassium channel openers. Bladder strips from obstructed animals showed significantly lower maximal efficacy (E(max)) and sensitivity to stimulation by ATP and carbachol, but not to those evoked by serotonin, compared to age-matched controls. Tissue strips from obstructed animals also showed a 2.5-fold increase in the potency and significantly reduced maximum response following K+ depolarization. With respect to spontaneous activity, bladder strips from control strips demonstrated little spontaneous phasic activity at all preloads examined. In contrast, bladder strips from obstructed animals showed large preload-dependent increases in spontaneous phasic activity at preload values of 16-32 g. The potencies of K(ATP) channel openers to relax carbachol-evoked contractions showed a good 1:1 correlation (r(2)=0.90) between obstructed and control bladder strips. These studies demonstrate that obstructed pig bladders show enhanced spontaneous phasic activity especially at elevated preloads, which may underlie unstable myogenic bladder contractions reported in cystometrographic measurements in vivo. The impaired responses to electrical field stimulation could be attributed to reduced efficacies and/or lower sensitivities of muscarinic and purinergic signaling pathways. K(ATP) channel sensitivities remain essentially unimpaired in the obstructed bladder and could be effectively modulated by openers with potential for the treatment of overactive bladder secondary to outlet obstruction.

1-[2-[4-(6-fluoro-1H-indol-3-yl)-3,6-dihydro-1(2H)-pyridinyl]ethyl]-3-isopropyl-6-(methylsulphonyl)-3,4-dihydro-1H-2,1,3-benzothiadiazine-2,2-dioxide (LY393558) is a potent inhibitor of [3H]5-hydroxytryptamine ([3H]5-HT) uptake into rat cortical synaptosomes (pIC(50)=8.48+/-0.12). It produces a dextral shift of the 5-HT dose-response curves for the binding of GTPgamma[35S] to human 5-HT(1B) (pK(b)=9.05+/-0.14) and 5-HT(1D) (pK(b)=8.98+/-0.07) receptors and inhibits the contractile response of the rabbit saphenous vein to the 5-HT(1B/D) receptor agonist, sumatriptan (pK(b)=8.4+/-0.2). In addition, it is an antagonist at the 5-HT(2A) (pK(i)=7.29+/-0.19) and 5-HT(2B) (pK(i)=7.35+/-0.11) receptors. Presynaptic autoreceptor antagonist activity was demonstrated by its ability to potentiate the K(+)-induced outflow of [3H]5-HT from guinea pig cortical slices (pEC(50)=7.74+/-0.05 nM) in which the 5-HT transporter had been inhibited by a maximally effective concentration of paroxetine. It is concluded that LY393558 should be an effective antidepressant with the potential to produce an earlier onset of efficacy than selective serotonin uptake inhibitors.

The stimulation of terminal 5-HT(1B/1D) autoreceptors limits the effects of selective serotonin reuptake inhibitors on extracellular levels of 5-hydroxytryptamine (5-HT, serotonin) in vivo. Microdialysis studies show that acute oral administration of LY393558-a 5-HT reuptake inhibitor and antagonist at both the human 5-HT(1B) and 5-HT(1D) receptor-in the dose range 1-20 mg/kg, increases extracellular levels of 5-HT in both the guinea pig hypothalamus and rat frontal cortex. In both species, the levels of 5-HT that were attained were higher than following an acute, maximally effective dose of fluoxetine (20 mg/kg orally), reaching approximately 1500% in the guinea pig hypothalamus and 700% in the rat frontal cortex. In both species, the response to LY393558 (10 mg/kg p.o.) was impulse dependent, being absent in the presence of tetrodotoxin delivered at 1 microM via the microdialysis probe. The sensitivity to tetrodotoxin contrasted with the effects seen with DL-fenfluramine. Studies in rats showed that the microdialysate 5-HT concentration achieved in the frontal cortex after an acute challenge with LY393558 (5 mg/kg p.o.) was significantly greater than following a chronic regime of fluoxetine treatment (10 mg/kg/day orally for 21 days). Moreover, in rats chronically treated with LY393558 (5 mg/kg/day orally for 21 days), the mean basal concentration, 24 h after the final pretreatment dose, was of the same magnitude as that following chronic fluoxetine. However, in contrast to the response seen in fluoxetine-pretreated animals, a challenge dose of LY393558 still elicited a further increase in extracellular 5-HT in LY393558-pretreated animals. LY393558 is a potent 5-HT reuptake inhibitor and 5-HT(1B/1D) receptor antagonist. Microdialysis studies show that acute oral administration increases extracellular levels of 5-HT, by an impulse-dependent mechanism, above those produced by a maximally effective dose of fluoxetine, and in rats to levels only achieved following chronic fluoxetine treatment. Its neurochemical profile in vivo suggests that it may be a more effective antidepressant with the potential for producing an earlier onset of clinical activity than selective serotonin reuptake inhibitors.

The potential antidepressant, LY367265 (1-[2-[4-(6-fluoro-1H-indol-3-yl)-3, 6-dihydro-1(2H)-pyridinyl]ethyl]-5,6-dihydro-1H,4H-[1,2, 5]thiadiazolo[4.3.2-ij]quinoline-2,2,-dioxide) has been shown to have a higher affinity for the 5-hydroxytryptamine (5-HT) transporter (K(i)=2.3 nM) and 5-HT(2A) (K(i)=0.81 nM) receptor than the clinically effective antidepressant, nefazodone. It is a potent inhibitor of [3H]5-HT uptake into rat cortical synaptosomes (IC(50)=3.1 nM) and shows selectivity over that for [3H]noradrenaline (IC(50)>1000 nM). It potentiates potassium-induced [3H]5-HT outflow from prelabelled guinea pig cortical slices both in the presence (EC(50)=950 nM) and absence (EC(50)=250 nM) of a saturating concentration of the 5-HT transport inhibitor, paroxetine, indicating a low level of activity at the 5-HT(1B/1D) autoreceptor. These studies indicate that LY367265 is a putative antidepressant which, because of its 5-HT(2A) receptor antagonist activity, has the potential to produce less sleep disturbance and sexual dysfunction than selective serotonin uptake inhibitors.

In this study, quantitative autoradiography was used to determine the selectivity of RP 62203, a novel naphtosultam derivative, for 5-HT2 receptors in vitro and ex vivo, using [125I]7-amino-8-iodo-ketanserin ([125I]AMIK) and [3H]mesulergine as radioligands. The density of [125I]AMIK or [3H]mesulergine binding sites was determined by quantitative image analysis. In in vitro experiments, RP 62203 displaced [125I]AMIK from 5-HT2 receptors with an IC50 of 0.21 nM in rat frontal cortex. Its affinity for 5-HT1C receptors was 100-fold lower (IC50 25 nM versus [3H]mesulergine in rat choroid plexus). RP 62203 showed moderate affinity for alpha 1-adrenoceptors in the rat thalamus (IC50 14 nM) and for histamine H1 receptors in the guinea-pig cerebellum (IC50 13 nM). The tetrabenazine sites were not affected by RP 62203 at a concentration of 30 nM. In ex vivo experiments, RP 62203 was about 4 times more potent than ritanserin in displacing [125I]AMIK from 5-HT2 receptors (ED50 0.58 mg/kg p.o.). A dose of 10 mg/kg of RP 62203 did not displace [3H]mesulergine from 5-HT1C receptors or [125I]AMIK from alpha 1-adrenoceptors and tetrabenazine sites in the rat brain and from histamine H1 receptors in the guinea-pig brain. These results demonstrate that RP 62203 specifically recognizes 5-HT2 receptors in rodent brain.

There are only a few ligands available for labelling brain receptors simply because in vivo binding requires more severe experimental conditions than in vitro binding. We now describe the in vivo binding properties of [3H]RP 62203, a new potent and selective 5-HT2 antagonist. After intravenous injection into rats, [3H]RP 62203 accumulated predominantly in brain regions containing 5-HT2 receptors, with a frontal cortex/cerebellum ratio of 6 to 7. A good correlation was obtained between the regional distribution of [3H]RP 62203 in the brain and the density of 5-HT2 receptors measured in vitro. In vivo binding of [3H] RP 62203 was saturable in the frontal cortex but not in the cerebellum. The Bmax in the frontal cortex was equal to 42.5 fmol/mg, thus in the same range as was found in vitro. The 5-HT2 selectivity was ascertained by displacement (prevention) experiments; 5-HT2 antagonists or the agonist 2,5-dimethoxy- 4-iodophenylisopropylamine could prevent specific labelling of [3H]RP 62203 only in brain regions containing 5-HT2 receptors. Interestingly, the radioactivity remaining in various brain regions after displacement with pipamperone corresponded exactly to that measured in the cerebellum, with or without pipamperone. In conclusion, [3H]RP 62203 possesses striking properties of in vivo binding which make it a suitable candidate for examining 5-HT2 receptors in human brain by positron emission tomography scanning.

The influence of the novel 5-hydroxytryptamine2 (5-HT2) receptor antagonist, RP 62203, on serotoninergic neurotransmission was investigated using an in vivo electrophysiological technique. For this purpose, the ability of RP 62203 (1-3 mg/kg i.p.) to block the inhibitory responses of prefrontal cortical neurons induced by electrical stimulation of the median raphe nucleus was examined in anesthetized rats. In addition, the effects of RP 62203 on the dopaminergic inhibitory responses induced in the prefrontal cortex by stimulation of the ventral tegmental area and on the enhancement of these responses by lysergic acid diethylamide (LSD, a 5-HT2 receptor agonist, 30 or 60 micrograms/kg i.p.), were analyzed. RP 62203 blocked the inhibition induced by median raphe nucleus stimulation in a dose-dependent and reversible fashion. In contrast, RP 62203 failed to affect the inhibitory responses induced in prefrontal cortical cells by stimulation of the ventral tegmental area. However, the enhancement by LSD of the inhibition induced by stimulation of the ventral tegmental area in the prefrontal cortex was blocked by RP 62203. These results demonstrate that RP 62203 selectively blocks serotoninergic, but not dopaminergic, neurotransmission in the prefrontal cortex.